The Mycota

Edited by
K. Esser and IW. Bennett

Springer-Verlag Berlin Heidelberg GmbH

The Mycota
I Growth, Differentiation and Sexuality
Ed. by IGH. Wesseis and F. Meinhardt

II Genetics and Biotechnology

Ed. by U. Kück

III Biochemistry and Molecular Biology

Ed. by R. Brambl and G Marzluf

IV Environmental and Microbial Relationships

Ed. by D. Wicklow and B. Sädersträm

V Plant Relationships
Ed. by G. Carroll and P. Tudzynski

VI Human and Animal Relationships

Ed. by D.H. Howard and ID. Miller

VII Systematics and Evolution

Ed. by D.I McLaughlin, E.G. McLaughlin, and P.A. Lemke t

VIII Biology of the Fungal Cell

Ed. by R.I Howard and N.A.R. Gow

IX Fungal Associations
Ed. by B. Hock

X Industrial Applications
Ed. by H.D. Osiewacz

XI Agricultural Applications
Ed. by F. Kempken

XII Human Fungal Pathogens

Ed. by IE. Domer and GS. Kobayashi
The Mycota
A Comprehensive Treatise
on Fungi as Experimental Systems
for Basic and Applied Research

Edited by K. Esser and IW. Bennett

XI
Volume Editor:
Agricultural Applications

F. Kempken

With 44 Figures, 2 in Color, and 29 Tables

Springer
Series Editors
Professor Dr. Dr. h.c. mult. KARL ESSER
Allgemeine Botanik
Ruhr-Universität
44780 Bochum, Germany
Tel.: +49(234)32-22211
Fax: +49(234)32-14211
e-mail: Karl.Esser@ruhr-uni-bochum.de
Professor Dr. JOAN W. BENNETI
Department of Cell and Molecular Biology
Tulane University
New Orleans, Louisiana 70118
USA
Tel.: +1(504)865-5546
Fax: +1(504)865-6785
e-mail: jbennett@tulane.edu

Volume Editor
Professor Dr. Frank Kempken
Christian-Albrechts-Universität zu Kiel
Botanisches Institut
Abteilung Botanische Genetik und Molekularbiologie
Olshausenstr. 40
24098 Kiel, Germany
Tel: +49(431) 8804274
Fax: +49(431) 880 4248
e-mail: fkempken@bot.uni-kiel.de

ISBN 978-3-642-07650-3 ISBN 978-3-662-03059-2 (eBook)

DOI 10.1007/978-3-662-03059-2

Library of Congress Cataloging-in-Publication Data

The Mycota. Includes bibliographical references and index. Contents: 1. Growth, differentiation, and sexuality/editors,
J.G.H. Wesseis and F. Meinhardt - 2. Genetics and biotechnology. 1. Mycology. 2. Fungi. 3. Mycology - Research.
4. Research. 1. Esser, Karl, 1924-. H. Lemke, Paul A., 1937- . QK603.M87 1994 589.2

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Series Preface

Mycology, the study of fungi, originated as a subdiscipline of botany and was a descrip-
tive discipline, largely neglected as an experimental science until the early years of
this century. A seminal paper by Blakeslee in 1904 provided evidence for self-
incompatibility, termed "heterothallism", and stimulated interest in studies related to
the control of sexual reproduction in fungi by mating-type specificities. Soon to follow
was the demonstration that sexually reproducing fungi exhibit Mendelian inheritance
and that it was possible to conduct formal genetic analysis with fungi. The names
Burgeff, Kniep and Lindegren are all associated with this early period of fungal
genetics research.
These studies and the discovery of penicillin by Fleming, who shared a Nobel Prize
in 1945, provided further impetus for experimental research with fungi. Thus began a
period of interest in mutation induction and analysis of mutants for biochemical traits.
Such fundamental research, conducted largely with Neurospora crassa, led to the one
gene: one enzyme hypothesis and to a second Nobel Prize for fungal research awarded
to Beadle and Tatum in 1958. Fundamental research in biochemical genetics was
extended to other fungi, especially to Saccharomyces cerevisiae, and by the mid-1960s
fungal systems were much favored for studies in eukaryotic molecular biology and
were soon able to compete with bacterial systems in the molecular arena.
The experimental achievements in research on the genetics and molecular biology
of fungi have benefited more generally studies in the related fields of fungal bio-
chemistry, plant pathology, medical mycology, and systematics. Today, there is much
interest in the genetic manipulation of fungi for applied research. This current
interest in biotechnical genetics has been augmented by the development of DNA-
media ted transformation systems in fungi and by an understanding of gene expression
and regulation at the molecular level. Applied research initiatives involving fungi
extend broadly to areas of interest not only to industry but to agricultural and envi-
ronmental sciences as weB.
It is this burgeoning interest in fungi as experimental systems for applied as weIl
as basic research that has prompted publication of this se ries of books under the title
The Mycota. This title knowingly relegates fungi into aseparate realm, distinct from
that of either plants, animals, or protozoa. For consistency throughout this Series of
Volumes the names adopted for major groups of fungi (representative genera in paren-
theses) are as folIows:

Pseudomycota
Division: Oomycota (Achlya, Phytophthora, Pythium)
Division: Hyphochytriomycota

Eumycota
Division: Chytridiomycota (Allomyces)
Division: Zygomycota (Mucor, Phycomyces, Blakeslea)
Division: Dikaryomycota
VI Series Preface

Subdivision: Ascomycotina
Class: Saccharomycetes (Saccharomyces, Schizosaccharomyces)
Class: Ascomycetes (Neurospora, Podospora, Aspergillus)
Subdivision: Basidiomycotina
Class: Heterobasidiomycetes (Ustilago, Tremella)
Class: Homobasidiomycetes (Schizophyllum, Coprinus)

We have made the decision to exclude from The Mycota the slime molds whieh,
although they have traditional and strong ties to mycology, truly represent nonfungal
forms insofar as they ingest nutrients by phagocytosis, lack a cell wall during the assim-
ilative phase, and clearly show affinities with certain protozoan taxa.
The Series throughout will address three basic questions: what are the fungi, what
do they do, and what is their relevance to human affairs? Such a focused and com-
prehensive treatment of the fungi is long overdue in the opinion of the editors.
A volume devoted to systematies would ordinarily have been the first to appear
in this Series. However, the scope of such a volume, coupled with the need to give
serious and sustained consideration to any reclassification of major fungal groups, has
delayed early publication. We wish, however, to provide a preamble on the nature of
fungi, to acquaint readers who are unfamiliar with fungi with certain characteristics
that are representative of these organisms and which make them attractive subjects
for experimentation.
The fungi represent a heterogeneous assemblage of eukaryotic microorganisms.
Fungal metabolism is characteristically heterotrophic or assimilative for organic car-
bon and some none lernen tal source of nitrogen. Fungal cells characteristically imbibe
or absorb, rather than ingest, nutrients and they have rigid cell walls. The vast major-
ity of fungi are haploid organisms reproducing either sexually or asexually through
spores. The spore forms and details on their method of production have been used
to delineate most fungal taxa. Although there is a multitude of spore forms, fungal
spores are basically only of two types: (i) asexual spores are formed following mitosis
(mitospores) and culminate vegetative growth, and (ii) sexual spores are formed fol-
lowing meiosis (meiospores) and are borne in or upon specialized generative struc-
tures, the latter frequently clustered in a fruit body. The vegetative forms of fungi are
either unicellular, yeasts are an example, or hyphal; the latter may be branched to form
an extensive mycelium.
Regardless of these details, it is the accessibility of spores, especially the direct
recovery of meiospores coupled with extended vegetative haploidy, that have made
fungi especially attractive as objects for experimental research.
The ability of fungi, especially the saprobic fungi, to absorb and grow on rather
simple and defined substrates and to convert these substances, not only into essential
metabolites but into important secondary metabolites, is also noteworthy. The meta-
bolic capacities of fungi have attracted much interest in natural products chemistry
and in the production of antibiotics and other bioactive compounds. Fungi, especially
yeasts, are important in fermentation processes. Other fungi are important in the
production of enzymes, citric acid and other organic compounds as weH as in the
fermentation of foods.
Fungi have invaded every conceivable ecological niche. Saprobie forms abound,
especiaHy in the decay of organie debris. Pathogenic forms exist with both plant and
animal hosts. Fungi even grow on other fungi. They are found in aquatie as weH as soil
environments, and their spores may pollute the air. Some are edible; others are poi-
sonous. Many are variously associated with plants as copartners in the formation of
lichens and mycorrhizae, as symbiotic endophytes or as overt pathogens. Association
with animal systems varies; examples include the predaceous fungi that trap nema-
todes, the microfungi that grow in the an aerobic environment of the rumen, the many
 Series Preface VII

insectassociated fungi and the medically important pathogens affiicting humans. Yes,
fungi are ubiquitous and important.
There are many fungi, conservative estimates are in the order of 100000 species,
and there are many ways to study them, from descriptive accounts of organisms found
in nature to laboratory experimentation at the cellular and molecular level. All such
studies expand our knowledge of fungi and of fungal processes and improve our ability
to utilize and to control fungi for the benefit of humankind.
We have invited leading research specialists in the field of mycology to contribute
to this Series. We are especially indebted and grateful for the initiative and leadership
shown by the Volume Editors in selecting topics and assembling the experts. We have
all been a bit ambitious in producing these Volumes on a timely basis and therein
lies the possibility of mistakes and oversights in this first edition. We encourage the
readership to draw our attention to any error, omission or inconsistency in this Series
in order that improvements can be made in any subsequent edition.
Finally, we wish to acknowledge the willingness of Springer-Verlag to host this
project, which is envisioned to require more than 5 years of effort and the publication
of at least nine Volumes.

Bochum, Germany KARL ESSER

Auburn, AL, USA PAUL A. LEMKE
April 1994 Series Editors
Addendum to the Series Preface

In early 1989, encouraged by Dieter Czeschlik, Springer-Verlag, Paul A. Lemke and

I began to plan The Mycota. The first volume was released in 1994, other volumes
followed in the subsequent years. Also on behalf of Paul, I would like to take
this opportunity to thank Dieter Czeschlik, his coHeague Andrea Schlitzberger, and
Springer-Verlag for their help in realizing the enterprise and for their exceHent co-
operation for many years.
Unfortunately, after a long and serious illness, Paul A. Lemke died in November
1995. Without his expertise, his talent for organization and his capability to grasp the
essentials, we would not have been able to work out a concept for the volumes of the
series and to acquire the current team of competent volume editors. He was an out-
standing scientist interested in many fields. Together with the volume editors, authors,
and Springer-Verlag, I mourn the loss of a very good and reliable friend and colleague.
Since the first Volumes of The Mycota were weH accepted by the scientific com-
munity, the publisher suggested to extend this series. For Volumes X, XI and XII I was
able to win Joan W. Bennett as serial co-editor.

Bochum, Germany KARL ESSER

New Orleans, LA, USA JOAN W. BENNETI
July 2001
Volume Preface

The development of agriculture was an essential prerequisite to the establishment of

permanent settlements and eventually complex human society. At all times, and pos-
sibly even more so now, humanity depended on the annual crop yield, which may be
influenced by weeds, pathogens or poor weather conditions. Fungi are important plant
pathogens and can reduce yield significantly. In fact, many examples can be cited where
fungal pathogens have actually made history, e.g., the infections by the ergot fungus
Claviceps purpurea causing ergotismen in the middle ages or the disastrous Phytoph-
thora infections of potato in nineteenth century Ireland, leading to the emigration of
millions of Irish people to the United States of America. In addition, the occurrence
of mildew can severally spoil food and fodder and mycotoxins produced by these fungi
may cause illness or even death. Clearly, fungi had and still have a tremendous impact
on humanity. However, while only a minority of fungi are pathogens, many others can
be quite useful, e.g., to nutritionally enrich straw or to ferment food and drink.
In this volume the relevance of fungi for agriculture is discussed in 18 chapters,
wh ich are divided into four sections: (1) food and fodder production, (2) mycotoxins
and detoxification, (3) disease control, diagnostic, and management, and finally (4)
update on host-parasite interactions.
Chapters 1-3 discuss various aspects of food and fodder production, featuring the
application and potential of mushrooms, straw enrichment and food or crop spoilage.
The first article by Paul Horgan and Alan Castle provides insight into the use and
genetics of mushrooms, especially Agaricus. Dusan Jale contributed a chapter about
straw enrichment by fungi, giving many details about this field of research. Jan
Dijksterhuis and Rob Samson present the current knowledge about the important
problem of food and crop spoilage. I should add that other aspects of food production
have already been reviewed in Vol. X (Industrial Applications) of The Mycota and are
therefore not covered again here.
The second section contains two chapters which are devoted to knowledge about
the biosynthesis of mycotoxins and the use of fungi in organopollutant degradation.
Contamination with mycotoxins of course is a problem, particularly in humid climates,
and may increase when anti-fungal agents are not available or are rejected, as is the
case in organic farming. Nancy Keller and colleagues provide a detailed insight into
the synthesis of some mycotoxins. Organopollutant degradation, the second chapter in
this section, has a high potential for future pollution management and was written by
Daniel Cullen.
As fungal phytopathogens are of great concern in agriculture, a large section of
this volume deals with various aspects of biological control (three chapters), diagnos-
tics (two chapters) and disease management (three chapters). Fungal biological control
is covered by Yigal Elad and Stanley Freeman, insect control by Tariq Butt and weed
control by Harry Evans.
Disease control is another focus with emphasis on the example of Magnaporthe
grisea given by Nicholas Talbot. Another chapter, by Diana Fernandez and Thierry
Langin, deals with the use of repeated DNA and transposons as diagnostic tools, a
rather recent development. Finally, disease management is covered in three chapters
XII \1alurne Preface

dealing with important fungal pathogens such as Phoma, by Kerstin Voigt and
Johannes Wöstemeyer, Fusarium, by Kerstin Voigt, and rusts and powdery mildew by
Holger Deising and collaborators.
In the fourth section, host-parasite interactions are the main topic. This section
has been named "update on ..." to acknowledge work presented in a previous volume
of The Mycota (Vol. V). In this volume, five chapters present the current knowl-
edge in this important field discussing relevant issues like signal transduction by
Michael Bölker, avirulence determinants by Wolfgang Knogge, phytotoxins by Dan
Panacchione and colleagues, and cell wall degradation, by Jan van Kan and collabora-
tors. The final chapter, written by Jacques Mugnier, gives insight into the co-evolution
of pathogenic fungi and grass hosts.
As this volume is restricted in size, certainly not all aspects of fungal applications
in agriculture are covered. However, the 18 chapters provide an important insight into
this area of research and, I sincerely hope that it will serve as a guide for readers from
outside the field and as a valuable reference for those to this type of research.
Finally, I wish to express my gratitude to all contributors to this volume.

Kiel, Germany, April 2002 FRANK KEMPKEN

Volume Editor
Contents

Food and Fodder Production

1 Application and Potential of Molecular Appraaches to Mushrooms

PAUL A. HORGEN and ALAN CASTLE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

2 Straw Enrichment for Fodder Production by Fungi

DUSAN JALC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

3 Food and Crop Spoilage on Storage

JAN DIJKSTERHUIS and ROBERT A. SAMSON 39

Mycotoxins and Detoxification

4 Genetics and Biosynthesis of Aflatoxins and Sterigmatocystin

JULIE K. HICKS, KIMINORI SHIMIZU, and NANCY P. KELLER .............. 55

5 Molecular Genetics of Lignin-Degrading Fungi and Their Applications

in Organopollutant Degradation
DANIEL CULLEN .............................................. 71

Disease Control, Diagnostic, and Management

6 Biological Contral of Fungal Plant Pathogens

YIGAL ELAD and STANLEY FREEMAN ............................... 93

7 Use of Entomogenous Fungi for the Control of Insect Pests

TARIQ BUTT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111

8 Biological Control of Weeds

HARRY C. EVANS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135

9 Molecular Variability Studies of Magnaporthe grisea

and Their Application in Disease Contral
NICHOLAS 1. TALBOT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153

10 Transposable Elements in Fungal Pathogens: New Diagnostic Tools

DIANA FERNANDEZ and THIERRY LANGlN ........................... 171

11 Disease Management of Phoma Infections

KERSTIN VOIGT and JOHANNES W. WÖSTEMEYER . . . . . . . . . . . . . . . . . . . . . . . 193

12 Management of Fusarium Diseases

KERSTIN VOIGT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 217
XIV Contents

13 Disease Management of Rusts and Powdery Mildews

HOLGER B. DEISING, SVEN REIMANN, ANDREAS PEIL,
and W. EBERHARD WEBER . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 243

Update on Host-Parasite Interactions

14 Signal Transduction Pathways in Phytopathogenic Fungi

MICHAEL BÖLKER ............................................. 273

15 Avirulence Determinants and Elicitors

WOLFGANG KNOGGE 289

16 Fungal Phytotoxins
DANIEL G. PANACCIONE, R. D. JOHNSON, JACK B. RAs MUS SEN,
and T.L. FRIESEN. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 311

17 The Contribution of Cell Wall Degrading Enzymes to Pathogenesis

of Fungal Plant Pathogens
ARJEN TEN HAVE, KLAUS B. ThNBERGE, JACQUES A. E. BENEN,
PAUL TuDZYNSKI, JAAP VISSER, and JAN A. L. VAN KAN . . . . . . . . . . . . . . . . . 341

18 Coevolution of Pathogenic Fungi and Grass Hosts

JACQUES MUGNIER . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 359

Biosystematic Index. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 375

Subject Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 381

List of Contributors

J.A.E. BENEN (e-mail: jac.benen@algemeen.micr.wau.nl. Tel.: +31-317-483240,

Fax: +31-317-484011) Wageningen University, Laboratory of Microbiology,
Dreijenlaan 2, 6703 HA Wageningen, The Netherlands

M. BÖLKER (e-mail: boelker@mailer.uni-marburg.de, Tel.: +49-6421-2821536, Fax:

+49-6421-2828971) Universität Marburg, Fachbereich Biologie, Karl-von-Frisch-
Strasse 8, 35032 Marburg, Germany

T.M. BUTT (e-mail: t.butt@swansea.ac.uk, Tel.: +44-1792-295374, Fax: +44-1792-

295447) School of Biological Sciences, University of Wales Swansea, Singleton Park,
Swansea SA2 8PP, UK

A. CASTLE (e-mail: acastle@spartan.ac.brocku.ca. Tel.: +1-905-6885550 ext. 3598,

Fax: +1-905-6881855) Department of Biological Sciences, Brock University, St.
Catherines, Ontario, L2S 3A1, Canada

D. CULLEN (e-mail: dcullen@facstaff.wisc.edu, Tel.: +1-608-2319468, Fax: +1-608-

2319488) USDA, Forest Service, Forest Products Laboratory, One Gifford Pinchot
Dr., Madison, WI 53705, USA

H.B. DEISING (e-mail: deising@landw.uni-halle.de, Tel.: +49-345-5522660, Fax: +49-

345-5527120) Martin-Luther-University Halle-Wittenberg, Faculty of Agriculture,
Department of Plant Breeding and Plant Protection, Ludwig-Wucherer-Str. 2,
D-06099 Halle (Saale), Germany

J. DIJKSTERHUIS (e-mail: dijksterhuis@cbs.knaw.nl, Tel.: +31-30-2122654, Fax: +31-30-

2512097) Centraalbureau voor Schimmelcultures (CBS), Uppsalalaan 8,3584 CT
Utrecht, The Netherlands

Y. ELAD (e-mail: elady@netvision.net.il, Tel.: +972-3-9683580, Fax: +972-3-9683688)

Department of Plant Pathology, ARO, The Volcani Center, Bet Dagan 50250, Israel

H.C. EVANS (e-mail: h.evans@cabi.org, Tel.: +44-1491-829129, Fax: +44-1491-829123)

CABI Bioscience, Silwood Park, Ascot, Berks. SL5 7TA, UK

D. FERNANDEz (Diana.Fernandez@mpLird.fr, Tel.: +33-467-416287, Fax: +33-467-

416283) Unite Resistance des Plantes aux Parasites (UR 075), Institut de Recherche
pour le Developpement (IRD), 911 avenue Agropolis, BP5045, 34032 Montpellier,
France

S. FREEMAN (e-mail: freeman@netvision.net.il, Tel.: +972-3-9683537, Fax: +972-3-

9683543) Department of Plant Pathology, ARO, The Volcani Center, Bet Dagan
50250, Israel
XVI List of Contributors

T.L. FRIESEN (e-mail: friesent@fargo.ars.usda.gov, Tel.: +1-701-2391456, Fax: +1-701-

2391350) Department of Plant Pathology, North Dakota State University, Fargo,
North Dakota 58105, USA

IK. HICKS (e-mail: Lhicks31@hotmail.com, Fax: +1-919-6843036) Duke University

Medical Center, 322 CARL Building, Research Drive, Box 3546, Durham, North
Carolina 27710, USA

P.A. HORGEN (e-mail: phorgen@erin.utoronto.ca, Tel.: +1-905-8285424, Fax: +1-905-

5694738) Department of Botany, Erindale Campus, University of Toronto,
Mississauga, Ontario L5L 1C6, Canada

n JALC (e-mail: jalcd@saske.sk, Tel.: +421-55-6336251, Fax: +421-55-6782162)

Institute of Animal Physiology, Slovak Academy of Sciences, Soitesovej 4-6, 04001
Kosice, Slovak Republic

R.n JOHNSON (e-mail: rjohns19@wvu.edu, Tel.: +1-304-2933911, Fax: +1-304-2932872)

Division of Plant and Soil Sciences, West Virginia University, Morgantown, West
Virginia 26506, USA

N.P. KELLER (e-mail: npk@plantpath.wisc.edu, Tel.: +1-608-2629795, Fax: +1-608-

2632626) Department of Plant Pathology, University of Wisconsin-Madison, 1630
Linden Drive, Madison, Wisconsin 53706, USA

W. KNOGGE (e-mail: wolfgang.knogge@adelaide.edu.au, Tel.: +61-8-83036822, Fax:

+61-8-83037109) Department of Plant Science, Adelaide University, GIen Osmond,
SA 5064, Australia

T. LANGIN (e-mail: langin@ibp.u-psud.fr, Tel: +33-1-69153367, Fax: +33-1-69336424

Laboratoire de Phytopathologie Moleculaire, Institut de Biotechnologie des Plantes
(IEP), Bat. 630, Universite Paris-Sud, 91405 Orsay Cedex, France

I MUGNIER (e-mail: jacques.mugnier@agroservices.net, Tel.: +33-437-478800, Fax: +33-

472-430134) Laboratoires Agroservices Aventis Crop Science France, 11 avenue
Albert Einstein, F-69100 Villeurbanne, France

nG. PANACCIONE (e-mail: danpan@wvu.edu, Tel.: +1-304-2933911, Fax: +1-304-

2932872) Division of Plant & Soil Sciences, West Virginia University, 401 Brooks
Hall, Morgantown, West Virginia 26506, USA

A. PEIL (e-mail: peil@landw.uni-halle.de, Tel.: +49-345-5522682, Fax: +49-345-

5527222) Martin-Luther-University Halle-Wittenberg, Faculty of Agriculture,
Department of Plant Breeding and Plant Protection, Ludwig-Wucherer-Str. 2, 06099
Halle (Saale), Germany

IB. RASMUSSEN (e-mail: Jack_Rasmussen@ndsu.nodak.edu, Tel.: +1-701-2311027,

Fax: +1-701-2317851) Department of Plant Pathology, North Dakota State
University, Fargo, North Dakota 58105, USA

S. REIMANN (e-mail: reimann@landw.uni-halle.de, Tel.: +49-345-5522666, Fax: +49-345-

5527120) Martin-Luther-University Halle-Wittenberg, Faculty of Agriculture,
Department of Plant Breeding and Plant Protection, Ludwig-Wucherer-Str. 2, 06099
Halle (Saale), Germany
 List of Contributors XVII

R.A. SAMSON (e-mail: samson@cbs.knaw.nl, Tel.: +31-30-2122600, Fax: +31-30-

2512097) Centraalbureau voor Schimmelcultures (CBS), Uppsalalaan 8,3584 CT
Utrecht, The Netherlands

K SHIMIZU (e-mail: kshimizu@ myco.pf.chiba-u.ac.jp, Tel.: +81-43-2265927, Fax: +81-

43-2265927) Chiba University, Research Center for Pathogenic Fungi and Microbial
Toxicoses, 1-8-1 Jnohana, Chuo-ku, Chiba 260-8673, Japan

N.J. TALBOT (e-mail: n.j.talbot@exeter.ac.uk, Tel.: +44-1392-264673, Fax: +44-1392-

264668) School of Biological Sciences, University of Exeter, Washington Singer
Laboratories, Perry Road, Exeter EX4 4QG, UK

KB. TENBERGE (e-mail: tenberg@uni-muenster.de, Tel.: +49-251-8324812, Fax: +49-

251-8323823) Westfälische Wilhelms-Universität Münster, Institut für Botanik,
Schloßgarten 3,48149 Münster, Germany

A. TEN HAvE (e-mail: arjen.tenhave@fyto.dpw.wau.nl. Tel.: +31-317-483126,

Fax: +31-317-483412) Wageningen University, Laboratory of Phytopathology,
Binnenhaven 5, 6709 PD Wageningen, The Netherlands

P. TUDZYNSKI (e-mail: tudzyns@uni-muenster.de, Tel.: +49-251-8324998, Fax: +49-251-

8323823) Westfälische Wilhelms-Universität Münster, Institut für Botanik,
Schloß garten 3,48149 Münster, Germany

J.A.L VAN KAN (e-mail: jan.vankan@fyto.dpw.wau.nl, Tel.: +31-317-483126, Fax: +31-

317-483412) Wageningen University, Laboratory of Phytopathology, Binnenhaven 5,
6709 PD Wageningen, The Netherlands

J. VISSER FGT, PO Box 396, 6700 AJ Wageningen, The Netherlands

K VOIGT (e-mail: b5kevo@rz.uni-jena.de, Tel.: +49-3641-949321, Fax: +49-3641-

949322) Pilz-Referenz-Zentrum, Institut für Mikrobiologie, Friedrich-Schiller-
Universität, Neugasse 24,07743 Jena, Germany

W.E. WEBER (e-mail: weber@landw.uni-halle.de, Tel.: +49-345-5522680, Fax: +49-345-

5527222) Martin-Luther-University Halle-Wittenberg, Faculty of Agriculture,
Department of Plant Breeding and Plant Protection, Ludwig-Wucherer-Str. 2,06099
Halle (Saale), Germany

J.w. WÖSTEMEYER (e-mail: b5wojo@rz.uni-jena.de, Tel.: +49-3641-949310, Fax: +49-

3641-949312) Lehrstuhl für Allgemeine Mikrobiologie und Mikrobengenetik, Institut
für Mikrobiologie, Friedrich-Schiller-Universität, Neugasse 24,07743 Jena, Germany
Food and Fodder Production
1 Application and Potential of Molecular Approaches to Mushrooms
PAUL A. HORGEN 1 and ALAN CASTLE2
CONTENTS
1. Edible Mushrooms on a Global Scale . . . . . . .
A. Specialty Mushrooms . . . . . . . . . . . . . . . . . . . .
B. White Button Mushroom. Agaricus
bisporus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
11. The State of Breeding and Strain
Improvement at the Turn of the Century ....
A. Breaking Down Life Cycle Barriers ........
B. Genomics in Mushrooms . . . . . . . . . . . . . . . . .
C. The State of A. bisporus Nuclear DNA . . . . . .
1. Heterothallic Forms of A. bisporus
in Nature. . . . . . . . . . . . . . . . . . . . . . . . . . .
2. Somatic Contact and the Potential
for Breeding ........................
D. The Mitochondrial Genome and Its Potential
Role in Strain Performance . . . . . . . . . . . . . . .
E. Extrachromosomal Elements in Mitochondria
of Agaricus Species .... . . . . . . . . . . . . . . . . .
F. Transformation in Agaricus . . . . . . . . . . . . . . .
III. Genetic Improvement of A. bisporus:
Perspectives for the Future . . . . . . . . . . . . . . .
IV. Conclusions ...........................
References . . . . . . . . . . . . . . . . . . . . . . . . . . . .
J. Edible Mushrooms on aGIobai Scale
The information that will be discussed in this
chapter deals mainly with the white button mush-
room, Agaricus bisporus, but we will also present
some background information on other mush-
rooms. There are more than 38,000 kinds of mush-
rooms in the world and these vary considerably in
color, size and shape. Only a fraction of these
38,000+ types of mushrooms are grown or har-
vested for commercial purposes. The most signifi-
cant of these inc1ude the specialty mushrooms and
the white button mushroom.
1 Department of Botany, Erindale Campus, University of
Toronto, Mississauga, Ontario L5L 1C6, Canada
2 Department of Biological Sciences, Brock University, St.
Catharines, Ontario L2S 3A1, Canada
A. Specialty Mushrooms
3
3 Specialty or exotic varieties of mushrooms are
commercially grown in the United States, Western
4
Europe and the Orient, as well as in other parts of
4 the world. These exotic mushrooms inc1ude
4 Shiitake (Lentinula edodes), Maitake (Grifola
8 frondosa) , Nameko (Pholiota nameko), Enoki
8
(Flammulina velutipes), Pom Pom (Hericium eri-
8 naceus), Oyster (Pleurotus spp.), Portabella,
Crimini (Agaricus bisporus) , and others. World-
9 wide production has steadily increased over the
9 last 30-40 years as these varieties have gained in
popularity. Shiitake, Portabella, and Oyster are the
12 most popular, followed by the Enoki, Maitake,
12
Nameko and Pom Pom (Molin 1995). USDA
13 (United States Department ofAgriculture) figures
14 have indicated that the volume of sales for com-
14 mercially grown specialty mushrooms in the US
market has tripled over the last decade. The value
of the sales rose in 1999 for Shiitake mushrooms
to 8.24 million pounds, Oyster mushrooms to 3.53
million pounds and all other specialty mushroom
sales totaled 1.20 million pounds (USDA
Mushroom Industry Report 1999).
Growers and breeders of specialty mush-
rooms and of A. bisporus are faced with very
similar ehallenges. These inc1ude identifying novel
substrates for growth and production, optimizing
bioeonversion of these substrates, controlling dis-
eases and pests, developing characteristics of agro-
nomie importance, inc1uding improved rate of
production and post-harvest quality, and offering
mushroom consumers increased choice. In addi-
tion, interest in the medicinal properties of spe-
cialty mushrooms is growing. Molecular genetic
approaches to the challenges and opportunities
confronting the specialty mushroom industry par-
allel efforts on A. bisporus. Moleeular eharaeteris-
ties are being developed and mapped relative to
desired traits; novel strains and crosses are veri-
fied with these markers and transformation pro-
cedures are optimized and applied to specific
The Mycota XI
Agricultural Applications
Kempken (Ed.)
© Springer-Verlag Berlin Heidelberg 2002
4 p.A. Horgen and A. Castle
situations. The reader is directed to the following
references for details and specific goals of research
programs on specialty mushrooms (Royse 1997;
Chen et al. 2000; Honda et al. 2000; Lee et al. 2000;
Ramirez et al. 2000).
B. White Button Mushroom, Agaricus bisporus
The cultivation of the white button mushroom is
by far the most successful component of the mush-
room industry worldwide. Production in 1999 was
over 2 million metric tons with a retail value in
excess of US $10 billion (Faostat Database 2000).
The his tory of mushroom cultivation is extensive,
dating back to the seventeenth century, when the
industry first started in France (van Griensven
1988; Khush et al. 1995). During the last 300 years,
three major events have dramatically affected the
mushroom industry to position it where it is as we
enter the new millennium.
• The first was the development of mushroom
spawn in the late 1800s. This allowed for a new
companion industry to develop (the spawn
industry which is the "seed" component of
mushroom production), which ensured the
delivery of "pure, disease-free" strains (van
Griensven 1988; Khush et al. 1995).
• The second was the study and development of
effective composting technology, which really
was advanced in the 1940s and 1950s. This is an
area where the industry still expends consider-
able resources as we enter the new century.
Because of the semi-sterile nature of mushroom
cultivation, there is a prevailing feeling within
the white button mushroom industry that virtu-
ally all problems that develop can be managed,
controlled and solved by manipulation of the
compost and the growth environment.
• The third most significant development in the
button mushroom industry was the selection of
the Horst V1 (and also U3) spawn strains in
1978 by Gerda Fritsche (see review, Fritsche
1991). Because of the nature of the A. bisporus
life cycle, strain improvement and breeding
have been problematic over the last three cen-
turies. The most successful and dramatic
improvement in A. bisporus resulted from the
least complex of breeding strategies, selection
and maintenance of phenotypic variants. This
important advancement dramatically illustrated
the potential that a sustained breeding effort
(Fritsche 1983) could bring to this enormously
profitable industry.
A complete review of these three developments is
described by Khush et al. (1995). Although the
sustained breeding effort that Fritsche described
has not occurred, this article will deal mainly
with the development and application of modern
genetic approaches to mushroom strain improve-
ment which could greatly add to this effort. We will
mainly focus on Agaricus bisporus, but have also
provided some background information of spe-
cialty mushrooms. For general reviews of mush-
room breeding, see Raper (1985); Fritsche (1991);
Horgen and Anderson (1993); Khush et al. (1995);
Stoop and Mooibroek (1999); and Honda et al.
(2000). Recent references on breeding Agaricus
bisporus include Kerrigan (2000), Loftus et al.
(2000) and Sonnenberg (2000). All are included in
the Proceedings of the 15th International Con-
gress on the Science and Cultivation of Edible
Fungi (Van Griensven 2000). Numerous other
papers on specific aspects of the cultivation and
breeding of several mushroom species are
included.
11. The State of Breeding and
Strain Improvement at the Turn
of the Centuty
A. Breaking Down Life eyde Barriers
Early advances in the und erst an ding of A.
bisporus breeding and genetics were made by
Charles Miller, Carlene Raper, and Tim Elliott
(see review by Khush et al. 1995). The most serious
issue relating to the difficulties in breeding A. bis-
porus during the last century has been the secon-
darily homothallic life cyde of the fungus. Most
basidiospores receive two post-meiotic nuclei
(reviewed by Khush et al. 1995). Furthermore, all
of the prevailing data suggests that post-meiotic
events are non-random (Royse and May 1982)
such that non-sister nuclei, i.e., those that contain
non-sister chromatids, are preferentially packaged
into the binucleate basidiospores (Khush et al.
1995). In addition, all data collected to date
suggest that crossing-over events in meiosis are
extremely rare in A. bisporus. The combination of
non-random post-meiotic nuclear packaging and a
greatly reduced frequency of crossing-over results
 Application and Potential of Molecular Approaches to Mushrooms
in over 90% of Iod that are heteroallelie in both
parents and offspring (see review by Khush et al.
1995). The mating type locus is also maintained in
a heteroallelic state and most basidiospores
produce a fertile mycelium upon germination. At
no point in the life cycle, with the exception of
relatively rare basidiospores, does this species
produce a haploid, monokaryotic cell which can be
readily crossed with an appropriate breeding
partner. These data would all suggest that tradi-
tional breeding strategies with the button mush-
room are extremely slow and laborious, but
nevertheless could result in improved strains
(Fritsche 1983; Raper 1985).
After the pioneering work of Miller, Raper
and Elliott, the first major advancement in the
development of a modern breeding pro gram came
in identifying stable genetie markers that could be
followed in crosses. The classic isozyme work of
May and Royse (1981) and Royse and May (1982)
were the first studies in which multiple markers
were presented. This work resulted in the identifi-
cation of approximately 10 useful isozyme loci
(Khush et al. 1995). The development and adop-
tion of DNA-based markers greatly increased the
number of characters that could be exploited
(Castle et al. 1987,1988; Loftus et al. 1988; Horgen
and Anderson 1993). Restriction fragment length
polymorphisms (RFLPs), and later randomly
amplified polymorphie DNA (RAPDs ) and
amplified fragment length polymorphisms
(AFLPs), both genetic markers produced by the
polymerase chain reaction (PCR), potentially pro-
vided an unlimited number of unique markers
(Khush et al. 1992). These genetically undefined
markers were so on followed by gene markers,
which continue to be added each year
(Sonnenberg et al. 1996, 1999; Ospina-Giraldo et
al. 2000). There are now several hundred genetic
markers deposited in GenBank (for a list of
accession numbers, access the website <url>http://
www.ncbi.nlm.nih.gov/Genbank/</url> and
search the protein or nucleotide database with
the keyword "Agaricus"). An initial genetic map
was published of the A. bisporus genome
(Kerrigan et al. 1993) with RFLP, RAPD, and
isozyme markers. The map continues to evolve
and expand with the addition of other defined
genetic loci (Horgen et al. 1996; Imbernon et al.
1996; Callac et al. 1997).
A second important advancement in moving
the button mushroom towards a rational breeding
strategy was the increase in our collective under-
5
standing of the meiotic process. The secondarily
homothallic life cycle was described by early
studies prior to the establishment of DNA-based
markers (Evans 1959; Miller 1971) which were
used to elucidate the unusual events occurring
surrounding meiosis. Studies using RFLP markers
(Summerbell et al. 1989; Allan et al. 1992) and
PCR-based markers (Khush et al. 1992) recon-
firmed that the isolates of A. bisporus studied
favored a life style termed "intramixus" by
Kerrigan (1990). Because meiosis was so unusual
in this species, Allen et al. (1992) hypothesized
that perhaps it rarely occurred during the pro duc-
tion of basidiospores. Their results, however,
instead established that meiosis does predominate
(Allen et al. 1992).1t was suggested that low levels
of recombination are common and that non-
random chromosome segregation occurs. In the
most detailed study of meiosis to date, Kerrigan et
al. (1993) reported that chromosome segregation
in meiosis I is random. We have established that
independent assortment occurs by the non-
parental genotypes observed in homokaryotic
spores, or in protoplast regenerates of germinated
heterothallic spores (Summerbell et al. 1989; Allen
et al. 1992; Khush et al. 1992; Kerrigan et al. 1993).
Non-sister pairings of post-meiotic nuclei appear
to be preferentially packaged during basidiospore
production in commercial, wild-collected and
hybrid isolates (Khush et al. 1995). The net result
of the A. bisporus life cycle is the maintenance of
the parental genotype through what Kerrigan has
called pseudo-c1onal Iineages (Kerrigan 1990).
Heteroallelism predominates at the mating-type
locus, and the binucleate spores are self-fertile
(Khush et al. 1995).
A third important development that occurred
was the development of pulse field gel elec-
trophoresis (PFGE) which resulted in resolution
of A. bisporus chromosomes (Royer et al. 1991;
Sonnenberg et al. 1991; Horgen et al. 1996) and the
publication and expansion of the genetic map
(Kerrigan et al. 1993). A. bisporus heterokaryons
(dikaryotic state) possess 13 pairs of chromosomes
(Fig. 1.1) ranging in size from 1.4Mb to 3.65Mb
(Horgen et al. 1996; Sonnenberg et al. 1996). The
genetic map, proposed by Kerrigan et al. (1993),
suggested a number of linkage groups that could
be associated with the chromosome-sized DNAs.
Although the physical map for A. bisporus is
developing slowly, several loci have been placed
on specific chromosomes, and a partial represen-
tation is shown in Table 1.1. The mating-type locus
6 p.A. Horgen and A. Castle

cn
M
C
'"
cn
C
Table 1.1. Some mapped genetic elements in Agaricus bis-
porus. Data were obtained from Sonnenberg et al. (1996);
Callac et al. (1997); Horgen et al. (1997); Kerstern et al.
(1997); PWJ De Groot et al. (1998)
MB MB Chromosome Specific genetic elements
n39 n97
3.65 c:::=>......c:::=> I 3.60 I. plnl7, pln31, plnl48, plnl50, MAT,
11 3.44 BSN, tefA,pkiA, PR2, PR5, PR6, PR7
3.14 11 c:::=>", <:::::> 11. atpD, p4n6, pln21
3.06 III·IV c:::=> .. <:::::> IU·IV 3.06
III. p33n5, p33n16, pgkA, hypA, htbA glnA
IV. rpaB, pruA, hypA, htbA, p33n25", pln105
V. p33n25", EST 2, xinA
,<:::::> V 2.57 VI. plnI25,p33nl0,hhfA,hhfB
2.49 V·VI c:::::::x:';C:::> VI· VII 2.32
VII. gdhB, EST3, pln37 b, p33n7
VIII. pln55, ntbB, rs13A
,,',~ IX 2.13
2.23 VII C:::=>' , IX. s15a, 141A, ES Tl , rDNA, gtiA, abs15n
2.11 VIII c::>-,: X. p1n36 b, gdhA, cell
:"'e:::=> VIII 1.90 XI. lcel, EST5, cel3
1.86 IX c::::;/ ,,<:::::> X 1.81 XII. EST4, p4n14 a
1.75 X e:::=>" ,<:::::> XI 1.66 XIII. EST6, gdpA, p4nl4 a, ab280, p4n33, put hy
1.68 XI e:::=>"
1.51 XII c:::=> -' <:::::> XII 1.51 aChromosome placement differs between studies.
XIII c:::=>" <:::::> XIII 1.44
1.45 bDiffers from Kerrigan et al. (1995).

Fig. 1.1. Karyotype and chromosome separation for the

two parents of the VI cultivar. The two parental
homokaryons of the VI cultivar are n39 and n97; the
CHEF separations of the homologous chromosomes are
shown on the right- and left-hand sides of the figure. Beside
each lane are the size determinations for the homologues
and in the center is a diagrammatic representation of the
13 homologues of each parental homokaryon. Data were
compiled from Horgen et al. (1997) and Sonnenberg et al.
(1996)
was mapped to linkage group land chromosome
I (Xu et al. 1993; Xu 1996), with the nearest
marker being 36 cM away. Recombinophobic
regions of chromosomes have been identified, and
the mating-type locus (MAT) appears in this
region in the middle of chromosome I (Callac et
al. 1997). Matings between European and North
American tetrasporic isolates of A. bisporus
resolved a higher level of recombination, and
the linkage of the MAT locus with the BSN
(basidiospore number) locus (Imbernon et al.
1996). In addition, Callac and coworkers utilized
SCAR markers and described a tight linkage
group for chromosome I (Callac et al. 1997). The
overall implication of the studies comparing sec-
ondarily homothallic isolates with heterothallic
isolates is that recombination is more frequent in
the tetrasporic heterothallic isolates (Callac et al.
1997).
One striking observation obtained from the
PFGE studies is that the chromosomes of Agari-
cus are highly polymorphie (Royer et al. 1991,
1992). Eleven of the thirteen chromosomes differ
in size in the two parental nuc1ear types that con-
stitute the V1 heterokaryon (Fig.1.1). The size dif-
ferences would arise from an addition, deletion or
translocation event. A reasonable expectation is
that inversions would also be present. Both trans-
mission genetic and DNA hybridization analyses
have provided some evidence for the presence of
these chromosomal re arrangements in some
strains of A. bisporus. For example, the recom-
binophobic region of chromosome I led Callac
et al. (1997) to infer that the strain studied by
Kerrigan et al. (1993) was structurally heteroge-
neous in chromosome I, i.e., carried an inversion.
Stronger evidence was reported by Evans (1959)
with his observation of dicentric bridges and acen-
tric fragments produced by crossing-over within a
paracentric inversion. In addition, the mapping of
the transposon-Iike element Abr} to different
chromosomes in the parental nuc1ear types of the
Horst V1 strain (Sonnenberg et al. 1999) may be
explained by translocation events or by transposi-
tion. The frequency of these differences may not
be substantially different from that which is
observed with numerous other fungal species (see
Zolan 1995); however, the consequences of these
aberrations on strain instability and on the devel-
opment of new mushroom strains are potentially
profound and warrant further consideration.
Map-based studies relating to strain instabil-
ity and gene placement suggested that the number
 Application and Potential of Molecular Approaches to Mushrooms
of co pies of the rDNA cistrons in A. bisporus
was highly variable resulting in chromosomal
polymorphism (Horgen et al. 1996; Sonnenberg
et al. 1996, 2000). Furthermore, data from un-
stable isolates of the U1 cultivar suggested that
a number of chromosomal abnormalities could
be associated with strain degeneration (Horgen
et al. 1996).
The production of new strains through a
breeding program would be hampered by inver-
sions and translocations. Inversions were first
described as "suppressors of crossing-over" since
a crossing-over event in an inversion heterozygote
in the inverted region results in loss of genetic
information and, usually, non-viability of the
recipient cell. The recovery of novel recombinant
genotypes in a mushroom breeding program
would be dramatically affected by this type of
aberration (Callac et al. 1997). The theoretical
effects of a single reciprocal translocation on
homokaryon and heterokaryon formation are
shown in Fig. 1.2. If we consider the simplest situ-
ation when no crossing-over occurs, then adjacent
segregation should occur in about 50% of all
meioses and 90-100% of all heterokaryotic
A.
=Nl=::::J9 ~~==Tl=
T2
~ ~ N2
B.
Heterokaryon
Segregation Haploid Characteristics
Pattern Characteristics Non-sister llairing
Alternate NIN2 orTlT2 NIN2TlT2
fuH complement fuH complement
Adjacent 1 NIT2orN2Tl NIN2TlT2
duplicate/deficient fuH complement
Adjacent 2 NITI orN2T2 NIN2TlT2
duplicate/deficient fuH complement
Fig. 1.2. Expected meiotic pairing pattern in astrain het-
eromorphie for a reciprocal translocation involving chro-
mosomes 1 and 2. A Expected meiotic pairing pattern in a
strain heteromorphie for a reciprocal translocation involv-
ing chromosomes 1 and 2. Ni Normal configuration for
chromosome 1; N2 normal configuration for chromosome 2;
Tl chromosome 1 carrying a terminal segment norma11y
carried by chromosome 2; T2 chromosome 2 carrying a
terminal segment norma11y carried by chromosome 1.
B Expected chromosome segregation patterns for recipro-
cal translocation in the absence of crossing-over in the inter-
stitial regions, i.e., the regions between the centromeres and
the break points. The normal expectation is that alternate
7
basidiospores should be genetically normal while
the remainder would be duplicate/deficient and
probably non-viable. From a breeding perspective,
this outcome would be acceptable. However, the
main problem caused by translocations is realized
when individual haploid nuclei are examined. All
haploids from adjacent segregation will not have
a fun genetic complement and should show debil-
itation to a greater or lesser extent depending on
the segments involved in the translocation. Since
alternate segregation would result in genetically
normal haploids, approximately 50% of an
haploid meiotic products should be duplicate/defi-
cient. About half of all heterokaryons recovered
from basidiospores should contain haploid nuclei
that would not function wen without a com-
plementary nuclear type. The recovery of
homokaryons necessary for breeding from these
strains would be difficult or impossible. All of this
analysis is hypothetical and based on the supposed
existence of these aberrations. The inability to
regenerate protoplasts from both homokaryons of
selected heterokaryons is consistent with this
hypothesis (Stockton and Horgen 1993). A con-
certed effort on the development of a saturation
Heterokaryon
Characteristics
Sister Pairing
NININ2N2orTITIT2T2
fuH complement
N IN IT2T2 or N2N2Tl Tl
duplicate/deficient
NINITITlorN2N2T2T2
duplicateldeficient
segregation would occur 50% of the time, adjacent 1 would
be elose to 50% and adjacent 2 would be relatively rare. The
term "full complement" means that a11 of the genetic infor-
mation is present in the correct amount and the strain
should be viable and fu11y functional. "Duplicate/dejicient"
me ans that some information is present in too many copies
and other information is missing. For example, a haploid
with the NI T2 genotype would have two copies of the chro-
mosome 1 information contained in the break point to
telomere segment and would have no copies of the infor-
mation carried norma11y in the break point to telomere
region of chromosome 2. Duplicate/deficient strains would
likely be non-viable or severely debilitated
8 P.A. Horgen and A. Castle
map of the Agaricus genome will allow unequivo-
cal determination of the presence and the effects
of such aberrations. If they are present, then the
development of sets of strains with similar chro-
mosome structure will bypass the described
concerns. Numerous studies relating to natural
populations of A. bisporus (see Kerrigan et al.
1995, 1998) provide the basis for the genetic
resources to facilitate this type of breeding
approach.
B. Genomics in Mushrooms
Whereas functional genomics has moved forward
at a very rapid rate for a number of other com-
mercially important fungi including Ustilago
maydis, Saccharomyces cerevisiae, Aspergillus sp.
and Neuropora crassa, much less effort has been
extended to further bioinformatics in the com-
mercially important A. bisporus. With the excep-
tion of the work by Monquet et al. (1999) on
Agaricus bisporus resistance to bacterial blotch,
the expansion of information on quantitative trait
loci has not advanced much in the last decade.
However, a number of genes have been sequenced
and described for Agaricus bisporus (search
GenBank). A number of these genes, along with
other loci, have been placed on the 13 homologues
of the VI strain (Table 1.1). Developmentally reg-
ulated genes that are associated with fruit body
formation have been described by De Groot et al.
(1997). Four different stages were identified in the
transition from vegetative to mature fruit body: it
was shown that nine genes could be induced to
specially change during the production of froit
body primordia (De Groot et al. 1997). Nitrogen
was shown to post-transcriptionally regulate
glutamate synthetase (Kerstern et al. 1997). Fur-
thermore, a compost-utilizing enzyme gene for
xylanase was isolated and characterized (PWJ De
Groot et al. 1998). In Agaricus bisporus, there are
at least two hydrophobin genes that are differen-
tially expressed and associated with fruit body
development (De Groot et al. 1996; Lugones et al.
1996; De Groot et al. 1999). In arecent study,
Sonnenberg et al. (1999) describe a 300-bp mobile
repetitive element with transposon characteristics
in the genome of the VI hybrid. This element
exists as 15 copies in the haploid genome that
show little nucleotide variation within tradition al
and between current mushroom cultivars
(Sonnenberg et al. 1999). On the other hand, high
levels of variability were observed in field isolates
of A. bisporus with greatest variation being
between secondarily homothallic isolates and the
four-spored heterothallic isolates (Sonnenberg et
al. 1999). In arecent study, the initial establish-
ment of an expressed sequenced tagged library
consisting of several hundred genes associated
with fruit body development has been initiated
(Ospina-Giraldo et al. 2000).
C. The State of A. bisporus Nuclear DNA
As suggested above, all indicators would suggest
that the cultivated mushroom's genetic material
goes through meiosis, but behaves differently
from most other eukaryotic organisms (reviewed
by Khush et al. 1992). In arecent study by Binz
et al. (1998), it was demonstrated that the extent
of cytosine methylation in the DNA of A. bisporus
and other homobasidiomycetes was considerably
higher than in other fungi, and indeed most
other eukaryotes. The levels were approximately
4 % in A. bisporus as opposed to levels of approx-
imately 1.5% in other groups of filamentous fungi
and approximately 2 % in heterobasidiomycetes
(Binz et al. 1998). The exceptions to these high
levels of 5-methylcytosine were degenerate
isolates of A. bisporus, where it had already been
shown that dramatic changes in the genome had
occurred (Horgen et al. 1996). Furthermore,
these authors related the high levels of DNA
methylation to the extreme difficulties researchers
had encountered in attempting to develop a
workable "fungal" transformation system in
A. bisporus (Binz et al. 1998). One cannot help but
suggest that the mechanisms in operation that
seem to keep the genetic material in mushrooms
shielded from the kinds of genetic re arrangements
characteristic of eukaryotic meiosis may be
related to the physical state of the genetic
material. The state of cytosine methylation is now
documented (Binz et al. 1998) and may contribute
to this phenomenon.
1. Heterothallic Forms of A. bisporus in Nature
All of the difficulties described above with respect
to the secondarily homothallic life cycle were
found to be absent in selected populations of
A. bisporus in nature. Callac et al. (1993) described
 Application and Potential of Molecular Approaches to Mushrooms
a population of heterothallic forms with four-
haploid basidiospores in the Sonoran Desert of
California. The existence of this population and
perhaps other populations with a heterothallic life
cycle offers new opportunities to mushroom
breeders (Fig. 1.3). This tetrasporic form is
completely interfertile with bisporic forms of
A. bisporus (Callac et al. 1993). With the het-
erothallic forms of A. bisporus a tradition al
genetic breeding approach is possible, as weIl as
the possibility of crossing with secondarily
homothallic forms (Fig. 1.3). More recent work on
the four-spored forms has led to the description
and partial genetic analysis of the locus in A. bis-
porus that controls basidiospore number (BSN;
Imbernon et al. 1996). This new germplasm is
available for breeding and strain development
should the industry choose to expand the
resources to move it forward.
2. Somatic Contact and the Potential
for Breeding
Xu et al. (1996) demonstrated that an alternative
source in nature of recombinant genotypes of A.
bisporus exists as a result of somatic pairings of
heterokaryons. They also demonstrated that, in
the laboratory, heterokaryon x homokaryon pair-
ings could also result. Genetic analysis of these
pairings using RFLP loci suggested evidence for
genetic recombination (Xu et al. 1996). With the
mixing of nuclear types (at least four in a het-
erokaryon x heterokaryon pairing and at least
three in a heterokaryon x homokaryon pairing),
the possibility of pairing of new nuclear types and
deheterokaryotization, as weIl as genetic change
through mitotic chromosomal exchange, may
result. Indeed, this was suggested as a method that
contributes to genetic diversity in field isolates of
A. bisporus and other related basidiomycetes
(Saville et al. 1996, 1998; Xu et al. 1996). In a study
of strain instability of the V1 cultivar, Horgen et
al. (1996) demonstrated that deheterokaryotiza-
tion, somatic recombination, loss of heterozygos-
ity, chromosome length polymorphisms, and
chromosome translocations all are possible within
cultivated isolates of A. bisporus. With these
studies in mind, and the existence of heterothallic
forms of A. bisporus, Fig. 1.3 demonstrates the
multitude of opportunities that are now available
to mushroom breeders for developing improved
strains of A. bisporus.
9
D. The Mitochondrial Genome and
Its Potential Role in Strain Performance
The transmission of the mitochondrial (mt)
genome following somatic crosses or a complete
sexual cycle has been studied for a number of
species of higher fungi including Agaricus bisporus
(Hintz et al. 1988a; Jin et al. 1992; Jin and Horgen
1994). The pattern of inheritance is variable among
species of fungi, but A. bisporus appears to be
uniparental and is influenced by (1) the different
combinations of isolates mated, (2) the relative
contribution of cytoplasm for each isolate (anisog-
amous versus isogamous matings), (3) the method-
ology used to mate compatible isolates, and (4) the
spatial and temporalorigin of isolates mated. In
A. bisporus, we do not know whether inheritance of
a single mtDNA haplotype (uniparental inheri-
tance) is an entirely stochastic process, or subject
to some type of controlling mechanism. Possible
mechanisms of uniparental inheritance among
eukaryotes are discussed by Birky (1995) and may
occur at the prezygotic, fertilization, or zygotic
stage of development. Zygotic mechanisms include
both deterministic and stochastic processes, the
latter type including the random segregation of
organelles with cell division, or the random replica-
tion of organelle genomes. In the latter case, one
of the parental genomes may be replicated more
frequently by chance, which may happen with the
relaxed replication of cytoplasmic genomes (Birky
1994, 1995). The detection of only one mtDNA
haplotype in a heterokaryon of A. bisporus by
many studies does suggest that the segregation
of the parental haplotypes may not be a purely
stochastic process. De la Bastide et al. (1997) estab-
lished that a mitochondrial haplotype can exert
an influence on parameters of commercial impor-
tance in mushroom cultivation. Seven genetically
distinct mitochondrial DNA haplotypes were eva-
luated in different nuclear genetic backgrounds.
The growth of different heterokaryon pairs differ-
ing only in mitochondrial haplotype was evaluated
for three different growth parameters measured.
Both temperature-dependent and temperature-
independent differences were documented
showing that mt genotype can indeed influence
mushroom strain performance (de la Bastide et al.
1997).
The mitochondrial chromosome in A.
bisporus is a 136-kb molecule that has been
physically mapped for a number of restriction
10 p.A. Horgen and A. Castle

IJ C.e)
II~ O. ~UnlnUCleate
~

Heterothallic
• 0 .0. basidiospore •

basidlum meiosis
(b)

~t
('
I
r~Si~me~!ß ~-~(a)
J" =--'y
Secondari!y
homothallic
Agaricus
ba~~~fl~~~~~es

m~shroomS bisporus basid~os~ore

Sex not required 10 Jgermmatlon

camplete 11" ,e,-CY",C""e==- ____ ==..:...::=;.,....:....;,.__,--+_

" Hyphal Fusion (C)

Confrontatlon of
two cultures

•
6Y
~rf~~~~~u~:~~:n;~~~~ of
Nuclel could fuse like in a
normal mating

Compatible matin~g
! Mitotic recombination
can result in variation

* lI- •
e

j j
New
heterokaryons
New '" ce
heterokaryon 8>

Parental homokaryon

9 9
from wild collected
A. bisporus

New mushroom strain New rnushroom strain

Fig. 1.3. Three different approaehes for breeding Agaricus
bisporus. Genetieally unique eombinations ean result from
parental homokaryons reeovered from either: a the bis-
porie life eycle, b the tetrasporie or heterothallie life eycle,
and c somatie fusion of vegetative hyphae. The binuclear
basidiospore from seeondarily homothallie strains of A. bis-
porus are self-fertile and after germination are very similar
to the heterokaryon that went through meiosis. Generally,
less than 5% of the spores are uninucleate. The most effee-
tive way to earry out a mating from this life eycle is to
isolate parental homokaryons by protoplast produetion,
regeneration and typing with DNA analysis, or to labori-
ously seareh for haploid single spores. With the tetrasporie
heterothallie life eycle, haploid spores ean be utilized as
parental homokaryons to mate with eompatible parental
homokaryons reeovered from any of the methodologies
shown above. Finally, vegetative myeelium ean fuse either
homokaryon x heterokaryon or heterokaryon x het-
erokaryon. The resulting heterokaryons earrying more than
two nuclear types ean either (1) undergo deheterokaryti-
zation to beeome a new binuclear heterokaryon with new
nuclear pairing or (2) nuclei ean somatieally fuse in the
heterokaryon and mitotie reeombination ean oeeur, result-
ing in new nuclear genotypes, whieh ean pair to form
genetieally unique binuclear heterokaryons (funetional
dikaryons). A number of different combinations utilizing
all of these different life styles ean result in genetieally
unique individuals. Nuclear eombinations ean eome from
eommereial eultivars and wild-colleeted isolates with both
a seeondarily homothallie life eycle and a heterothallie life
eyde
 Application and Potential of Molecular Approaches to Mushrooms 11

Table 1.2. Selected summary of mitochondrial analyses during specific matings. Homokaryon origin: n97 (mt type 1) is
one of parents of U1 hybrid (Netheriands), n39 (mt type 3 is the other parent of U1 hybrid (Netherlands), JB-36 (mt
type 18) was isolated from a tetrasporic heterothallic Sonoran Desert isolate, JB-23 (mt type 24) is from tetrasporic het-
erothallic Sonoran Desert isolate, FS-20 (mt type 3) is a coastal California isolate, RWK 1548 (mt type 28) is an isolate
of Richard Kerrigan collected in Banff, Alberta. The nomenclature used to describe the mitochondrial haplotypes (mt
types) were discussed by Xu et al. (1998). These data will be presented in full in a manuscript in preparation by de la
Bastide and Horgen. P Parent; at least 20 progeny were analyzed for each cross

Resultant mitochondrial haplotype (% of matings)

Homokaryons crossed Parent 1 Parent 2 Heteroplasmon a Recombinantb

JB-23(P1) x n97(P2) 80% 10% 10% 0

FS-20 (P1) x n97 100% 0 0 0
JB-36 (P1) x JB-23(P2) 68% 0 0 32%
RWK1548(P1) x n39(P2) 60% 20% 5% 15%
JB-36(P1) x n39(P2) 0 60% 0 40%

aHeteroplasm refers to a cytoplasm which in this case contains both P1 and P2 mt haplotypes after successive
subculturing.
bRecombinant mt haplotype is based on the persistent appearance of new restriction fragments.

endonuc1ease sites and for some key mt genes
(Hintz et al. 1988b; Robison et al. 2000). It con-
tains a pair of large inverted repeated regions that
may contribute to mt haplotype variability in
natural populations (Jin and Horgen 1993). This
variability is high with at least 138 haplotypes rec-
ognized, whereas only two haplotypes have been
found in commercial cultivars (Xu et al. 1997,
1998). The ease with which different nuclear
genomic and mt haplotype combinations can be
produced by matings of compatible homokaryons,
and by fusion and regeneration of protoplasts, can
indeed provide another strategy for generating
new and interesting cultivars of A. bisporus (de la
Bastide et al. 1997).
One of the observations made as we have
evaluated the role of the mt genome in mating
reactions and strain performance is that a variety
of po ssibiliti es exist after a mating event. These
possibilities are determined by the genotype of the
isolate. Furthermore, selected mt haplotypes are
prone to exhibiting specific patterns in the F1
progeny. A number of general observations can be
made based on a limited number of measurements
of both commercial and field-collected isolates
showing different mt haplotypes:
• In most cases, after examining 20 progeny from
any specific cross, one of the two parental mt
haplotypes was generally transmitted to the F1
progeny.
• Under the conditions used for our matings,
we observed what appeared to be stable
heteroplasms in approximately one-half of the
crosses.
• Recombinant mt genotypes were observed in a
number of crosses, and one mt haplotype
studied, when mated, nearly always resulted in
a recombinant haplotype.
A preliminary analysis is shown in Table 1.2. What
was unique about this study was our protocol
for matings. Cultures were routinely maintained
on solid complete yeast medium (CYM; Raper
et al. 1972) and 4-week-old colonies of all
homokaryons were used as inoculum for cultures
in liquid CYM. For each homokaryon, 40 plugs
(5 mm diameter) of mycelium were taken from the
edge of each colony and were added to a blender
cup containing 50ml of liquid CYM. This mixture
was then blended for lOs at maximum speed in a
Waring blender and 5 ml of this mycelial slurry
were used to inoculate a 10 x 150mm Petri dish
containing 15 ml of liquid CYM. Following 2 weeks
of growth at 22°C, these liquid cultures were used
for crosses. For each pairing of homokaryons, one
Petri dish liquid culture of each homokaryon was
added to the same blender cup. This mixture was
blended at maximum speed for lOs and five agar
plates of compost extract medium (CEM; Xu et al.
1993; de la Bastide et al. 1997) were then inocu-
lated at ten points, each with 100,u1 of the mixed
mycelial slurry. Cultures were maintained at 22°C
and the single colonies that grew from each inoc-
ulation point were subcultured to new plates of
solid CYM by taking a sm all quantity of hyphae
from the edge of the colony. Colonies were sub-
cultured a total of three times onto fresh plates of
CYM; this was done to avoid a mixed sampie of
different mycelial types (homo- and heterokary-
12 P.A. Horgen and A. Castle
nue:
pEM:
ehr:
u-p: IIWITPSGLKISLSNIKY-
+ W +P GL+I K F +N+IHSLDA++
Fig.l.4. Alignment of portions of four putative mitochon-
drial RNA polymerase translation products in an Agarieus
isolate. An amino acid alignment of four unique computer-
translated RNA polymerase genes in Agarieus crocodilinus
is shown. Most species of Agarieus have at least three
genes: nue nuclear encoded mt RNA polymerase;pEM mt
plasmid RNA polymerase; ehr mt chromosomal pseudo
gene. A. eroeodilinus has at least one additional gene, u-p
unknown plasmid RNA polymerase. The nucleotide com-
otic mycelia) and to obtain only a single het-
erokaryon genotype. Far each cross, a total of 50
colonies were subcultured and 20 of these were
randomly selected and subjected to genetic analy-
ses (de la Bastide and Horgen, in prep.).
Our studies with the mitochondrion clearly
suggest that this genetic component of mushrooms
can be manipulated and can result in unique and
different genetic combinations involving nuclear
genomes mixed with specifically chosen mt haplo-
types. More detailed analysis of how these
nuclear/mitochondrial combinations affect strain
performance may be extremely beneficial to the
mushroom industry in the future.
E. Extrachromosomal Elements in Mitochondria
of Agaricus Species
Extrachromosomal DNA molecules or plasmids
have been discovered in a large number of eukary-
otic organisms and are quite common in plants
and fungi (Meinhardt et al. 1990). Both circular
and linear molecules exist but, in fungi, linear plas-
mids are often associated with the rnitochondrion
and often possess similar genetic elements
(Meinhardt et al. 1990). Plasmids were shown to
exist in the mitochondrion of Agaricus species by
Mohan et al. (1984). These viral-like entities were
established to have open reading frames that were
capable of encoding genes for both DNA replica-
tion and transcription (Robison et al. 1991). Most
interestingly, however, is that unlike other eukary-
otes, which do not seem to have any genes for
RNA polymerase in their mitochondrial chromo-
somes, sequences for a pseudo-gene characteristic
of a mitochrondrial RNA polymer ase exist in
Agaricus mitochondrial chromosomes (Robison
et al. 1991). There is direct evidence to demon-
position of the chromosomal, pEM, and unknown plasmid
are highly similar (24.2% G+C, 24.4% G+C, and 22.8%
G+C, respectively). The nucleotide composition of the
nuclear-encoded gene is much different (50.0% G+C).
Highlighted residues indicate conservation. There is an
overall 54.4% similarity at the amino acid level between
these four gene products. These data are from Olszewski
and Horgen (in prep.)
strate that RNA polymerase gene sequences are
nuclear encoded in yeast, N crassa, Arabidopsis,
and in humans (Masters et al. 1987; Chen et al.
1996; Hedtke et al. 1997; Tiranti et al. 1997). Good
sequence matches from nuclear encoded frag-
ments isola ted from a growing number of other
eukaryotes would suggest that this important gene
that is responsible for transcription in the mito-
chondrion has, indeed, during the evolution of
eukaryotes become situated on nuclear chromo-
somes (Cermakian et al. 1996). Again, there is
some uniqueness in the Agaricus species; most
have three mtRNA polymerase genes, and at least
one isolate possesses four genes (one nuclear, two
of which are encoded on different mt plasmids,
and one pseudo gene on the mt chromosome).
Figure 1.4 shows the alignment of a portion of the
putative proteins of these four genes. We believe
that studies within the genus Agaricus will provide
important information on the evolution of the
gene involved in transcription in mitochondria
and, indeed, may shed some valuable information
on the evolution of the organelle itself from its
suggested prokaryotic progenitor.
F. Transformation in Agaricus
Several methods for the development of an effi-
cient transformation system for A. bisporus have
been tried with little success over the past 10 years
or so. These attempts have included CaClz and
polyethylene glycol, electroporation and particle
bombardment procedures in several possible com-
binations with basidiospores, hyphal fragments or
protoplasts (Royer and Hargen 1991; Li and
Horgen 1993; Challen and Elliott 1994). In 1996,
van de Rhee et al. reported the recovery of trans-
formants from a homokaryon and from a het-
 Application and Potential of Molecular Approaches to Mushrooms
erokaryotic derivative of Horst Ul. Protoplasts
were transformed by electroporation with a mod-
ified hygromycin B resistance gene under the
control of a A. bisporus GPD promoter (van de
Rhee et al. 1996a). The transformation efficiency
was quite low at 0.1-0.5 transformants per micro-
gram of transforming DNA or 1-5 transformants
per 105 or 10 6 regenerated colonies. In a subse-
quent study, the frequency of homologous inte-
gration of transforming sequences (but not the
overall transformation efficiency) was increased
by the inclusion of tandem copies of B-glucanase
genes in the plasmid used for transformation (van
de Rhee et al. 1996b). A substantially different
approach based on established procedures for the
transformation of plants has also been applied
to fungi (Bundock et al. 1995; MJA De Groot
et al. 1998). A. bisporus was transformed by co-
incubating germinating basidiospores with cells of
Agrobacterium tumifaciens carrying plasmids
with a hygromycin resistance gene (MJA De
Groot et al. 1998). Transformation efficiency was
not reported or readily apparent from the data
since the frequency of basidiospore germination
was not included; however, it appeared to be quite
low. In arecent study, a more efficient procedure
based on these earlier studies was reported specif-
ically for A. bisporus genetic manipulation (Chen
et al. 2000). Two major modifications, which sub-
stantially improved efficiency, were noted. First,
the hygromycin resistance gene and a second
reporter gene (EGFP encoding the green ftuores-
cent protein from Aequorea victoria) were placed
under the control of the promoter for the glycer-
aldehyde-3-phosphate dehydrogenase (gpd) gene
of A. bisporus. Second, immature gill tissue was
used in pi ace of germinating basidiospores.
Transformants were stable through successive
sub-cultures and through fruiting trials (Chen
et al. 2000). Therefore, transformation technology
appears to be at a stage where genetic modifica-
tion of mushrooms for a wide array of applications
is feasible.
III. Genetic Improvement of A. bisporus:
Perspectives for the Future
At the beginning of the 1980s, it was generally feIt
that conducting any tradition al genetics with the
secondarily homothallic white button mushroom
was extremely problematic and time intensive if
13
possible at all. Tremendous gains in mushroom
biology and genetics occurred during the last
quarter of the twentieth century. Despite the exis-
tence of the tools described above, the global
mushroom industry has invested very little effort
or resources into research and development
leading towards adapting modern crop improve-
ment strategies to mushrooms. Almost without
exception, most of the world's producers at the
turn of the century are growing the U1 cultivar
or minor variants of this strain. The historical
danger of this monocultural practice in agriculture
is ominous (see, for example, Matossian 1989;
Shumann 1991). Grapes, corn and wheat are
three crops that have experienced "crashes"
during the last century where entire crops were
wiped out by fungal pathogens and tens of mil-
lions of dollars of losses were experienced by
growers of these crops. What these crops have,
however, is a rich history of breeding, and numer-
ous alternative cultivars waiting in the wings. This
luxury does not exist for the mushroom industry
at this time.
Furthermore, there is a prevailing feeling in
the global mushroom industry today that all
problems that develop with mushroom production
can be solved by further manipulation of the envi-
ronment. By increasing sanitation, and stressing
the environment with increased use of chemicals
to control pathogens, one sets up the ideal situa-
tion for the evolution of "super virulent"
pathogens. For example, benomyl resistance in
fungi is the result of a single gene mutation most
frequently in the ß-tubulin gene (Sheir-Neiss et al.
1978; Thomas et al. 1985). This mutation has
appeared in many pathogenic fungi, including Ver-
ticillium, formerly controlled by benzimidazole
fungicides. It is possible to generate a benomyl
resistant form of the virulent TH4 Green Mold
isolate that is resistant to at least four times the
current legal limit of application of the fungicide
(D. Rinker, University of Guelph Vineland,
Ontario, pers. comm.). While this mutant was
developed artificially in the laboratory, "natural"
benomyl resistant variants of Trichoderma
harzianum (TH1 type) have been recovered from
a Shiitake log in 1990 and in 1998 from an oyster
mushroom crop (D. Rinker, pers. comm.). It is
just a matter of time before a benomyl resistant
TH4 or TH2 strain appears spontaneously on a
mushroom farm. Finally, clonal propagation of the
monocultural strain will result in strain instability,
and degeneration, something that the industry has
14 P.A. Horgen and A. Castle
already experienced (Li et al. 1994; Heath et al.
1995; Horgen et al. 1996).
IV. Conclusions
The direction that mushroom industry R&D and
academic mushroom research takes in the early
part of this century will greatly affect the robust-
ness of the industry's production and sales capa-
bilities as the century progresses. Considerable
resources have been, and continue to be, put into
genomics and bioinformatics of green agricultural
crops. These efforts are funded both by industry
and governments. No such tradition exists for
mushrooms and, unless some leaders develop
within the industry, the future for the mushroom
industry will remain problematic. Mushroom sci-
entists within the industry who have the capabil-
ity to apply the new technologies to move the
industry forward spend a large percentage of their
time dealing with crisis management of the VI
monoculture. From a scientific and historical per-
spective, the development and success of the
mushroom industry is an interesting and perhaps
a unique case in Agri-business. From the 1960s on,
the industry has moved from what was a "cottage
industry" with small unsophisticated farms pro-
ducing most of the product to a highly sophisti-
cated and developed agro-business with larger and
fewer producers developing on aglobai scale. The
industry has been extremely successful to date, but
now exists by perpetuating a monocultural pr ac-
tice. Genetic manipulation through intensive
breeding pro grams utilizing all of the methodolo-
gies described in this review, and adapting all of
the new technologies as they evolve to mushroom
research, will position the industry for success in
the twenty-first century.
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2 Straw Enrichment for Fodder Production by Fungi
DUSAN JAU~
CONTENTS
I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . .
11. Characterization of Lignolytic Fungi .......
A. Fungal Degradation of Straw .............
B. Solid-State Fermentation. . . . . . . . . . . . . . . . .
1. Pre-selection of Fungi . . . . . . . . . . . . . . . . .
2. Temperature and pR .................
3. Moisture Content ....................
4. Nitrogen Content ....................
5. Composition of the Gas Phase . . . . . . . . . .
6. Agitation and Aeration . . . . . . . . . . . . . . . .
III. In Vitro Studies . . . . . . . . . . . . . . . . . . . . . . . .
A. Wheat Straw ..........................
1. Effect of P. cinnabarinus, P. chrysosporium,
C. stercoreus, and D. squalens . . . . . . . . . . .
2. Effect of S. rugosoannulata
and Pleurotus spp. . . . . . . . . . . . . . . . . . . . .
3. Effect of Tropical and Other Fungi ......
B. Rice Straw . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Effect of Fungi on Rice Straw ..........
2. Protein Supplementation of Fungi-Treated
Rice Straw . . . . . . . . . . . . . . . . . . . . . . . . . .
C. Barley Straw ..........................
D. Oat Straw ............................
E. Maize Stover . . . . . . . . . . . . . . . . . . . . . . . . . .
F. Other Straws and By-Products ............
IV. In Vivo Studies ........................
V. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . .
I. Introduction
Straw (wheat, barley, oat, rye, rice) represents
the main agricnltural crop residue in the world
and botanically belongs to the Graminae family.
Cereal straws are lignocelullosic material rich in
energy, low in crude protein and poor in pal-
atability. One characteristic of straw is that it
mainly consists of 60-70% carbohydrates (Jackson
1977). Lignin complexed with cellulose and hemi-
cellulose makes the carbohydrates of straw less
accessible to microbial attack in the rumen. Lignin
Institute of Animal Physiology, Slovak Academy of Sci-
ences, Kosice, Slovak Republic
acts as a barrier to the utilization of structural car-
19 bohydrates. Considering the quantitative impor-
19 tance of these crop residues, considerable efforts
20 have been made to upgrade their nutritive value
22
22 (Flachowsky et al. 1999).
22 Several physical treatments, such as pellet-
22 ing, grinding, steaming and irradiation (Walker
22 1984), as well as chemical treatments, particularly
22
24 alkali addition (Sundst(jl and Owen 1984), have
24 been tested in attempts to improve dry matter
24 digestibility and voluntary intake of straw. The
25
practical use of these treatments is limited by
safety concerns, costs and potentially negative
26 environmental consequences. As an alternative,
27 biological treatment may offer a more practical
29
30 and environment al approach towards enhancing
the nutrition al value of low-quality roughage.
30 The main problem of biological upgrading of low-
31 quality roughage into feed is to find suitable
31
32 microorganisms with metabolic patterns different
32 from those of the rumen flora and fauna for use in
33 the process of delignification and upgrading.
34 "Ideal microorganisms" for upgrading ligno-
35
cellulose crop residues into animal feed should
have a strong lignin metabolism with poor degra-
dation of cellulose and hemicellulose so that, in
the treated material, carbohydrates are preserved
for their utilization by the rumen microbes as an
energy source in ruminants (Zadrazil et al. 1996).
One potential to improve the nutritional value of
crop residues by selective delignification is the use
of fungi, which mineralize lignocellulose in nature.
Other pertinent reviews on straw enrichment by
fungi can be found in Zadrazil (1985), Agosin et
al. (1987), Gupta (1987) and Zadrazil et al. (1995).
11. Characterization of Lignolytic Fungi
Lignolytic microorganisms are mainly wood-
inhabiting fungi which are able to colonize differ-
ent plant residues. Different ecological groups of
The Mycota XI
Agricultural Applications
Kempken (Ed.)
© Springer-Verlag Berlin Heidelberg 2002
20
rot can be distinguished: sweet-rot, brown-rot,
and white-rot. Sweet-rot fungi only metabolize
soluble sugars or other easily digestible cell con-
stituents of crop residues. Brown-rot fungi pri-
marily decompose cellulose and hemicellulose at
an extremely low pH (2.0-3.0) and decomposed
material is characterized by lignin accumulation.
White-rot fungi are able to decompose and min-
eralize all plant cell constituents - cellulose, hemi-
cellulose and lignin. The use of white-rot fungi
for conversion of lignocellulose into feed has
been studied and the positive effect to the nutri-
tional value determined (Hartley et al. 1974;
Kaneshiro 1977; Zadrazil 1977, 1980; Latham
1979; Lindenfelser et al. 1979; Ibrahim and Pierce
1980; Zadrazil and Brunnert 1980,1981).
These investigations showed that (1) in vitro
digestibility of fungal substrates decreases at the
beginning of colonization by white-rot fungi and
increases afterwards (Zadrazil1977; Zadrazil and
Brunnert 1982); during the incubation period,
the contents of soluble substances are increased
(Lindenfelser et al. 1979); (2) the increase in
digestibility depends on the fungal species, culti-
vation time, temperature, composition of sub-
strate, etc. (Zadrazil and Brunnert 1981, 1982); and
(3) in vitro digestibility of lignocellulosic crop
residues by white-rot fungi decreases by addition
of inorganic nitrogen (Zadrazil and Brunnert
1980). Zadrazil (1985) tested 115 species and a
total of 235 different strains of white-rot fungi
(belonging to the group basidiomycetes) for
growth on wheat straw: 85 species (115 strains)
showed intensive growth and were tested at 22,25
and 30 oe for the decomposition rate of organic
matter and lignin, liberation of water-soluble sub-
stances from plant polymers and in vitro organic
matter digestibility. The different physiological
behavior shown by these fungi has been used to
divide them into four groups.
• Fungi which degrade little or no lignin and
decompose cellulose and hemicellulose selec-
tively. The in vitro dry matter digestibility
(IVDMD) of these substrates is decreased as
compared to the untreated substrate (typical
fungi of this group are Agrocybe aegerita, Flam-
mulina velutipes, and Volvariella volvacea).
• Fungi which decompose lignin selectively
and other substrate components (cellulose,
hemicellulose) only partially. IVDMD of these
substrates is increased (typical fungi of this
group are Dichomitus squalens, Abortiporus
D. Jale
biennis, Pleurotus spp., and Stropharia
rugosoannulata) .
• Fungi which decompose lignin, cellulose and
hemicellulose as group B, but IVDMD is
decreased or remains unchanged (some strains
of this group are Polyporus, Ganoderma, and
Pleurotus spp.).
• Fungi which decompose lignin and other sub-
strates (cellulose and hemicellulose) rapidly and
change digestibility of the substrate only partly.
A typical member of this group is Phane-
rochaete chrysosporium.
White-rot basidiomycetes degrade the polymers
cellulose, hemicellulose and lignin at different
rates and extent. In general, strains of white-
rot fungi are the most effective lignin degraders in
nature (Eriksson et al. 1990). White-rot fungi
produce extracellular lignin-modifying enzymes,
of which lignin-peroxidases (LiP) , manganese-
dependent peroxidase (MnP), laccase and H20z-
producing oxidases (aryl-alcohol oxidase and
glyoxaloxidase) are the most important (Kirk and
Farrell 1987; Buswell 1992). The oxidative enzymes
ligninase, manganese peroxidase and laccase may
be defined as phenoloxidases. Both ligninase and
mangane se peroxidase belong to the class of perox-
idases (Tuor et al. 1995).
According to their typical production patterns
of extracellular lignolytic enzymes, white-rot fungi
may be divided into four groups: (1) the LiP-
MnP group, (2) the MnP-Iaccase group, (3) the
LiP-Iaccase group, and (4) the laccase-AAO
group. AAO is aryl alcohol oxidase (Hatakka
1994). Studies on lignin biodegradation have
been carried out mostly using Phanerochaete
chrysosporium which is a typical representative of
the fungi belonging to the LiP-MnP group. P.
chrysosporium is a very efficient degrader of
lignin. Typical fungi producing (1) LiP-MnP are
Phlebia radiata, Coriolus versicolor and Trametes
gibbosa, (2) fungi producing MnP-Iaccase are
Panus tigrinus and Dichomitus squalens, (3)
LiP-Iaccase producing fungi are Junghuhnia sepa-
rabilima and Phlebia ochraceofulva, and (4)
fungi producing AAO are Pleurotus ostreatus and
Pleurotus sajor-caju.
A. Fungal Degradation of Straw
Plant cell walls contain three types of structural
polysaccharides: cellulose, hemicellulose, pectic
 Straw Enrichment for Fodder Production by Fungi
Table 2.1. Chemical composition of different straws as determined by the detergent fiber analyses (% of dry weight)
Roughage Cell walls Hemicellulose
Barley straw 81 27
Oat straw 73 16
Paddy straw 79 26
Wheat straw 80 36
Sorghum stover 74 30
Chickpea straw 62 20
Lucerne straw 69 19
Sugarcane bagasse 82 29
polysaccharides and lignin (Table 2.1). In straws
the polysaccharide composition is rather simple,
cellulose and hemicellulose being the predomi-
nant components. Smaller amounts of polysaccha-
rides containing mannose and galactose are also
present. Cellulose is the most abundant molecule
in nature and occurs in a large crystalline form,
organized as fibrils. Apart from the structural
polysaccharides, lignin is the main component
of straw (Theander and Äman 1984). Moreover,
as a gramineous plant, it contains three types of
lignin units (H, G, and S). Lignins are often classi-
fied according to the distribution of these three
elements. Lignin in the cell wall is the largest
source of phenolic material in plants (Jung and
Fahey 1983). Straw contains p-coumaric and
ferulic acid units that have partially common
structures with lignin monomers (Lapierre et al.
1989). The phenolic acids are ester-linked to the
carbohydrates and also ether-linked to lignin, thus
forming a stabilizing bridge in the cell walls
(Iiyama et al. 1990).
White-rot fungi are able to totally degrade
lignin into CO 2 and H 2 0 (Moreira et al. 1998).
They are able to perform different patterns of
degradation of lignocellulosic substrates depend-
ing on their species and on the environmental con-
ditions in wh ich the mycelium grows (Blanchette
and Reid 1986). This may result in perforations
in the straw wall, all three cell wall polymers,
lignin, cellulose and hemicellulose, being locally
removed at the same time in a simultaneous type
of degradation.
The variations in the pattern of degradation
performed by white-rot fungi have been corre-
lated with the enzymatic equipment of the fungi
(Ruel et al. 1994). From among the lignolytic
enzymes produced by white-rot fungi (Odier and
21
Cellulose Lignin Silica Reference
44 7 3 Fernandez Carmona and
Greenhalgh (1972)
41 11 3 Saxena et al (1971)
33 7 13 Sharma (1974)
39 10 6 Sharma (1974)
31 11 3 Sharma (1974)
30 10 2 Johnson and Pezo (1975)
38 11 1 Johnson and Pezo (1975)
40 13 2 Sharma (1974)
Artaud 1992, Thurston 1994) only lignin peroxi-
dase can directly oxidize nonphenolic lignin,
whereas in manganese-dependent peroxidase
and laccase mainly phenolic units are supposed
to degrade. Phenolic acids, mainly p-coumaric
acid and ferulic acid, serve to cross-link different
wall components (Jung 1989; Hartley et al. 1990),
thereby enhancing structural rigidity and also lim-
iting the availability of structural carbohydrates to
rumen digestion. Phenolic monomers inhibit the
rumen microorganisms (Hartley and Akin 1989)
and are negatively correlated to fiber digestibility
(Bohn and Fales 1989).
To increase the availability of structural car-
bohydrates for animal utilization, it is important to
break down the phenolic acids between the wall
components. The fungal rem oval of the cell wall
bound phenolic acids may be either initiated by
breaking the phenolic bridges between lignin and
structural carbohydrates or by directly attacking
the phenolic components. White-rot fungi are
capable of decomposing free phenolic monomers
in liquid media and removing the phenolic acids
from the cell walls (Chen et al. 1996). The mech-
anism by which fungi remove these phenolic acids
is not clear. If the phenolic-carbohydrate com-
plexes cannot be detected and do not accumulate
during fungal treatment, the fungi must remove
the phenolic acids either by breaking phenolic
ester or ether bonds or by directly attacking the
phenolic components. Fungal consumption of
the cell wall carbohydrates of straw does not pri-
marily depend on the fungal ability to degrade
lignin and other ligneous structural units as phe-
nolic acids. Utilization of the cell wall carbohy-
drates is more likely to be associated with the
fungal ability to consume the released carbohy-
drates for its growth.
22 D. laIe
B. Solid-State Fermentation
Solid-state fermentation (SSF) can be defined as
a process in which solid substrates are decom-
posed by fungi, which are able to grow on and
through the substrate (lignocellulose crop resi-
dues) under controlled environmental conditions
with the aim of producing a high-quality product.
During the growth of white-rot fungi on the
lignocellulosic substrate, the following phases are
observed: (1) colonization of the substrate, (2)
maturation of the fungal mycelium, (3) induction
of fruiting bodies, and (4) autolysis. Solid-state
fermentation of crop residues by white-rot fungi
often results in an improvement of in vitro dry
matter digestibility (Reid 1989). During coloniza-
tion on substrates, the fungi first convert easily
digestible polysaccharides into low molecular
weight sugars. Once the low molecular weight
sugars are depleted the fungus starts to starve and
then begins to degrade structural carbohydrates
and lignin (Eriksson 1988). During this phase the
cell wall carbohydrates are converted into cell sol-
ubles by fungal extracellular enzymes. Of all sub-
strates, biological treatment of wheat straw has
been the most widely studied crop residue. Solid-
state fermentation of wheat straw with white-rot
fungi is influenced by such factors as the species
of the fungus, substrate, temperature (Zadrazil
1980, 1985), moisture content (Zadrazil and
Brunnert 1980,1982), nitrogen content (Zadrazil
and Brunnert 1980) and composition of the gas
phase (Chiavari et al. 1989). All these factors
determine the sequence of degradation of the cell
wall polymers and final digestibility after solid-
state fermentation.
1. Pre-selection of Fungi
Various species of fungi, mainly basidiomycetes
(115 species,235 different strains), were tested for
their ability to grow on sterile straw substrate.
Some species (40) grew very slowly or were not
able to grow on wheat straw. This group includes
the species of Agaricus, Amanita, Armillariella,
Boletus, Cantharellus, Lactarius, Serpula and
other species (Zadrazil 1985). Fungi with good
growth performance on wheat straw (80 species,
115 strains) were tested for the lignin decomposi-
tion and in vitro dry matter digestibility during 60
days of incubation. The fungi that degraded lignin
selectively and caused an increase in in vitro
digestibility are listed in Table 2.2. On the other
hand, the fungi that decomposed lignin only to a
small extent and reduced the in vitro digestibility
of wheat straw are listed in Table 2.3.
2. Temperature and pH
Temperatures of 22, 25 and 30°C influenced
the decomposition of wheat straw by white-rot
fungi. A small number of the fungi reached their
growth and decomposition of lignin at 22°C
(Hymenochaete tabacina) or at 25°C (Flammulina
velutipes, Collybia radicata, Pleurotus serotinus) ,
but the majority of fungi showed best growth and
substrate decomposition at 30°C (Zadrazil 1985).
Higher temperatures have an adverse effect on
the process of fermentation and lead to less lignin
degradation as weIl as to decreased digestibility
of wheat straw. Temperatures of 35, 37 and 32-
40°C were optimal for the SSF of wheat straw
by P. chrysosporium (Parveen et al. 1983), by
Chaetomium cellulolyticum (Abdullah et al.
1985) and by Coprinus jimetarius (Flegel and
Meevootisom 1986), respectively. Lignin degrada-
tion by most fungi occurs at an acidic pH (Reid
1985).
3. Moisture Content
The water content (65-75 %) of the substrates
should be optimal for the growth of various fungi
(Zadrazil and Brunnert 1982; Babitskaja et al.
1986). A water conte nt of more than 75% resulted
in the reduction of the gas phase while a lower
water conte nt reduced fungal growth. The fungi
showed specific growth optima on wheat straw at
a solid/liquid ratio of 1: 3 (Zadrazil and Brunnert
1981).
4. Nitrogen Conte nt
Some fungi - Stropharia rugusoannulata, Lentinus
edodes and Pleurotus sp. Florida - were stimulated
during decomposition of wheat straw by the addi-
tion of lower values of NH 4NO J while Pleurotus
eryngii was inhibited by this nitrogen source
(Zadrazil and Brunnert 1980). Generally, lignin
removal from lignocellulosic crop re si du es was
suppressed by high nitrogen levels (Munoz et al.
1983).
5. Composition of the Gas Phase
Solid-state fermentation is affected by the pene-
tration and diffusion of gas through the substrate
 Straw Enrichment for Fodder Production by Fungi 23

Table 2.2. Infiuence of fungal species on liberation of water-soluble substances, lignin decomposition and in vitra
digestibility of wheat straw during a 60-day incubation period Zadrazil (1985). IVDMD of untreated straw was 40%

Fungus Temp. (0C) Decomposition Water-soluble Changes in in vitra

of lignin (%) substances (%) digestibility (%)

Abortiporus biennis 22 16.5 24.1 +21.6

(Za. AI) 25 17.1 21.1 +26.3
30 21.6 20.1 +23.5
Dichomitus squalens 22 11.3 27.3 +32.2
(Karst.) Reid 25 13.5 31.8 +28.3
(SBS 432.34) 30 17.2 28.1 +24.2
Ganoderma lucidum 22 11.2 12.8 +22.5
(w. Curt. Ex Fr.) Karst. 25 10.8 14.9 +25.7
(Za.) 30 8.3 10.9 +13.2
Inonotus hispidus 22 8.4 17.1 +24.4
(BuH. ex Fr.) Karst. 25 13.3 20.5 +29.5
(CBS 386.61) 30 15.9 20.8 +26.6
Inonotus rheades 22 1.1 12.5 +4.7
(Pers.) Karst. 25 3.2 15.4 +22.5
(CBS 277.33) 30 15.7 16.9 +36.1
Lentinus tigrinus 22 14.2 14.7 +19.3
(BuH. ex Fr.) Fr. 25 13.5 14.3 +15.3
(Za.) 30 13.1 15.1 +14.3
Phellinus igniarius 22 7.9 17.3 +13.2
Var. trivialis (Bres. ex 25 8.9 14.9 +10.2
Killermann) Niemelae (CBS 512.63) 30 7.9 16.1 +1.3
Phellinus robustus 22 14.1 13.5 +9.5
(Karst.) Bourd. & Galzin 25 15.8 16.1 +11.8
(CBS 389.72) 30 17.8 16.8 +12.5
Pleurotus eryngii 22 15.2 +15.3
(DC. ex Fr.) Oue!. 25 17.5 +22.9
(Za.4) 30 17.1 +23.2
Pleurotus ostreatus 22 7.0 17.2 +22.4
(Jacq. ex Fr.) Kummer 25 6.5 17.9 +15.6
(Si. P 409) 30 5.4 15.1 -1.2
Pleurotus sajor-caju 22 13.9 18.6 +20.6
(Fr.) Sing. (Singh) 25 14.4 17.4 +18.6
30 14.6 19.1 +20.1
Pleurotus sapidus M. 22 3.2 15.2 +22.3
25 3.7 17.5 +25.0
30 3.0 16.6 +30.0
Pleurotus sapidus 22 8.7 10.6 +15.3
(Schulz. apud Kalchbr.) 25 11.2 12.7 +19.1
Sacc. (Za. - Japan) 30 13.8 12.4 +7.7
Pleurotus serotinus 22 6.9 20.7 +25.8
(Schrad. ex Fr.) 25 10.7 22.2 +33.3
Kummer (Si. P 381) 30 4.6 13.3 +10.6
Polyporus brumalis 22 7.6 16.7 +20.8
(Si. P 375) 25 9.0 15.5 +20.0
30 7.6 13.7 +19.0
Sporotrichum 22 20.4 34.6 +16.8
pulverulentum 25 24.3 39.6 +14.3
(CBS 481.73) 30 24.2 37.7 +9.1
Stropharia rugoso 22 6.3 41.3 +9.5
annulata Farlow ex 25 9.1 38.9 +13.4
MurriH (Za. T 70) 30 11.7 38.7 +25.7
Trametes hirsuta 22 9.0 18.0 +20.0
(Wulfen ex Fr.) Lloyd 25 10.3 19.6 +11.5
(Si. T 241) 30 12.5 19.9 +6.5
24 D. laie

Table 2.3. Influence of fungal species on liberation of water-soluble substances, lignin decomposition and in vitra
digestibility of wheat straw during a 60-day incubation period Zadrazil (1985). IVDMD of untreated straw was 40%

Fungus Temp. (0C) Decomposition Water-soluble Changes in in vitra

of lignin (%) substances (%) digestibility (%)

Agrocybe aegerita 22 7.7 -30.0

(Briganti) Singer 25 8.1 -29.0
(Za. A.a. 1) 30 6.8 -32.1
Cerrena unicolor 22 9.6 9.5 -18.2
(BuB. Ex Fr.) MurriB 25 8.5 7.7 -22.5
(CBS 173.50) 30 9.8 10.5 -30.7
Collybia radicata 22 2.3 10.8 -13.0
(Relhan ex Fr.) Que!. 25 2.0 9.9 -20.0
(Sa. C 224) 30
Flammulina velutipes 22 0.9 36.1 -15.1
(Curt. ex Fr.) Singer 25 1.7 36.3 -18.8
(Za. St. 6) 30 0.4 37.3 -5.0
Peniophora duplex 22 0.4 10.3 -25.8
Burt. 25 0.0 9.0 -27.2
(CBS 286.58) 30 0.2 6.7 -24.2
Polyporus tomentosus 22 0.1 22.1 -13.2
(Si. P 340) 25 0.6 21.4 -10.3
30 1.8 20.0 -2.3
Poria rivulosa 22 14.0 17.4 +1.0
(Berk. ex Curt.) Cooke 25 16.9 10.2 -14.5
(CBS 434.48) 30 19.5 12.3 -7.4
Trametes zonata 22 10.3 20.5 -14.6
(Nees ex Fr.) Pilat 25 10.6 23.7 -10.4
(Sa. CS 356) 30 4.3 22.2 -11.8
Volvariella volvacea 22 23.2 -21.0
(BuB. ex Fr.) Singer 25 25.1 -25.4
(Chang) 30 25.6 -30.3

which removes volatile and gaseous metabolites the lignolytic activity (Agosin et al. 1986). Mixing
produced during fungal metabolism (Leisola et a1. of the solid substrates improves the oxygen supply
1983). Degradation of organic matter and lignin to the microorganisms and removes gaseous
was greater in the presence of oxygen than in the metabolites from the spaces in the substrate.
presence of air (Kamra and Zadrazil 1985,1986).
The influence of gas-phase composition on degra-
dation of the substrate and lignin as weIl as III. In Vitro Studies
changes in the in vitro digestibility of wheat straw
have been studied in the fungi Pleurotus sajor-
Different species of straw are highly variable in
caju, Pleurotus eryngii, Phanerochaete chrysospo-
their nutritive content. It is generally accepted
rium and Stropharia rugosoannulata (Kamra and
that oat straw is more digestible than barley straw
Zadrazil 1986; Zadrazil et a1. 1993; Puniya et al.
(45% vs. 40% of IVDMD) and barley straw has
1994).
proved to be more digestible than wheat straw
(40% vs. 36% of IVDMD) (White et a1. 1981).
6. Agitation and Aeration Most of the experiments conducted on the evalu-
ation of fungi-treated straw were performed by
Lignin biodegradation occurs efficiently only
either in vitro digestibility or by in sacco degrad-
under oxidative conditions. The lignolytic activity
ability estimations.
of several fungi - Lentinus edodes (Reid and Seifert
1982), Pleurotus sajor-caju (Kamra and Zadrazil
1986) and Pleurotus ostreatus (Reid et al. 1985) - A. Wheat Straw
were stimulated by oxygen and agitation of liquid
cultures to enhance biodegradability of wheat Wheat straw is usually available in greater quanti-
straw. However, very intensive agitation can inhibit ties than any other straw, but it is less desirable as
 Straw Enrichment for Fodder Production by Fungi
feed than other types of straw. Wheat straw
was used as a model for other lignocellulosic
substrates during fungal treatment by white-rot
fungi (ZadraziI1985). Wheat straw is a good model
for studying lignin distribution as being a grami-
neous plant it contains the three types of lignin
units. Lignin binds with the hemicellulosic compo-
nents of plant cell walls and, through covalent link-
ages and physical binding, prevents accessibility
to and biodegradation of plant carbohydrates by
cellulolytic and hemicellulolytic microorgan-
isms (Eriksson et al. 1990). A negative relation-
ship between lignin concentration and forage
digestibility was recognized to exist many years
ago (Sullivan 1959). White-rot fungi are the only
known organisms that are able to remove lignin to
any extent from plant cell walls, using plant poly-
saccharides as energy (Gold and Alic 1993).
Many white-rot fungi, such as Phanerochaete
chrysosporium, simultaneously degrade cellulose
and hemicellulose along with the lignin and often
leave a residue less biodegradable than the initial
lignocellulosic material (Jung et al. 1992; Akin et
al. 1993). Other white-rot fungi appear to be selec-
tive in their degradation of lignocellulose, mainly
degrading lignin and hemicellulose but leaving
a major part of cellulose undegraded (Eriksson
et al. 1990; Karunanandaa et al. 1992).
Zadrazil (1985) tested approximately 200
strains of white-rot fungi for their effect on wheat
straw and found increases in digestibility ranging
from 15-32 units in dependence on temperature
and the period of fermentation. This first extensive
screening of fungi was characterized by studying
the following parameters: speed of the substrate
(wheat straw) decomposition, liberation of water-
soluble substances, lignin decomposition and in
vitro digestibility of wheat straw. This study
showed that for upgrading of wheat straw, fungi
with high lignolytic activity and a low cellulose
and hemicellulose metabolism are needed. In
addition, accumulation of digestible, non-toxic
substances is desirable together with the rapid
growth and high substrate colonization ability of
fungi (ZadraziI1985).
During fungal fermentation of lignocellulosis
by white-rot fungi, the relative amount of nitro-
gen in the substrate increases due to carbon loss.
However, an increase in the nitrogen conte nt
did not correlate with the in vitro digestibility.
Also, the amount of lignin decomposition by
fungi does not correlate with the increase of in
vitro digestibility (Zadrazil and Brunnert 1980).
25
For lignin decomposition fungi require carbohy-
drates as an energy source.
1. Effect of P cinnabarinus, P chrysosporium,
C. stercoreus, and D. squalens
Studies aimed at estimating the changes in the
digestibility of fungi-treated straw are summa-
rized in Table 2.4. Lignin bio degradation and in
vitro dry matter digestibility (IVDMD) have been
investigated during solid-state fermentation of
wheat straw by eight strains of fungi (Agosin and
Odier 1985). Only two strains of fungi - Cyathus
stercoreus and Dichomitus squalens - facilitated
the highest improvement of IVDMD after 20 and
15 days of incubation with low dry matter losses
(15-20%; Table 2.4). These fungi had very differ-
ent patterns of lignin degradation and were char-
acterized by a rather slow growth rate on wheat
straw.
Agosin et al. (1985) also studied the mode
of fungal decay of wheat straw by the white-rot
fungi Pycnoporus cinnabarinus, Phanerochaete
chrysosporium and Cyathus stercoreus. All the
constituents of straw-cellulose, hemicellulose and
lignin of wheat straw were degraded by the white-
rot fungi studied, although differences in the rela-
tive extent of degradation of different components
were evident among the strains. P chrysosporium
degraded hemicelluloses and cellulose in wheat
straw at similar rates. P cinnabarinus and C. ster-
coreus degraded cellulose poorly and, therefore,
the hemicelluloses were extensively degraded by
the fungi supporting growth and lignin degrada-
tion. P chrysosporium increased IVDMD by
ab out 50% after a 35% weight loss (IVDMD of
wheat straw was particularly low - 24.6%). P
cinnabarinus and C. stercoreus increased IVDMD
by about 64 and 94% with a limited dry weight loss
of 12 and 18% after 7 and 13 days of incubation,
respectively. As compared with polysaccharides,
lignin was preferentially degraded by all three
fungi.
The highest degradation rate of wheat straw
was observed with the fungus P chrysosporium
(32 % loss of dry matter) after 15 days of incuba-
tion. The weight losses observed for C. stercoreus
and D. squalens were similar (20-22%). The fungi
C. stercoreus and D. squalens degraded hemicel-
lulose in preference to cellulose, whereas P
chrysosporium degraded cellulose and hemicellu-
lose non-selectively. This is the first information
indicative that the digestibility of wheat straw
26 D. laIe

Table 2.4. Digestibility of wheat straw after solid-state fermentation by white-rot fungi

Organisms Fermentation Change in Ref

period (days) digestibility

Fram To

Stropharia rugosoannulata 79 40 55-69 Zadrazil (1977)

Lentinus edodes 60 40 77 Zadrazil and Brunert (1981)
Abortipus biennis 26 40 79 Zadrazil and Brunert (1980)
Dichomitus squalens 15-30 38 63-68 Agosin and Odier (1985)
Cyathus stercoreus 15-30 38 63-68
Phanerochaete chrysosporium 15 40 51 Agosin et al. (1986)
Pleurotus pulmonarius 80 30 58.0 Moyson and Verachtert (1991)
Pleurotus sajor-caju 80 30 59.0
Lentinus edodes 60 30 43
Coprinus jimetarius 30 38 49 Gupta et al. (1992)
Pleurotus ostreatus 21 40 50 Tripathi and Yadav (1992)
Pleurotus sajor-caju 40 15 46 Zadrazil et al. (1993)
Phanerochaete chrysosporium 30 28 43 Puniya et al. (1994)
Polyporus ciliatus 30 47 53 laie et al. (1994)
Trametes gibbosa 30 41 57 laIe et al. (1996)
Pleurotus ostreatus 30 44 51 laIe et al. (1997)
Daedalea quercina 30 41 59
Hericium clathroides 30 41 56
Phelinus laevigatus 30 41 50
Inonotus andersonii 30 41 51
Inonotus obliquus 30 41 52
Inonotus dryophilus 30 41 56

improved after white-rot deeay indueed by
both solubilization and inereased biodegradabil-
ity of the residual eell wall eomponents. Addi-
tionally, these fungi inereased IVDMD by about
22% (C stercoreus, D. squalens) or 11% (P.
chrysosporium).
2. Effeet of S. rugosoannulata and Pleurotus spp.
The fungi Pleurotus sajor-caju, Pleurotus eryngii
and Stropharia rugosoannulata were also used for
biological degradation of wheat straw (Kamra and
Zadrazi11986; Zadrazil and Kamra 1989; Zadrazil
et al. 1991; Zadrazil and Puniya 1994). The effeet
of the gaseous atmosphere (partial pressure of
earbon dioxide and oxygen), the effeet of light
on the degradation of organic matter, lignin and
on ehanges of the in vitro digestibility of wheat
straw fermented by white-rot fungi were studied.
The loss of organic matter, lignin and in vitro
digestibility (Tilly and Terry 1963) were estimated
after 40days of ineubation.
The results of these experiments revealed that
gaseous metabolites including earbon dioxide are
inhibitory to the growth of white-rot fungi and
lignin degradation. In all three fungi tested, lignin
degradation was more effieient in an oxygen
atmosphere than in the presenee of air, while
degradation of organic matter was not affeeted.
Substrate ehanges with respeet to the inerease in
digestibility were mueh greater in the dark than in
the light. The inereases in IVDMD were approxi-
mately 21 and 14%, respeetively, and losses of
lignin were 27 and 43% in wheat straw deeayed
by P. eryngii and P. sajor-caju in an oxygen
atmosphere. The penetration and diffusion of gas
into the substrate, which removes volatile and
gaseous metabolites produced during fungal
metabolism, is a very important faetor affeeting
the proeess of solid-state fermentation (Leisola et
al. 1983).
It also known that fungi of Pleurotus spp. are
primarily organie matter decomposers and can
efficiently decompose lignoeellulose without any
chemie al and physical pretreatment. Solid-state
fermentation of wheat straw by white-rot fungi of
the genus Pleurotus has been shown to increase
its mmen digestibility (Lindenfelser et al. 1979;
Me Queen and Reade 1983) and susceptibility
to enzymatic saeeharifieation (Hatakka 1983).
Optimum cultural and nutrition al conditions for
solid-state fermentation of wheat straw were
determined with the fungus Pleurotus ostreatus
(Tripathi and Yadav 1992). The fungus behaves
 Straw Enrichment for Fodder Production by Fungi
optimally under the following fermentation con-
ditions: pR 5.5; initial moisture 55%; temperature
22°C; period 21 days; substrate turning frequency;
no addition of free carbohydrates; urea level 1 %.
A maximum increase in IVDMD (10.4%) accom-
panied by a 2.7% degradation of lignin (g per 100
g dry matter of original straw) was attained under
the above conditions.
During screening of basidiomycetes for wheat
straw delignification, considerable lignin degrada-
tion with a limited effect on cellulose was attained
with Pleurotus eryngii. Among lignolytic enzymes
produced by white-rot fungi only lignin pero-
xidase can directly oxidize nonphenolic lignin
models, whereas Mn-dependent peroxidase and
laccase were detected und er SSF conditions;
however, the high laccase and aryl-alcohol oxi-
dase found in Pleurotus are supposed to degrade
mainly phenolic units. No lignin peroxidase was
detected under SSF conditions, but high laccase
and aryl-alcohol oxidase were found in Pleurotus
eryngii (Martinez et al. 1994).
Some white-rot fungi belonging to the genus
Pleurotus are characterized by their ability to pref-
erentially remove lignin with only a minor attack
on cellulose. Valmaseda et al. (1991) found pref-
erential degradation of lignin in solid-state fer-
mentation of wheat straw by P. ostreatus. The
attack on lignin degradation was more selective
with P. eryngii removing alm ost 50% of the lignin
with a very limited attack on cellulose, which was
less than 15% of the initial content. Kamra and
Zadrazil (1986) have also reported the preferen-
tial removal of wheat straw lignin by P. eryngii.
Other strains of Pleurotus, i.e., Pleurotus pul-
monarius and Pleurotus sajor-caju, were also used
during biological delignification of wheat straw.
These fungi halved lignin concentration (trom 12.1
to 5.6%) and doubled enzymatic digestibility of
wheat straw (trom 29.8 to 59.2%) within 12days
of incubation. After incubation, hemicellulose
concentration was halved, but the cellulose
content of the wheat straw remained constant
(Moyson and Verachtert 1991).
The other fungi of the Pleurotus spp. - Pleu-
rotus sajor-caju, Pleurotus sapidus, Pleurotus cor-
nucopiae, Pleurotus ostreatus (Tsang et al. 1987)
and Pleurotus eryngii (Zadrazil 1997) - form
edible fruit bodies and commercial use of Pleuro-
tus mushrooms is economically attractive. The
average yield of mushrooms was 3.6%, with me an
of weight loss 18%. Lignin losses (11 %) were
lower than cellulose (20%) and hemicellulose
27
(50%) losses in residual straw after mushroom
harvest. Cellulase digestibility was generally lower
than that of the original straw (Tsang et al. 1987).
The yield of fruit bodies (dry matter) of selected
Pleurotus strains - Pleurotus ostreatus, eryngii,
sajor-caju, sapidus, spodoleucus, and squarosulus
- on wheat straw fluctuated between 0 and 11.6%
(Zadrazil 1997), but the losses of organic matter
were considerably higher (about 40%). The sum-
marized results of the experiments mentioned
above revealed that some fungal strains belonging
to the genus Pleurotus very intensively degraded
hemicellulose over cellulose together with consid-
erable degradation of lignin (Table 2.5). Losses of
dry matter varied from 18-19%.
The fungus Phanerochaete chrysosporium
degraded the cell wall constituents (cellulose
and hemicellulose) at similar rates but with high
weight losses. All fungi mentioned in Table 2.5
increased the IVDMD of wheat straw. The
increase in digestibility must be offset against a
loss of organic matter. To obtain the maximum
change in the digestibility of substrates using
white-rot fungi aperiod of 4-5 weeks is needed.
Because of dry matter losses, the fermentation
period should be reduced to a few weeks; this
maximizes the total digestible dry matter. Studies
using lignin degradation and improvement of in
vitro dry matter digestibility of wheat straw by
white-rot fungi have constituted the evaluation for
the effectiveness of biological treatment.
3. Effect of Tropical and Other Fungi
Capelari and Zadrazil (1997) screened 72 species
and strains of tropical fungi for lignin degradation
and their ability to increase or decrease the in
vitro digestibility of wheat straw (25 and 30°C,
30 and 60days of incubation). Twenty-two strains,
including species of Gymnopilus, Irpex, Lentinus,
Oudemansiella, Peniophora and Psilocybe,
decomposed more than 50% of lignin and 10 of
them increased in vitro digestibility of the sub-
strate by 30 units. The highest degradation of
lignin was observed with Lentinus crinitus (80%,
60days) and the highest increase in in vitro
digestibility was caused by Peniophora utriculosa
(36 units, 30days). Screening of different species
of white-rot fungi by evaluation of the changes in
the lignin and polysaccharide contents of the
wheat straw cell walls and investigation of the
effect of wheat straw treatment by white-rot fungi
was carried out in experiments by Jale et al. (1997,
28 D. Jale

Table 2.5. Loss of structural components of wheat straw during decay by white-rat fungi

Fungus Time Losses (%)

(days)
Dry matter Cellulose Hemicellulose Lignin Reference

Phanerochaete chrysosporium 12 36.9 41.2 42.3 44.5 Agosin et al. (1985)

Cyathus stercoreus 22 34.5 36.8 40.8 52.2
Pycnoporus cinnabarinus 30 31.2 30.4 38.5 41.3
Phanerochaete chrysosporium 30 65.9 61.38 64.7 62.0 Moyson and
Verachtert (1991)
Pleurotus sajor-caju 80 19.6 12.7 59.40 63.0
Lentinus edodes 60 17.1 22.5 43.6 38.0
Pleurotus pulmonarius 80 19.1 7.4 58.1 62.0
Pleurotus ostreatus 20 18.0 17.0 57.0 13.0 Tsang et al. (1987)
Pleurotus sajor-caju 10 18.0 17.4 33.0 11.0
Pleurotus sapidus 18 18.0 23.0 48.0 31.0
Pleurotus cornucopiae 16 18.0 16.0 50.0 5.0
Pleurotus eryngii 80 18.0 14.0 43.0 47.0 Camarera et al. (1994)
Phanerochaete chrysosporium 30 39.0 63.0 50.0 45.0
Daedalia quercina 30 43.0 49.0 59.0 65.0 Jale et al. (1998)
Hericium clathroides 30 12.0 6.0 27.0 41.0
Phelinus laevigatus 30 24.0 20.0 26.0 36.0
Inonotus andersonii 30 24.0 24.0 32.0 19.0
Inonotus obliquus 30 24.0 22.0 30.0 31.0
Inonotus dryophilus 30 22.0 21.0 31.0 2.0

Table 2.6. Chemical composition (%) and in vitra dry matter digestibility (IVDMD) (%) of wheat straw after fungal
treatment

Variable Cellulose Hemicellulose Lignin IVDMD

UWS 46.2 15.5 8.8 41.4

D. quercina 41.9+++ 11.2+++ 5.Y++ 59.2+
H. clathroides 49.4++ 13.9+ 5.9+++ 56.3+
I. andersonii 46.4 15.1 8.6 51.4+
1. dryophilus 46.5 13.J+ 11.1+++ 55.9+
1. obliquus 47.7 15.4 8.1++ 52.0+
L. tigrinus 44.1 12.6++ 12.1 +++ 33.1 ++
P. laevigatus 48.J++ 15.2 7.4+++ 50.2+
UWS 41.2 25.0 8.9 44.0
L. ulmarium 42.0 21.2+++ 8.0 36.0+++
P. ostreatus 36.1 +++ 14.9+++ 7S 51.0+++
P. ostreatus (mutant) 39.1 ++ 18.4+++ 6.2+++ 54.0+++
P. brumalis 38.0+++ 19.4+++ 6.3+++ 40.1+
P. ciliatus 34.2+++ 11.2+++ 11.1++ 50.1 +++
T. gibbosa 38.1+++ 9.4+++ 6.5+++ 57.0+++

+P< 0.05; ++P < 0.01; +++P < 0.001, UWS - untreated wheat straw.

1998). Altogether, 13 different species of basid-
iomycetes were tested (Table 2.6).
The inoculated substrate was incubated at 28
oe for 30days. Treatment with ten species of
white-rot fungi significantly reduced the hemicel-
lulose content in wheat straw. Other fungi -
Inonotus andersonii, Inonotus obliquus and Pheli-
nus laevigatus - caused only sm all differences in
the hemicellulose conte nt of decayed straw. The
cellulose content was (1) reduced in straw treated
with six species of fungi, (2) unchanged with five
species of fungi, and (3) increased with two species
of fungi. The lignin content was reduced after
fungal treatment of wheat straw by most of
the fungi. Fungal treatment with L. tigrinus, 1.
dryophilus and P. ciliatus increased the lignin
content of wheat straw since these fungi removed
a relatively greater amount of digestible wall poly-
saccharides than lignin. Fungal treatment of wheat
straw significantly increased IVDMD in ten
 Straw Enrichment for Fodder Production by Fungi
decayed sampies while it decreased IVDMD in
straw decayed by three species of fungi (Table
2.6). The increase in IVDMD in decayed straw is
usually related to a decrease in the lignin content.
Losses of dry matter and cell wall constituents
(cellulose, hemicellulose and lignin) were deter-
mined only in straw inoculated with D. quer-
cina, H.clathroides, P.laevigatus, 1. andersonii, 1.
obliquus, and 1. dryophilus (Table 2.5). Out of the
three fractions (cellulose, hemicellulose, lignin)
hemicellulose and lignin revealed the largest
proportionate loss after inoculation with D.
quercina, C. clathroides, P. laevigatus, and 1.
obliquus. The other two fungi caused the largest
proportionate loss in cellulose and hemicellulose
content. The dry matter losses were similar; only
the fungus D. quercina caused a high dry matter
loss in wheat straw. Screening of 13 white-rot fungi
showed that 9 strains of the fungi effectively
enhanced the digestibility of wheat straw. A
lower effectiveness of wheat straw treatment was
observed with the fungi L. tigrinus, L. ulmarium
(decreased IVDMD) and P. brumalis (43% dry
matter loss).
Biological approaches which utilize fungi to
upgrade wheat straw into ruminant feed were
applied using an artificial rumen (Rusitec).
The semicontinuous culture system Rusitec
(Czerkawski and Breckenridge 1977) can provide
information on ruminal fermentation patterns,
microbial protein synthesis and nutrient digestibil-
ity with relatively small quantities of feed as
compared to in vivo experiments. This system is
especially useful when generation of a large quan-
tity of pretreated material is not desirable, as in
the case of biological delignification.
To study the effect of fungi-treated straw on
rumen fermentation in vitro a semicontinuous fer-
menter (Rusitec) was used. Wheat straw treated
with the fungi P. ciliatus, L. tigrinus, P. ostreatus, P.
ostreatus-mutant and T. gibbosa, together with
barley (80:20%), were used as experimental diets
in the artificial rumen. Untreated wheat straw
together with barley served as the control diet.
The fermentation characteristics during in vitro
experiments in Rusitec showed that the fungi-
treated straws did not have a higher nutrition al
value and were comparable to untreated wheat
straw. The fermentation of diets containing fungi-
wheat straw treated with three strains of the
fungus Pleurotus tuber-regium (Jale et al. 1999)
was of a similar nature.
29
B. Rice Straw
Rice or paddy straw is produced in large quanti-
ties and is unique in several ways. The sterns are
more digestible than the leaves, a finding in con-
trast to other straw species (Coxworth et al. 1981).
Rice straw contains more silica (12-16%) and less
lignin (6-7%) than other straws having a conte nt
of 3-5% silica and 10-12% lignin. The biogenic Si
content may reduce biodegradability by lowering
the cell wall digestibility (Van Soest 1981, 1994).
Other studies have shown that Si in rice straw
may inhibit degradability and reduce palatability
(Doyle and Chanpongsang 1990). The function
of silica is to increase the degree of resistance
against pathogens and against Iod ging of the
plants. In addition to this, the potassium content
in rice straw supports the plant resistibility, it influ-
ences the proportion of extractable silica, and may
affect the degradability of rice straw (Shen et al.
1998).
The biological treatment of rice straw by
white-rot fungi could have a major impact on the
utilization of this crop residue in developing coun-
tries where rice straw is currently fed to animals
untreated or treated with chemicals. For example,
urea treatment of rice straw has been combined
with fungal treatment by the alkalophilic fungus
Coprinus fimetarius. This treatment, the so-called
Karnal process, was characterized by a 30-day
ensiling of rice straw sprayed with urea, inocula-
tion with 3% Coprinus fimetarius culture and
incubation of the substrate for 14days at 35-
45°C (Gupta 1987). Fungally treated straw was
degraded to a lesser extent than untreated or
urea-treated straw. Karunanandaa et al. (1992)
studied the degradation of rice straw by applying
selected white-rot fungi and using loss of organic
matter, disappearance of cell wall constituents and
in vitro dry matter digestibility as indices of rumen
microbial degradation. Solid-state fermentation of
rice straw for 30 days with four white-rot fungi -
Cyathus stercoreus, Dichomitus squalens and two
strains of Phanerochaete chrysosporium (cellulase
- less mutant and a wild-type) - caused the dif-
ferent DM losses of rice straw (Table 2.7).
The highest DM losses were caused by the
strain P. chrysosporium and were primarily due
to extensive hemicellulose, cellulose and lignin
degradation. The weight los ses for C. stercoreus
and D. squalens were lower. All fungi showed lig-
nolytic activity, but D. squalens did not degrade
30 D. Jale

Table 2.7. Losses of cell wall constituents from rice straw after 30d incubation by white-rot fungi

Fungus Weight loss of OM Cellulose Hemicellulose Lignin IVDMD Reference

Rice straw
P. chrysosporium (wild) 31.7 48.8 51.5 33.3 -9.7 Karunanandaa
et al. (1992)
P. chrysosporium (mutant) 33.7 50.6 53.2 37.7 -11.6
Dichomitus squalens 9.4 5.9 11.8 ND -16.5
Cyathus stercoreus 6.8 ND 33.6 47.1 +17.1
Rice leaf
P. chrysosporium 25.0 24.5 56.1 37.9 -8.0 Karunanandaa
et al. (1995)
C. stercoreus 10.0 ND 52.1 54.7 +11.0
Pleurotus sajor-caju 10.3 2.1 52.2 53.6 +8.0
Rice stern
P. chrysosporium 8.9 11.4 7.5 ND -21.2
C. stercoreus 5.3 4.7 11.8 ND -19.4
P. sajor-caju 11.9 2.4 52.4 48.1 +4.4

rice straw lignin, perhaps due to its poor growth
on the material. A higher degradation rate of rice
straw lignin was observed with C. stercoreus. Both
Phanerochaete strains degraded lignin at a similar
rate and to a lesser extent than C. stercoreus. The
latter was the most effective fungal strain and con-
siderably enhanced the potential nutritional value
of rice straw with a minimalloss in DM.
1. Effect of Fungi on Rice Straw
Rice straw differs considerably from other cereal
straws such as barley, wheat and oats in that it has
a very high proportion of leaf-to-stem content
(60-40%) (Theander and Äman 1984) and the
leaves and sterns are similar in digestibility (Walli
et al. 1988). Karunanandaa et al. (1995) studied
the effect of three white-rot fungi - P. chrysospo-
rium, C. stercoreus, and P. sajor-caju - on the
botanical parts of rice straw. After solid-state fer-
mentation for 30days, C. stercoreus and P. sajor-
caju increased the IVDMD of the leaves (Table
2.7) by selective degradation of hemicellulose
in contrast to cellulose. On the other hand, P.
chrysosporium degraded cellulose and hemicellu-
lose indiscriminately in the leaves and lowered
their IVDMD.
The lower digestibility was primarily due to
extensive degradation of the structural carbo-
hydrates hemicellulose and cellulose by P.
chrysosporium. The remaining structural carbohy-
drates are probably more recalcitrant to rumen
microbial digestion. In rice straw 43% of total cel-
lulose is present in the crystalline form (Han
1978). Sterns decayed by P. chrysosporium and C.
stercoreus became less digestible as compared to
the control (18.5 and 20.3% vs. 39.7%, respec-
tively), although the hemicellulose and cellulose in
the sterns were poorly degraded after solid-state
fermentation. Only P. sajor-caju improved the
IVDMD of the sterns as compared to the control
(44.1 vs. 39.7%).
This study has demonstrated that, compared
with the sterns, the quality of rice leaves can be
selectively improved by fungal treatment with C.
stercoreus. On the other hand, digestibility of the
sterns decayed by C. stercoreus was decreased. The
reason for the inhibition of stern digestibility is not
known but an insignificant amount of hemicellu-
lose and cellulose was consumed during SSF. This
was due to poor or no growth of this fungus on the
rice stern fraction. The authors suggest that a
longer period than 30 days of SSF is required for
P. chrysosporium and C. stercoreus for rice sterns
to degrade lignin and structural carbohydrates.
These fungi mayaiso produce some metabolites
on the rice stern that are inhibitory to rumen bac-
teria and reduce the digestibility of a partially
decayed substrate. The other factors besides
energy are limiting for further improvement in the
digestibility of fungi-treated material.
2. Pro tein Supplementation of Fungi-Treated
Rice Straw
Rice straw, like other cereal straws, is very low in
emde protein (CP) eontent. Pro tein supplementa-
tion of fungi-treated straw may improve the effi-
ciency of ruminal microbial fermentation and
nutrient digestibility. Karunannandaa and Varga
 Straw Enrichment for Fodder Production by Fungi
(1996) studied the effect of protein supplementa-
tion of fungi-treated rice straw by C. stercoreus on
ruminal microbial protein synthesis and nutrient
digestibility using the continuous culture system.
Four fermenters consisting of four 1-1 glass vessels
with devices to maintain the an aerobic condition
at a constant temperature of 39°C were used for
the fermentation of diets which consisted of fungi-
treated or control rice straw and concentrates
at a ratio of 75:25% on a DM basis. This study
revealed that fungal treatment with C. stercoreus
enhanced fiber digestibility by increasing the
availability of cellulose but decreasing CP avail-
ability for rumen microbial digestion. Soluble
compounds appear to arise during SSF of rice
straw, without regard to whether it is from rice
straw or from fungus-inhibited dietary protein
degradation by ruminal microorganisms. This inhi-
bition could be due to bin ding of dietary protein
with lignocellulosic complexes or direct inhibition
of proteolytic microorganisms by compounds
secreted by white-rot fungi.
C. Barley Straw
Barley straw is usually ranked second to oat straw
by feeders although many cultivars of barley have
been characterized as straw of higher quality than
some cultivars of oats. Barley straw as a substrate
for fungal treatment by white-rot fungi has been
used to a very limited extent. Asiegbu et al. (1994)
used five white-rot fungi - Coriolus versicolor,
Pleurotus sajor-caju, Phanerochaete chrysospo-
rium, Chaetomium cellulolyticum and Tricho-
derma harzianum - to enhance the nutritional
value of barley straw. The moistened barley
straw (70% of moisture content) was inoculated
with the fungi T. harzianum, P sajor-caju, and C.
versicolor and incubated for aperiod of 14days
at 28 oe. The fungi C. cellulolyticum and P
chrysosporium were incubated at 37 and 40°C,
respectively.
The fungi-treated barley straw was fermented
in the semicontinnous culture system Rusitec. The
results proved that output of carbon dioxide was
higher with barley straw treated with C. versicolor,
P. sajor-caju and P. chrysosporium. No marked dif-
ferences in methane production were observed
between straws treated with different white-rot
fungi. In contrast, fungal treatment with T.
harzianum, C. celulolyticum and P sajor-caju led
to decreased IVDMD of straw, whereas P
31
chrysosporium and C. versicolor enhanced
IVDMD. Other experiments with fungal treat-
ment of barley straw were carried out by Khazaal
et al. (1990) and Khazaal et al. (1993). Attempts
to use ligninase to delignify barley straw and
thereby improve digestibility were not successful.
It was assumed that the P chrysosporium ligninase
used and/or the conditions of treatment (enzyme
concentration, pH or temperature ) might have
been unsuitable. There was no effect of ligninase
on either organic matter digestibility (OMD) or
detergent fiber composition of barley straw during
rumen fermentation in vitro. It was concluded that
the straw contained compounds that inactivated
ligninase and did not cause ligninase degradation.
Some phenolic compounds of straw can oxidize
lignin ase, resulting in temporary inactivation of
the enzyme (Harvey and Palmer 1990).
D. Oat Straw
Oat straw is preferred by most cattle producers for
feeding. The fact that oats do not have awns, as do
some cultivars of wheat and barley, is another
reason why feeders prefer to use oat straw.
Eriksson (1981) determined the in vitro organic
matter digestibility (IVOMD) of 67 sampies of oat
straw and found values ranging from 40-68%, i.e.,
an average value of 54 %. This was a higher
average than found for barley, wheat or rye straw
in the same study. Less information has been
published ab out fungal treatment of oat straw
by white-rot fungi. Jung et al. (1992) evaluated
five white-rot basidiomycetes - Phanerochaete
chrysosporium, Scytinostroma galactinum, Phlebia
tremelosa, Phelinus pini and Pholiota mutabilis -
for their ability to improve ruminal degradation of
oat straw in vitro. The fungi were inoculated on
oat straw for 30 days at 28°C and 90% relative
humidity. Dry matter losses after fungal treatment
of oat straw were noted for P chrysosporium
(30.6%), P pini (9.2 %) and P tremelosa (14.7%).
Lignin was removed from oat straw by P.
chrysosporium and P. tremelosa. Cell wall poly-
saccharides were removed from oat straw by
fungal activity. Only P. chrysosporium (+9%) and
P tremelosa (+5%) increased the IVDMD of oat
straw. The other fungi decreased IVDMD of oat
straw by ab out 2-5 units. P. chrysosporium seems
to be able to improve the quality of oat straw but
the loss of DM limits the practical benefit of this
increased digestibility.
32 D. Jalc

E. Maize Stover times more effectively than P. brevispora, indicat-

ing that lignin degradation alone cannot account
The nutrient content and digestibility of maize for the IVDMD enhancement and that degrada-
stover is higher than that of many other straws. tion of PCA and FA may be important.
Maize stover is the most abundant crop residue in Hemicelluloses were preferentially utilized by
the world (994 x 106 Mg annually; USDA 1989). all the fungi. The fungal preference for hemicellu-
lose over lignin may be indicative of the need for
The feeding valne of this crop residue is very poor
because of the low concentration of digestible drya more readily digestible energy source prior
matter and pro tein. Fungal treatment of maize to lignin degradation. Cellulose was extensively
stover by white-rot fungi has not been adequately degraded only by P. chrysosporium (69% loss
investigated. Karunanandaa et al. (1992) studied after 28days of incubation), while maize stover
the degradation of maize stover by selected white-colonized by the other two fungi retained more
rot fungi using loss of organic matter, disappear-than 84 % of the original cellulose. The results
ance of cell wall constituents and in vitro dry of this study suggest that the enhancement of
matter digestibility (IVD MD) as indices of rumen digestibility of maize stover colonized by white-rot
microbial degradation. fungi is regulated by various factors including
Four white-rot fungi - Cyathus stercoreus, degradation of structural carbohydrates, phenolic
Dichomitus squalens and two strains of Phane- acids and lignin. The development of fungal bio-
rochaete chrysosporium (cellulase - less mutant conversion technology to upgrade the feeding
and wild-type) - were used. After solid-state fer-value of fibrous crop residues in animal feed
mentation for 30days, C. stercoreus increased requires the understanding of the impact of fungal
IVDMD by about 15 units by selective degrada- treatments on cell wall phenolic acids.
tion of hemicellulose as opposed to cellulose. The Chen et al. (1996) investigated the degrad-
highest IVDMD was obtained with minimalloss ability of six monomeric phenolic acids, p-
of dry matter (33 gkg-1). In contrast, both strainscoumaric acid (PCA), ferulic acid (FA), caffeic
of P. chrysosporium (wild and mutant) degraded acid (CA), vanillic acid (VA), syringic acid (SA)
cellulose and hemicellulose to a similar extent andand p-hydroxybenzoic acid (PHBA), in a liquid
thus lowered the IVDMD for maize stover. The nutrient medium and the degradation of cell wall
results of this experiment identified C. stercoreusbound phenolic acids in maize stover by two
as the most effective fungal strain for enhancing strains of white-rot fungi - P. chrysosporium and
the potential nutritional value of maize stover. P. brevispora. Both P. chrysosporium and P. bre-
Chen et al. (1995) studied the changes of in vitro vispora were able to degrade the phenolic acids
dry matter disappearance (IVDMDe) and degra- in a cell wall bound form and in the free acid
dation of cell wall constituents in maize stover form. Fungal degradation of these phenolic acids
treated with three white-rot fungi - Cyathus ster- depended on the fungal strains and whether the
coreus, Phlebia brevispora and Phanerochaete acids were present in the wall bound or in the free
chrysosporium. form. P. brevispora appears to be a better fun-
Solid-state fermentation of maize stover for gal strain when used for bioconversion of crop
28days at 27°C improved IVDMD by about 10.5 residues into more digestible feed due to its supe-
and 11.4 units for P. brevispora and C. stercoreus,rior ability to disturb the cell wall structure by
respectively. On the other hand, growth of P. extensively removing cell wall phenolic acids.
chrysosporium reduced IVDMD by ab out 11.1
units. All fungi degraded cell wall phenolic acids -
p-coumaric acid (PCA) and fernlic acid (FA) - but F. Other Straws and By-Products
P. chrysosporium appeared to be the least ef-
fective in degrading PCA and FA. Phenolic In addition to the species of straw mentioned
acids, mainly p-coumaric and ferulic acid, serve to above, other straws and by-products have been
cross-link different wall components (Jung 1989; used for fungal treatment with white-rot fungi
Hartley et al. 1990) thereby enhancing structural (Table 2.8). Adefinition of a by-product feedstuff
rigidity and also limiting the availability of the is a product that has value as an animal feed and
structural carbohydrates for rumen digestion. is obtained during the harvesting or processing of
Conversely, P. chrysosporium degraded lignin 1.6 a commodity in which human food or fiber is
times more effectively than C. stercoreus and 1.4 derived (Fadel1999). The solid-state fermentation
 Straw Enrichrnent for Fodder Production by Fungi 33

Table 2.8. Loss of organic matter and lignin and changes in IVDMD of different agricultural by-products treated with
fungi

Substrate Fungus Loss of Loss of Changes in Reference

OM(%) lignin (%) IVDMD a

Reed sterns Stropharia rugosoannulata 22.8 33.9 16.1 Zadrazil (1980)

Rape straw 28.0 58.2 48.2
Sunflower stalks 24.4 46.8 -2.3
Reed sterns Pleurotus sp. Florida 29.5 55.8 15.0 Zadrazil (1980)
Rape straw 30.1 67.6 37.1
Sunflower stalks 27.4 60.3 20.8
Reed sterns Pleurotus cornucopiae 26.2 35.5 32.6 Zadrazil (1980)
Rape straw 44.5 68.1 27.8
Sunflower stalks 43.2 59.6 22.9
Reed sterns Agrocybe aegerita 8.4 2.0 -13.7 Zadrazil (1980)
Rape straw 24.0 36.0 -6.7
Sunflower stalks 17.7 13.4 -8.7
Pea straw Ganoderma lucidurn ND ND 8.0 Ibrahirn and Pierce (1980)
Cotton stalks Pleurotus ostreatus 16.0 30.0 29.0 Kerern and Hadar (1995)
Sugarcane bagasse Pleurotus gigantea ND ND 7.0 Ibrahirn and Pierce (1980)
Pleurotus sp. P7 16.6 30.7 5.2 Zadrazil and Puniya (1995)
Agrocybe aegerita 8.4 5.0 -22.4
Pleurotus eryngii 10.6 29.0 16.3
Pleurotus sp. PI 15.7 25.4 7.6
Kuehneromyces mutabilis 12.9 25.4 8.9

aIVDMD of untreated substrates (%): reed sterns (29.9), rape straw (34.1), sunflower stalks (41.4), pea straw (40.0),
sugarcane bagasse (32.0), cotton stalks (24.0), ND, not determined.

of rape straw and by-products - reed sterns, sun-
flower stalks and sugarcane bagasse - was carried
out at 25 oe for aperiod of 1-60 days. Pleurotus sp.
Florida, Pleurotus cornucopiae, Pleurotus sp. PI,
P7, Pleurotus eryngii, and Stropharia rugosoan-
nulata showed good lignin decomposition and
increase of in vitro organic matter digestibility.
The highest decomposition rate was found with P.
cornucopiae on rape and sunflower stalks.
Decomposition of lignin to only a sm all ex-
te nt and decrease of the in vitro digestibility
was observed with Agrocybe aegerita. The white-
rot fungi Pleurotus sajor-caju, Polyporus hirsutus,
Stropharia rugosoannulata and Ganoderma
lucidum degraded lignin of sugarcane bagasse
selectively and significantly increased in vitro
digestibility (Kewalramani et al. 1988; Kamra et al.
1993). One of the main crop residues are cotton
stalks which are a suitable substrate for degrada-
tion by Pleurotus ostreatus. Growth of P. ostreatus
on cotton stalks was fast er than that on wheat
straw (Platt et al. 1981). Utilization of cotton
stalks as a substrate for the solid-state process by
P. ostreatus was analyzed (Kerem et al. 1992) and
the effect of Pleurotus on cotton stalk digestibility
examined. After 21 d of solid-state fermentation,
the substrates (untreated and P. ostreatus treated
cotton stalks) were incubated in the rumen using
Dacron bags. The disappearance of organic matter
from the Dacron bags containing treated cotton
stalks was significantly greater (22%) than from
those containing untreated stalks (10%).
p. ostreatus was also used for solid-state fer-
mentation of cotton stalks and the effect of man-
ganese on lignin and cellulose degradation studied
(Kerem and Radar 1995). Preferential degrada-
tion of lignin was enhanced by the addition of
Mn(II) to cotton stalks at a concentration ranging
from 30-620 f.1g of Mn per g. The in vitro digestibil-
ity value of fermented cotton stalks was 53 % of
the dry matter. Addition of 150 and 600 f.1g of Mn/g
to the cotton stalks resulted in a digestibility value
of 61.4 and 65.4%, respectively. Enhancement of
preferentiallignin degradation could be a result of
either increased activity of the lignolytic enzymes
or production of Mn(III) which might preferen-
tially degrade the aromatic structure of the ligno-
cellulose complex.
IV. In Vivo Studies
The majority of the experiments conducted on the
evaluation of fungi-treated straw have been per-
formed by either in vitro digestibility or in sacco
34 D. Jale
degradability estimations. Several studies have
been conducted in the past to identify the species
of white-rot fungi for their ability to improve the
quality of lignocellulosic substrates as ruminant
feedstuffs (Zadrazil and Reiniger 1988; Reid
1989). Most of these studies were preliminary and
have used lignin degradation and improvement of
in vitro dry matter digestibility (IVD MD) as an
index to evaluate the effectiveness of biological
treatment. Information on in vivo digestibility of
Iignocellulosic materials treated with white-rot
fungi is scarce because of the difficulties and lack
of technology to produce large quantities of fungi-
treated material. Cereal straws as renewable
sources of energy play an important role in animal
feeding. According to Chang (1993),2.3 x 106 tons
of spent straw after harvesting of mushroom
strains of the genus Pleurotus (compost) are pro-
duced in the world per annum and this amount
increases every year in line with the expansion in
mushroom production. World mushroom pro duc-
tion according to the Food and Agriculture Orga-
nization of the United Nations in the year 1998
was about 2.2million tons/year (Kües and Liu
2000).
Some authors suggest that straw (compost)
degraded by enzymes during mushroom produc-
tion can be more easily digested by ruminants
(Yang et al. 1993). It contains more free sugars,
more protein, less cellulose and lignin with an
increased content of ash as compared to the orig-
inal material. The results of the feeding experi-
ment in lambs with spent wheat straw after
harvesting of Pleurotus sajor-caju revealed that
the digestibility coefficients of various nutrients
were slightly lighter than with untreated wheat
straw (Calzada et al. 1987).
A similar conclusion was made when a fungi-
treated rice straw diet was fed to sheep (Chandra
et al. 1991). Paddy straw left after harvesting P.
sajor-caju when replaced at a level of 50% of
untreated straw intake in the buffalo did not affect
the digestibility of nutrients, but a 100% replace-
ment had a negative effect on nutrient availability
to animals (Dhanda et al. 1994). Adamovie et al.
(1998) used spent wheat straw after Pleurotus
ostreatus mushrooms (compost) in cattle fee ding
where compost formed 10 and 17% of dietary dry
matter. It was found that animals would not
consume a ration mixed with more than 17% DM
from the compost. The utilization of dry matter
per kg of gain tended to decrease with the
increased amount of compost in the diet. The
results of experiments when spent wheat straw
was fed to steers (Henics 1987) proved the inhi-
bition of ammonia-N utilization by ruminal
microbes.
On the whole, soluble compounds formed
during SSF (from the substrate or from the
fungus) appear to inhibit dietary protein degrada-
tion by ruminal microorganisms. This inhibition
could be due to the binding of dietary pro tein with
lignocellulosic complexes or to direct inhibition
of proteolytic microorganisms by compounds
secreted by white-rot fungi. However, to estimate
the influence of feeding fungi-treated straw on the
physiology and performance of ruminants, long-
term feeding experiments with a larger number of
ruminants are needed.
v. Conclusions
The main problem associated with the biological
upgrading of straw into feed is to find suitable
microorganisms for application in a cheap large-
scale process of delignification and upgrading. Lig-
nolytic microorganisms are mainly white-rot fungi
that are able to colonize different plant residues
and to increase the digestibility of the substrate.
At present, the white-rot fungi that form edible
fruit bodies - Agaricus, Volvariella, Lentinus, and
Pleurotus spp. - are commercially cultivated
worldwide. About 300 strains of basidiomycetes
have been screened for their ability to degrade
lignin and change the in vitro dry matter
digestibility of various crop residues, mainly straw.
Among the fungal strains tested, some species
of Pleurotus, Ganoderma, Stropharia, Polyporus,
Cyathus, Dichomitus, Lentinus, Sporotrichum,
Trametes and Phlebia have proved to degrade
lignin selectively and also to increase the in vitro
digestibility of different species of straw. The
increase in digestibility must be offset against loss
of organie matter (Zadrazil et al. 1995). Phane-
rochaete chrysosporium was the most effective
species for removing lignin and increasing the
digestibility of straw. Large losses of dry matter
occur since the fungal metabolism of the crop
residues imposes a serious constraint on practical
application. The feeding experiments conducted
with spent straw after harvesting mushrooms were
characterized by decreases in the digestibility
coefficients of various nutrients and average daily
gains in ruminants. Direct conversion of straw into
 Straw Enrichmcnt for Fodder Production by Fungi
ruminant feed by white-rot fungi with the present
knowledge is not economically feasible. New bio-
logical approaches utilizing fungi for bioconver-
sion of lignified crop residues under optimized
solid-state fermentation conditions will have to be
used. At present, molecular biological approaches
are applied to study fungal lignocellulose degra-
dation with the advent of the polymerase chain
re action (PCR). PCR-based strategies are used
to better understand the physiology and molecu-
lar biology of fungal lignocellulose degradation
(Reddy and D'Souza 1998) and will be an expand-
ing field in future research.
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3 Food and Crop Spoilage on Storage
JAN DIJKSTERHUIS 1 and ROBERT A. SAMSON2
CONTENTS
I. Introduction: Food Is an Ecological
Resource .............................
11. Infection of Living Crops:
Post-Harvest Diseases . . . . . . . . . . . . . . . . . . .
A. Anthracnose as an Example of Complex
Crop-Fungus Interaction . . . . . . . . . . . . . . . . .
B. Opportunistic Infections .................
111. Processed Foods: Spoilage as Colonisation
of a Medium ..........................
A. The Food Production Chain ..............
B. During Storage ........................
C. Association of Fungal Species
with Food Products .. . . . . . . . . . . . . . . . . . . .
IV. Food Preservation: Creation of Adverse
Environments .........................
A. Stress Resistance and Accumulation
of Compounds . . . . . . . . . . . . . . . . . . . . . . . . .
1. Osmotolerance ......................
2. Conidia and Phenotypic Plasticity .......
3. Trehalose as a Stress Protectant . . . . . . . . .
4. Is There a Preference for a Certain Protective
Compound? ........................
B. Heat Resistance . . . . . . . . . . . . . . . . . . . . . . . .
1. Fungal Propagules and Heat Resistance ..
2. Heat Resistance and
the (Micro)environment . . . . . . . . . . . . . . .
3. Endogenous Factors
and Heat Resistance . . . . . . . . . . . . . . . . . .
4. Ascospore Dormancy
and (Heat) Activation . . . . . . . . . . . . . . . . .
C. Alternative Methods for Sterilisation
of Food Products . . . . . . . . . . . . . . . . . . . . . . .
V. Mycotoxins ...........................
VI. Conc1usions ...........................
References . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1 Wageningen Centre for Food Sciences (WCFS), P.o. Box
557,6700 AN, Wageningen, The Netherlands
1 Agrotechnological Research Institute (ATO), P.O. Box
17,6700 AA Wageningen, The Netherlands
2 Centraalbureau voor Schimmelcultures (CBS),
Uppsalalaan 8,3584 CT Utrecht, The Netherlands
Present address: 1. DUKSTERHUIS, Centraalbureau voor
Schimmelcultures (CBS), Uppsalalaan 8, 3584 CT Utrecht,
The Netherlands
I. Introduction: Food Is an
Ecological Resource
39
39 As all places where organisms develop, the food
product can be regarded as an ecological resource.
40
40 When fungi colonise such environments, spoilage
of food and crops is the result. The interactions
40 between spoilage of food and fungi have been
41
41
described by Pitt and Hocking (1997) and Samson
et al. (2001). Prior to spoilage the fungi can be
41 present on or inside the crop in low numbers or as
survival structures. Spoilage fungi are also intro-
42
duced to an empty habitat when the food is
43 treated by pasteurisation treatments. Food prod-
43 ucts include two main groups, namely the living
44 crop and the processed food. Colonisation of food
44
products, therefore, is very diverse. This chapter
45 will evaluate different fungal-food relationships.
46 At first, the relationship between the living crop
46 and fungi will be illustrated. Then the association
47 of fungi with different types of processed food will
be described. Different preservation techniques
47 make the food product a difficult environment to
colonise, although it is also a rieh medium. So,
47
fungi that can survive adverse conditions as for
48 example high osmolarity and he at can still suc-
49 cessfully enter the food. Different aspects of stress
49
49
resistance are addressed in this chapter including
osmotolerance, protective compounds inside cells
and heat-resistant structures. Successful growth in
the food product may lead to the production of
toxic metabolites, which is an issue that needs our
continuous awareness with respect to food safety.
11. Infection of Living Crops:
Post-Harvest Diseases
The relation between fungi and living agricultural
crops can be plant-pathogenic in nature and
include complex communication between parasite
The Mycota XI
Agricultural Applications
Kempken (Ed.)
© Springer-Verlag Berlin Heidelberg 2002
40 1. Dijksterhuis and R.A. Samson

and host. Other fungi cause opportunistic infec- colonisation ofthe crop, enzymes that are released
tions of fruit, vegetables and fiower bulbs and by the growing hyphae dissolve the plant cell wall
enter cracks or wounds on the surface of crops. and the tissue be comes macerated. Indeed, fungi
are able to synthesise and secrete a wide variety
of polysaccharide-degrading enzymes. The extent
A. Anthracnose as an Example of Complex
and variability of these enzymes have recently
Crop-Fungus Interaction
been reviewed (De Vries 2000; De Vries and
Visser 2001) for the fungus Aspergillus niger. This
Members of the fungal genus Colletotrichum
fungus causes serious opportunistic infections in
(teleomorph; Glomerella) cause anthracnose
onions and hyacinth bulbs.
disease in various fruits and vegetables. In the case
In citrus fruit the fungi Penicillium italicum
of infection of the climacteric fruit of the tomato
and P. digitatum (blue and green rot of citrus
with Colletotrichum gloeosporioides, conidia actu-
respectively) cause the most serious and wide-
ally wait for the right moment to infect. The spores
spread rots of these crops. Other opportunistic
form specialised infection structures, the so-called
fungi on these fruits are Alternaria alternata,
appressoria, that penetrate the plant cells only
Aspergillus niger, Fusarium spp., Geotrichum can-
when the host (tomato) has ripened. Here, the
didum and Trichoderma viride (Snowdon 1990).
plant hormone ethylene that is produced by the
Penicillium hirsutum infects fiower bulbs (from e.g.
tomato during senescence is the trigger for proper
iris, tulip and lily) and invades wounds on the
infection (Flaishman and Kolattukudy 1994). C.
surface of the bulb and kills it when it reaches the
gloeosporioides also reacts on other chemical trig-
root disc (Smid et al. 1993). For tomatoes, oppor-
gers, namely the host surface wax in the case of
tunistic fungi often develop first on the remnants
anthracnose of avocado fruit (Podila et al. 1993).
of the leaves present on the fruit (the so-called
Conidia germinated with long germ tubes on a
calix) and then colonise the tissues of the fruit
glass surface but formed appressoria in the pre-
(Smid et al. 1996).
sence of extracted avocado wax. Analysis of wax
Careful handling of crops directly after har-
fractions showed that differentiation of appresso-
vesting is vital for the quality of the product. The
ria was maximal in the presence of long-chain fatty
more sm all wounds that are introduced by rough
acid alcohols. That this aspect of communication
treatment of the crop, the more damage is seen as
is also very specific is illustrated by the inhibition
a result of post-harvest diseases. Indeed, conidia
of appressorium formation by wax from other
of P. digitatum are stimulated to germinate in the
fruit Gade wax). Finally, the formation of appres-
presence of volatiles that surround wounded
soria is dependent on the presence of a surface
oranges (Eckert and Ratnayake 1994). In the
(Kim et al. 1998), a feature that is observed in
1970s it was recognised at international confer-
other parasitic fungi as weH. Different thig-
ences that increased amounts of food could be
motropic (sense-reactive) and chemical signalling
made available by protecting it more effectively
pathways cooperate in the differentiation process.
after harvest than by pouring more effort into
Wax- and ethylene-dependent pathways are not
production (see Snowdon 1990).
identical, but share two proteins that must be
phosphorylated (Kolattukudy et al. 1995). Sig-
nalling is also time-critical and must be transient;
dephosphorylation of these enzymes is also crucial 111. Processed Foods:
and, when blocked (by caliculin A, Kolattukudy et Spoilage as Colonisation
al. 1995), developmental sequences disrupt and ofa Medium
abnormal structures are formed.
Processed food compared to living crops is more
B. Opportunistic Infections like a complex medium that can be colonised and
therefore spoiled by invading organisms. Spoilage
Opportunistic fungi infect crops without the help fungi can be present in the basic components of
of specialised infection structures but enter cracks the product or can be introduced into the food
or wounds on the surface of the crop. They can during the food production chain and subsequent
also enter via the dying leaves of the fiower before storage (inoculation via the surroundings, most
the fruit is fully grown (Snowdon 1990). During probably the air). Often fungi are discouraged to
 Food and Crop Spoilage on Storage
develop as a result of preservation techniques
such as fermentation, addition of salts or high
concentrations of sugars, drying technologies or
addition of preservatives. The ecological niehe
of processed food products therefore can be
regarded as an extreme environment.
A. The Food Production Chain
Fungal spoilage organisms can develop in the food
production chain consequently building up
considerable biomass (and many propagules) in
certain areas of the chain and then acting as a
source of infection of food products that are newly
introduced into the chain. In this way a "house
flora" can develop inside certain factories; e.g.
Penicillium roqueforti that causes spoilage in rye
bread factories and Fusarium oxysporum in dairy
products. Geotrichum candidum was known as
the "machinery mould" or "dairy mould" and is
responsible for slime building in processing
equipment and off-smells in finished products
(Wildman and Clark 1947).
B. During Storage
The density of fungal spores in the (indoor) air
varies greatly and is certainly dependent on the
ability of fungi to form large numbers of them. The
propagules then enter food and crops and can
cause damage. In the case of Penicillium expan-
sum infecting apples, high spore densities in the air
are probably caused by growth of the mould in
high concentrations on rotten organic materials
in orchards (Börner 1963). Fresh food products
can be regarded as empty habitats as a result of
antimicrobial treatment during processing.
Fungi also develop inside buildings (where
storage occurs) and their proliferation is then
often related to leakage, flooding, condensation
and humidity problems. Occupants may also con-
tribute to mould growth by means of activities
gene rating humidity or obstructing venting of the
building. Interestingly, the fungal flora of indoor
environments is similar to that found on food
(Flannigan et al. 2001).
Especially in an indoor atmosphere, relatively
large numbers of conidia ensure colonisation of
newly introduced food products. Previous indoor
food spoilage may grossly enhance the inoculum
pressure on newly introduced food products. For
41
Fig. 3.1. Scanning electron micrographs of a conidial head
of Aspergillus niger showing the abundance of dry conidia
produced from phialides
example, in Dutch cheese warehouses, Penicillium
dis color commonly occurs and can cause serious
spoilage when poor hygienic conditions increase
the sporulation of this fungus.
Massive production of conidia can be
regarded as a vital strategy for dispersion of a
number of important food-borne fungi. The order
Eurotiales includes many relevant food spoilage
fungi (see Samson et al. 2001); for example, the
genera Paecilomyces, Penicillium and Aspergillus.
Paecilomyces forms relatively simple conidio-
phores, Penicillium forms relatively compact
complex conidiogenous cells and Aspergillus
forms highly specialised conidiophores that can
form enormous numbers of propagules (Berbee et
al. 1995; Fig. 3.1, A. niger). With respect to food
spoilage, Aspergillus is more suitable for tropical
areas compared to Penicillium, which is observed
more in temperate areas.
C. Association of Fungal Species
with Food Products
Fungal growth occurs under favourable conditions
for each species, adaptability determining species
domination. The specific reason why a particular
species dominates in a certain food product is
often not known, but is certainly correlated with
characteristics and properties of the product. Food
parameters also are surprisingly restrictive to the
spectrum of species which are able to grow and
thus spoil the individual food types. Normally, less
than ten and often one to three species are respon-
sible for spoilage (Frisvad and Filtenborg 1988,
1993). Heavy contamination from ecological
42 1. Dijksterhuis and R.A. Samson

Table 3.1. Associated spoilage mycobiota of different foods (based on Simmons 1986, 1992, 1993, 1994, 1995, 1999a,b;
Simmons and Roberts 1993; Filtenborg et al. 1996)

Foods Fungal species

Ci trus frui ts: Alternaria citri; A. tangelonis; A. turkisafria; A. colombiana; A. perangusta; A.

interrupta; A. dumosa; Penicillium digitatum; P. italicum; P. ulaiense
Pomaceous and stone fruits: Penicillium expansum; P. crustosum; P. solitum; Alternaria gaisen; A. mali; A.
tenuissima group; A. arborescens group; A. infectoria group; Cladosporium
spp.
Garlic and onions: Penicillium allii; P. albocoremium; P. glabrum; Petromyces alliaceus; Botrytis
aclada
Potato tubers: Fusarium sambucinum; F. coeruleum
Tomatoes: Alternaria arborescens; Stemphylium spp.; Penicillium olsonii
Yam tubers: Penicillium sclerotigenum
Wheat and rye grain, field condition: Fusarium culmorum; F. graminearum; F. avenaceum; F. equiseti; F. poae; F.
tricinctum; Alternaria triticimaculans; A. infectoria; A. oregonensis; A.
triticina; A. triticicola; A. tenuissima group; Cladosporium herbarum;
Epicoccum nigrum; Stemphylium spp.; Ulocladium spp.; Drechslera spp.;
Botrytis spp.; Penicillium spp.; Claviceps purpurea
Wheat and rye grain, stored condition: Penicillium aurantiocandidum; P. cyclopium; P. freii; P. hordei; P.
melanoconidium; P. polonicum; P. verrucosum; P. aurantiogriseum; P.
viridicatum; Aspergillus flavus; A. niger; A. candidus; Eurotium spp.;
Alternaria infectoria group
Cereals in airtight storage: Paecilomyces variotii; Scopulariopsis candida; Penicillium roqueforti; Candida
spp.; Byssochlamys fulva; B. nivea
Acid preserved cereals: Penicillium glandicola (= P. granulatum); P. roqueforti; Paecilomyces variotii;
A. flavus; A. candidus; A terreus; Monascus ruber
Spices: Aspergillus flavus; A. tamarii; A. niger; A. ochraceus; A. candidus; A.
versicolor; Eurotium spp.; Wallemia sebi; Penicillium islandicum; P.
neopurpurogenum; P. citrin um
Nuts: Penicillium commune; P. crustosum; P. discolor; P. solitum; P. funiculosum; P.
oxalicum; P. citrinum; Asfergillus flavus; A. wentii; A. versicolor; Eurotium
spp.; Alternaria infectoria group
Rye bread: Penicillium roqueforti; P. paneum; P. carneum; P. corylophilum; Eurotium
repens; E. rubrum; Paecilomyces variotii; Monascus ruber
Cheese: Penicillium commune; P. nalgiovense; P. atramentosum; P. nordicum;
Aspergillus versicolor; Scopulariopsis fusca; S. candida; S. brevicaulis
Fats, margarine, etc.: Penicillium echinulatum; P. commune; P. solitum; P. spinulosum; Cladosporium
herbarum
Fermented sausages: Penicillium nalgiovense; P. olsonü; P chrysogenum; P. nordicum; P solitum; P
oxalicum; P. commune; P. expansum; P. miczynskii; P. brasilianum
Pasteurised foods: Byssochlamys fulva; B. nivea; Hamigera reticulata; Neosartorya fischeri; N
glabra; N spinosa; Eupenicillium lapidosum; Talaromyces macrosporus; T.
bacillosporus; Paecilomyces variotii
Low water activity foods: Eurotium chevalieri; E. herbariorum; E. amstelodami; Wallemia sebi;
Aspergillus penicillioides; A. restrictus; Eremascus albus; E. fertilis;
Xeromyces bisporus; Scopulariopsis halophilica; Chrysosporium inops
(sensu Pitt); C. farinicola; C. fastidium; C. xerophilum; Polypaecilum pisce
Indoor environment: Aspergillus niger; A. versicolor; A. sydowii; Penicillium chrysogenum; P.
brevicompactum; P. glabrum; Eurotium herbariorum; Emericella nidulans;
Alternaria alternata; Ulocladium chartarum; Scopulariopsis brevicaulis;
Wallemia sebi

niehes where the mould has developed mayaiso classes of food products and associated fungi (see
determine the predominance of a fungal species also Filtenborg et al. 2001).
on food. Furthermore, critical species are often
completely different for each food type. As far as
fungi in foods are concerned this discovery is fairly IV. Food Preservation: Creation
new (Frisvad and Filtenborg 1988), and is due to
the development of new mycologieal methods and
of Adverse Environments
taxonomy of food-borne moulds with an empha-
sis on the genera Penicillium, Aspergillus and Several yeasts (Debaryomyces hansenü, Zygosac-
Fusarium. Table 3.1 shows a survey of different charomyces rouxii and bailü) that are notorious
 Food and Crop Spoilage on Storage
for spoiling food products are very osmotolerant
and can grow in environments with extremely high
concentrations of sugar and salto Ecologically, D.
hansenii is a marine fungus (Clipson and Jennings
1992) but it is also isolated from penguin drop-
pings, anthills and soils. It spoils brine foods and
contaminate fruit powders. Zygosacchamyces
species, including the extremely osmotolerant
species rouxii and bailii, cause major food
spoilage.
Other fungi can grow at very low water activ-
ities (Eurotium amstelodami, Wallemia sebi and
Xeromyces bisporus). E. amstelodami is known
for spoilage in corn silos where it can develop at
15-16% moisture levels. W sebi grows on dried
figs and dates; X bisporus is the most xerophilie
fungus known to date.
Fungi that are present on food products or
are introduced via the food production chain are
inactivated by heat treatments. Spoilage can occur
after pasteurisation treatments when certain heat-
resistant fungi survive high temperatures. Fungi
such as Byssochlamys nivea and B. fulva, Neosar-
torya fischeri and Talaromyces macrosporus can
survive temperatures of 85°C for many hours! For
these fungi, the sexual ascospores exhibit this
resistance. Storage of food products can also intro-
duce adverse conditions; cooled storage intro-
duces new selection criteria for psychrotolerant
fungi (often bel onging to the genera Alternaria,
Fusarium, Penicillium and Cladosporium.
A. Stress Resistance and the Accumulation
of Compounds
1. Osmotolerance
The number of yeast species that are involved in
food spoilage is only small compared to their total
number and include extreme osmotolerant organ-
isms. D. hansenii grows at sodium chloride con-
centrations of 2 M with a specific growth rate of
0.14 (Prista et al. 1997). Z. rouxii can still grow in
875 g sugar/l and at pH 2.5 (Membre et al. 1999)
or in 3.1M NaCl (Hosono 2000). In addition, Z.
bailii is also extremely tolerant to organic acids
such as sorbic and benzoic acid (Steels et al. 2000).
Generally, these yeasts prefer simple sugars as
nutrients.
In order to counteract water loss due to a
hyperosmotic environment many organisms accu-
mulate compatible solutes (Jennings and Burke
1990). The term compatible means that high intra-
43
cellular concentrations of solute are compatible
with enzyme functioning. Sugars and polyols are
regarded as more compatible solutes than sodium
chloride.
During mid-exponential growth, D. hansenii
accumulates glycerol up to approx. 35 % dry
weight of the cells (Adler et al. 1985). At the
beginning of the stationary phase, a major portion
of the glycerol leaks out of the cells. Simultane-
ously, glycerol is actively taken up by the cell and
metabolised. Growth at high salinity did not affect
the apparent Km of the glycerol transport but
caused a twofold increase in V max (Adler et al.
1985). Remarkably, the type of solute may change
with the growth phase; arabinitol (arabitol) is the
major solute present in the cell at the stationary
growth phase (Adler and Gustafsson 1980). Z.
rouxi also accumulates high levels of glycerol, but
is better able to retain it inside the cell (Hosono
2000).
The moderate osmotolerant fungus Geot-
richum candidum is an important spoilage organ-
ism (diary, vegetables and fruit) and can heavily
contaminate food production chains. It forms
numerous one-celled arthrospores and cultures
show some resemblance with yeasts in develop-
ment and morphology. Arabitol, a sugar alcohol,
accumulates in Geotrichum species in 1M NaCl.
It reaches amounts above 30-40% dry weight,
which decreases slightly at the stationary phase of
culturing. One species of Geotrichum (out of five) ,
however, accumulated mannitol as a compatible
solute in similar amounts. Mannitol is also formed
during the stationary growth phase without salt
(Luxo et al. 1993).
During mid-exponential growth, D. hanseni
accumulates high concentrations of sodium
(approx. 750mM) and potassium (300mM, Prista
et al. 1997). It differs from S. cerevisiae because
these high concentrations do cause less toxicity to
the cello During salt stress, Z. rouxi expresses a
proton-ATPase in combination with a Na+/H+
antiporter to remove sodium ions from the cell
(Watanabe et al. 1991,1995). A S. cerevisiae strain
that was very sensitive to salt stress was made
more osmotolerant with the antiporter genes
(Zsod2 and Zsod22) from Z. rouxi (Iwaki et al.
1998). When the antiporter was deleted from Z.
rouxii the organism could not grow on medium
with high salt concentration, but was still able to
develop on very high concentrations of sugar
(Watanabe et al. 1995).
In order to compare different compatible
solutes in S. cerevisiea, strains were constructed
44 1. Dijksterhuis and R.A. Samson

that accumulated mannitol or sorbitol instead of pounds inside the conidia. Optimal conditions
glycerol. Despite these compounds being present might result in stronger spores, which can be
in similar concentrations to glycerol, only restric- stored for longer times and used for biological
tive protective action was observed under salt control of insect pests. However, more generally,
stress (Shen et al. 1999). Glycerol apparently is the viability of the conidia of many fungal species
specific in its protective action during osmotoler- might be highly inftuenced by the conditions
ance in baker's yeast. In this study, transformed during conidiogenesis. The principles found in the
strains that were not able to synthesise glycerol entomopathogenic species mayaiso apply for the
were treated with the mutagen EMS and salt- species occurring on food.
tolerant mutants were selected. One mutant, For Metarrhizium anisopliae, conidia contain
designated sr 13, was very well able to survive 0.7 mannitol up to 9-13 % of the dry cell weight
M NaCl. These cells showed some accumulation (Hallsworth and Magan 1996). The mannitol
of glycerol and sorbitol, but had decreased tre- content of spores was much lower when spores
halose levels. However, potassium levels had were formed below pH 4 and 15 oe. Conidia of two
increased c1early compared to sodium levels and other fungal species, Beauveria bassiana and
it is conc1uded that the ratio between Na+ and K+ Paecilomyces farinosus, behaved differently. The
is important for tolerance (Gaxiola et al. 1996). mannitol content of these spores dropped when
Besides handling of ion concentrations and the culture aged and at temperatures above 20 oe.
prevention of water loss by compatible solute In addition, mannitol levels were lower, namely
accumulation, osmotolerance inc1udes other 2-6% of the dry weight in these conidia. Other
aspects. Z. rouxii alters the fluidity of the mem- solutes, mainly trehalose, arabitol, erythritol and
brane; ergosterol levels increased nearly three- glycerol, were present in these two species varying
fold during extreme salt stress (Hosono 2000). In with growth conditions. Generally, younger cul-
baker's yeast, an osmotic shock with salt leads to tures formed conidia with maximal amounts of
the up-regulation of 186 genes and the downreg- solute at moderate pH and temperature. When the
ulation of another 100. At least 300 genes of the water activity was lowered outside cultures of B.
total of 6000 are involved in the response of the bassiana and P farinosus, trehalose contents
cell and this is only at 45 min following the shock c1early dropped to zero at aw = 0.94. Under these
(Rep et al. 2000). Different signalling (MAP conditions, the lower molecular weight polyols
kinase ) pathways for general stress, osmotic shock, were present in the conidia at higher concentra-
pseudohyphal growth and the pheromone tions (Hallsworth and Magan 1994).
pathway share compounds and perform cross-talk
(see e.g. O'Rourke and Herskowitz 1998). This
3. Trehalose as a Stress Protectant
makes it c1ear that the cells solve problems raised
by external stress in a muItidisciplinary way. It is widely suggested that trehalose accumulation
is an important factor in yeast heat tolerance and
protection of cell components (Wiemken 1990).
2. Conidia and Phenotypic Plasticity
Both membranes (Crowe et al. 1984) and proteins
Conidia are major dispersible cells of teleomor- (Hottiger et al. 1994) are stabilised by trehalose.
phic fungi and have to survive dry conditions De Virgilio et al. (1994) measured thermotoler-
during transport through the air. In these cells ance and trehalose contents in S. cerevisiae fol-
accumulation of polyols or trehalose (a disaccha- lowing a he at shock at 40 oe. They found that
ride) is observed. Conidia of different fungal mutants without trehalose synthase (i1tspl) that
species often contain high levels of mannitol did not accumulate trehalose had no acquired
and/or trehalose (e.g. Aspergillus oryzae, A. thermotolerance. In addition, a neutral trehalase
nidulans; Horikoshi and Ikeda 1966; D'Enfert defective mutant (i1nthl) that exhibits very slow
and Fontaine 1997). Hallsworth and Magan (1994, mobilisation (degradation) of trehalose shows a
1996) state that phenotypic plasticity is an impor- slower decrease in thermotolerance after a he at
tant factor in optimisation of the resistance of shock. Kim et al. (1996) suggest that another
spores of three insect pathogenic fungi. This trehalase, the acidic vacuolar trehalase, is more
means that different environmental conditions important for degradation of trehalose and its
during cultivation of the fungus strongly inftuence disruption leads to better protection against
the composition of (putative) protective com- dehydration, freezing and ethanol shock. As a
 Food and Crop Spoilage on Storage
result of its deletion the trehalose concentration
in the cells stays higher and has a protective effect.
In addition, a correlation between trehalose
concentration and drought resistance and cryoto-
lerance of the cells is observed (Gadd et al. 1987;
G6linas et al. 1989). Drought and cryotolerance of
yeast cells is relevant in the food industry while
yeasts are widely used and stored in dried or
cooled conditions.
However, trehalose is not the only factor
involved in protection against these stresses.
Retention of trehalose at amounts of 10% dry
weight after the onset of fermentation (a factor
that enhances trehalose mobilisation) in S. cere-
visiae did not prevent a dramatic loss in he at
tolerance in the cells (Van Dijck et al. 1995).
Cryotolerance could be surprisingly low with even
high trehalose concentrations when culturing
conditions of yeasts were different. Further, only
small differences in trehalose contents caused
large differences in cryotolerance (G6linas et al.
1989). During culturing yeast accumulates tre-
halose inside cells and maximum contents are
observed within 6 days. Drought tolerance,
however, is only maximal after 12 days, indicating
that this stress resistance is increasing to its
maximum while the concentration of trehalose is
maximal. Z. bailii shows a thermotolerance at pH
6.5 in the presence of sorbic acid without trehalose
accumulation (Cheng et al. 1999).
In S. cerevisiae more protecting principles are
combined, as is shown in the study of Elliott et al.
(1996) where both the heat shock protein Hsp 104
and trehalose accumulation act synergistieally in
acquired thermotolerance. Disruption of one of
both gave only a marginal decrease in survival of
the cells at 50°C, but a double mutant showed
highly sensitive cells.
Here it is important to realise that trehalose
as a protectant in vegetative cells has to act in a
highly aqueous environment. However, high
concentrations of the compound are needed for
protection. Accumulation of protective com-
pounds is an important principle in both bacterial
and fungal heat-resistant cells. Bacterial spores
realise proteetion in different ways. First, intimate
associations between proteins and DNA prevent
UV-damage (Setlow 1992). In addition, high con-
centrations of minerals (calcium) and dipicolinic
acid are present outside the nucleoid (Nicholson
et al. 2000). Thirdly, low water contents in spores
are thought to add to the resistance of the spores
(Nicholson et al. 2000). A combination of low
45
water conte nt and very high trehalose (above 15 %
cell weight) contents might be an important factor
in heat resistance in the fungus T. macrosporus
(I Dijksterhuis, unpubl. results).
4. Is There a Preference for a Certain
Protective Compound?
Are there specific differences between the com-
patible solutes that make them better for a certain
type of proteetion? We have already observed the
role of glycerol in S. cerevisiae during osmotoler-
ance and the dominance of mannitol and trehalose
in fungal conidia and ascospores. Trehalose is asso-
ciated with longevity in ascospores (ascospores
of T. macrosporus that contain a high level of
trehalose can be stored for more than 2 years,
Beuchat 1992) and conidia (in the case of ento-
mopathogenic fungi, Halsworth and Magan 1996).
Trehalose also accumulates under drought stress;
the fungus Serpula lacrymans accumulates not
only trehalose, but also arabitol during growth
over Perspex (Brownlee and Jennings 1981).
Both trehalose and mannitol can act in meta-
bolie futile cycles, where compounds are synthe-
sised and degraded at the same time. Hottiger et
al. (1987) observed quick accumulation of tre-
halose after heat shock. The activities of both the
anabolie trehalose synthetase and the catabolic
trehalase were strongly enhanced. The turnover of
trehalose in the cell was very high although total
amounts of the compound remained stable in the
cell. These futile cycles may serve as an overflow
mechanism when metabolism slows down during
the appearance of stress. Mannitol formation and
degradation, for instance, lower the amount of
reduction equivalents formed during intensive
growth. The trehalose synthetase complex may act
as a regulator of glycolysis in the yeast S. cerevisiae
to prevent collapse of phosphate levels inside
the cytoplasm by lowering the internal glucose
concentration (Thevelein and Hohmann 1995;
Teusink et al. 1998). This role is less obvious in the
yeast Schizosaccharomyces pombe and the
filamentous fungus Aspergillus niger (Wolshek
and Kubicek 1997).
Feofilova (1992) states that trehalose has both
metabolie (overflow mechanism and storage com-
pounds during dormancy) and protective func-
tions (stabilisation of pro teins and membranes).
In this view the reason why or which compound
accumulates may originate from the need for
metabolie homeostasis in the cello
46 J. Dijksterhuis and R.A. Samson

Table 3.2. Heat-resistant moulds in fruit products; spoilage incidents from Scholte et al. (2001)

Species Isolated from Reference

Byssochlamys fulva Canned strawberries Olliver and Rendie (1934)

Byssochlamys nivea Canned strawberries Put and Kruiswijk (1964)
Eurotium herbariorum Grape jams/jellies Splittstoesser et al. (1989)
Neosartorya fischeri Canned strawberries Kavanagh et al. (1963)
Apple juice Scott and Bernard (1987)
Talaromyces flavus Apple juice van der Spuy et al. (1975)
Fruit juice King and Halbrook (1987)
Pineapple/grapefruit juice Scott and Bernard (1987)
Paecilomyces variotii Fruit concentrates, pectin Unpublished data
Talaromyces trachyspermus Dairy products Unpublished data
Fusarium oxysporum Dairy products Unpublished data
Neosartorya pseudofischeri Dairy products Unpublished data
Monascus ruber Fermented olives S. Panagou (per. comm.)

B. Heat Resistance

Heat-resistant fungi can survive the pasteurising

heat treatment of food products. Subsequent
germination causes spoilage of canned and pas-
teurised fruit products (for examples of outbreaks,
see Table 3.2). Fungi that cause damage worth
millions of dollars in the fruit-juice industry are
Byssochlamys nivea (fulva) , Talaromyces flavus
(macrosporus), Neosartorya fischeri, and Eupeni-
cillium brefeldianum (as reviewed by Tournas
1994). These are soil-borne fungi, and fruits that
develop in contact with soil (like strawberries) are
Fig.3.2. Thick-walled hyphae of a heat-resistant strain of
more prone to contamination. Paecilomyces variotii

1. Fungal Propagules and Heat Resistance

Fungal survival structures can be regarded as more
or less heat resistant compared to vegetative cells.
A variety of fungal survival structures including
conidia, sclerotia, chlamydospores and ascospores
can survive he at treatments between 55 and 95 oe.
The spoilage flora of bakery products includes
the species Penicillium roqueforti, P. expansum, P.
citrin um and Aspergillus flavus. Conidia of these
fungi have D-values (times needed to kill 90% of
cells) between 3.5 and 230min at 54-56°C (Bröker
et al. 1987; Spicher 1991). Yeast ascospores isola ted
from soft drinks and fruit products (mainly Saccha-
romyces cerevisiae, Z. bailii and chevalieri strains)
had D 60 values that were 25-350 times higher than
those of the corresponding vegetative cells (Put
and De long 1980).
Ascospores of heat-resistant fungi are extra-
ordinarily heat resistant and show D 90 values
of several minutes (Beuchat 1986). Most
studies have been carried out on the genera
Byssochlamys, Talaromyces and Neosartorya.
Byssochlamys nivea was described as early as the
1930s (Olliver and Rendie 1934). In 1963, a heat-
resistant Aspergillus (teleomorph, Neosartorya
fischeri) was isolated from canned strawberries
(Kavanagh et al. 1963). Two heat-resistant
Penicillium species were isolated from flash-
pasteurised apple juice (van der Spuy et al. 1975)
that later became known as Talaromyces (flavus
and macrosporus). In addition, other fungal
species have since been identified as heat-resistant
(e.g. Talaromyces trachyspermus; Enigl et al.
1993). Surprisingly, fungal structures other than
ascospores, namely thick-walled hyphal fragments,
chlamydospores (Paecilomyces variotii, Fusarium
spp., Samson, unpubl. data; see Fig. 3.2) and
 Food and Crop Spoilage on Storage
sclerotia (e.g. in Eupenicillium) , can exhibit
extreme thermoresistance (Splittstoesser and
King 1984).
2. Heat Resistance and the (Micro )Environment
Many studies have shown that heat-resistant
ascospores show decimal reduction of 1.5-11 min
at 90 oe. Heat resistance can vary with different
factors. These include the presence of sugar
(Splittstoesser and Splittstoesser 1977; Beuchat
1988a; King and Whitehand 1990), the influence of
pH and the presence of organic acids.
The pH of the medium influences heat resis-
tance and different species may show some speci-
ficity in this respect. B. fulva showed maximum
he at resistance within a pH range between 2.5
and 4.5 while N fischeri showed no changes in
resistance in a broader range of pH (3-5.5;
Splittstoesser and Splittstoesser 1977). Ascospores
of T. macrosporus exhibited a slight increase in
he at resistance between pH 3.6 and 6.6 (King and
Whitehand 1990).
Several organic acids eounteraet beat resis-
tanee of ascospores, but only at low values of pH
(lower than 4). This is most prominent for fumaric
acid for all three species (Splittstoesser and
Splittstoesser 1977; Conner and Beuchat 1987a;
Beuchat 1988b). Benzoic and sorbic acid also had
clear effects on T. flavus and N. fischeri (Beuchat
1988b; Rajashekhara et al. 1998). Tartaric and
malic acid, however, are reported to enhance he at
resistance of B. fulva. Conner and Beuchat
(1987a) state that pH, type, and molarity of the
undissociated form of the organic acid act syner-
gistically with heat to inactivate ascospores. The
different organic acids present in several fruit
juices may cause differences in heat inactivation.
N fischeri is more he at resistant in apple juice
compared with grape juice (Conner and Beuchat
1987a) and both B. fulva and N fischeri are much
more heat sensitive in cranberry juice compared
with grape, apple or tomato juice (Splittstoesser
and Splittstoesser 1977).
A combination of different factors may lead
to some of the puzzling variations in heat resis-
tance observed in the literature. N fischeri in
O.lM phosphate buffer (pH 7.0) exhibited a far
higher heat resistance than in grape jellies with
large amounts of cane sugar at pH 3.1-3.3
(Beuchat and Kuhn 1997). B. nivea and fulva and
N fischeri were approx. twice as heat resistant in
47
tomato juice (pH of 4.2) as in phosphate buffer
(pH 7.0, O.lM; Kotzekidou 1997).
3. Endogenous Factors and Heat Resistance
Not only factors outside the spore relate to the
resistance of the ascospore. Heat resistance of
the ascospores positively correlates to the age of
tbe eulture for N. fischeri, T. flavus and B. nivea
(Conner and Beuchat 1987b; Beuchat 1988a;
Casella et al. 1990). The eultivation temperature
of the spore-gene rating colony is also important
(Conner and Beuchat 1987b; King and Whitehand
1990).
Furthermore, King and Whitehand (1990)
report higher resistance of T. macrosporus when
the fungus was grown on solid medium. T. flavus
showed higher heat resistance when grown on
oatmeal agar compared to malt extract agar
(Beuchat 1988a). In several studies different iso-
lates of the fungi were studied and heat resistance
also varied between isolates in B. nivea, T.
macrosporus and flavus and N fischeri (Bayne
and Michener 1979; Beuchat 1986; King and
Whitehand 1990).
Conner et al. (1987) investigated the nature of
beat resistanee. They studied younger (11 days)
and older (25 days) ascospores of N fischeri with
different he at resistance (D S2 of approx. 23
and >60min respectively). Ascospores showed
changes in the inner cell wall region at the lateral
ridge during aging. They observed qualitative
differences in extractable proteins, but did not
observe changes in fatty acid or lipid content.
Older spores contained 2.8% (dry weight) of
mannitol and 0.6% of trehalose that could not be
measured inside 11-day-old spores. Polyols and
disaccharides may play an important role in he at
protection. Recently, very high concentrations of
trehalose (up to 15-20% of the cell weight) were
measured inside ascospores of T. macrosporus and
this compound was quickly degraded after activa-
tion of the cells (1. Dijksterhuis et al. , unpubl.
results).
4. Ascospore Dormancy and (Heat) Activation
Sussman and Halvorson (1966) defined two types
of dormancy. Exogenously dormant cells showed
delayed development due to unfavourable chemi-
calor physical conditions. Constitutive dormaney
includes an innate property of the dormant stage
48 J. Dijksterhuis and R.A. Samson
as a metabolie block, a barrier to the penetration
of nutrients or the produetion of a self-inhibitor.
Ascospores of the heat-resistant fungi described
here need a robust physical signal such as a short
heat treatment for the breaking of dormancy. In
most of the studies conducted on heat-resistant
fungi, heat activation is observed where the
number of viable counts after a short heat treat-
ment is increased by several log cycles (e.g.
Eurotium herbariorum at 60 oe, Splitstoesser et al.
1989; Fig. 3.1).
For ascospores of Talaromyces flavus activa-
tion is observed at 80 oe and, at 85 oe, activation is
followed by killing (Beuchat 1986). At lower tem-
peratures, activation fails and only low numbers of
germinated spores are observed. Remarkably, the
speed of activation seems to increase with higher
temperatures in the case of T. flavus. N fischeri
exhibits constants rates of heat activation between
70 and 85°e (King and Halbrook 1987; Fig. 3.1).
In the latter study, there is an indication that the
speed of activation increases between 80 and
90°e. Katan (1985) describes T. flavus isolates
that are activated after heating for 30min at 53 oe
in both soil sampies and in liquid, which might
indicate that different isolates also exhibit marked
variation in this respect.
For N fischeri the dormant state can be
broken by a drying treatment of 18h at 40 0 e
(Beuchat 1992), but T. flavus ascospores did not
showarelease of dormancy. After a subsequent
storage period, however, under dry conditions
(a w = 0.23) of 20months or more the spores did
not need he at aetivation anymore and germinated
after wetting. Splittstoesser et al. (1993) found
large variations in the number of ascospores of
N fischeri that were successfully aetivated after
he at treatment compared to the microscope count
of the cells.
The nature of activation of heat-resistant
ascospores is unknown. Heat activation might
serve an ecological function when soil fungi are
confronted with fire. The activated germinating
spores may quickly fill the empty ecological niches
formed after the fire. This phenomenon is weH
known with plant seeds (e.g. Eucalyptus).
However, it is also possible that heat activates
spores because it addresses the same mechanisms
as those occurring in nature. These extreme char-
acteristics mayaiso serve a very long shelf life of
the ascospores; they can be still viable for up to 17
years in the case of T. flavus (Nagtzaam and
Bollen 1994).
C. Alternative Methods for Sterilisation
of Food Products
Pasteurisation and sterilisation are important
methods to prevent spoilage in food. More
recently, the call for the development of a new
generation of non-thermal preservation tech-
niques has become even stronger (Barbosa-
Gmovas 1998; Smelt 1998). Heat treatments
influence the firmness and taste of the food
product and reduce the amount of vitamins in the
food. An alternative method such as high pressure
results in the death of microorganisms, but the
structure and vitamin content of the food stay
relatively unchanged.
Fungal survival structures are typically inacti-
vated at 250 and 350 MPa. For instance, sporan-
giospores of Phycomyces blakesleeanus die at
250MPa (Thevelein et al. 1979), the osmotolerant
yeast Zygosaccharomyces bailii cannot stand
5 min at 345 MPa (Palou et al. 1997), and 90% of
the ascospores of Saccharomyces cerevisiae are
killed after 11 min at 300 MPa (Zook et al. 1999).
High-pressure resistance of the cells in these
studies is relatively independent of pH, but
enhanced by the presence of sugars.
Heat-resistant fungi show extreme resistance
against high pressures (Butz et al. 1996, Palou et
al. 1998). These fungi cause spoilage outbreaks in
food, especially fruit products (see Tournas 1994),
and it is these products in particular that are
treated commercially with high pressure
(Barbosa-Glnovas 1998). Most studies in this
respect are performed with B. nivea. Pressures
over 600 MPa in combination with temperatures
above 60 oe are needed for the inactivation of
these cells (Butz et al. 1996). Oscillatory treat-
ments (Palou et al. 1998) that include more, short
cycles (1 s) of pressurisation inactivate ascospores
more rigorously.
A pressure-resistance of this kind is only
known from bacterial spores that are killed at or
above 800Mpa (Nakayama et al. 1996). Very
recently, we have noted that high-pressure treat-
ments (400-800 MPa) induce germination of he at -
resistant and dormant ascospores of Talaromyces
macrosporus (J. Dijksterhuis, unpubl. results). This
is very relevant for high-pressure "sterilisation"
and is already known for bacterial spores (at
100-250 MPa, elouston and Wills 1970; Gould and
Sale 1970; Raso et al. 1997). At moderate pres-
sures (approx. 250MPa), activation is foHowed by
killing of the germinating spore when its stress-
 Food and Crop Spoilage on Storage
resistance decreases (Wuytack et al. 1998, 2000).
At higher pressures, the germination sequence
of the spore is blocked. Interestingly, these
spores had become sensitive to heat, but remained
pressure-resistant. Indeed, a correlation between
heat resistance and pressure resistance of spores
among different Bacillus species was not observed
in other studies (N akayama et al. 1996; Raso et al.
1998).
V. Mycotoxins
There are many reviews on mycotoxins and the
reader is referred to Miller and Trendholm (1994),
Miraglia et al. (1998), Sinha and Bhatnagar (1998),
Watson (1998) and De Koe et al. (2001) for a more
detailed account. Frisvad and Thrane (2001)
provide an overview of the most common fila-
mentous fungi with their toxic metabolites. There
are a quite a few definitions of mycotoxins.
Gravesen et al. (1994) defined mycotoxins as sec-
ondary metabolites produced by filamentous fungi
that in sm all concentrations can evoke an acute or
chronic disease in vertebrate animals when intro-
duced via a natural route. Janzen (1977) considers
that secondary metabolites are of great ecolo-
gical significance, e.g. protection against other
microbes, insects and other animals. The number
of toxic species is large; in fact, it is a question
whether any naturally existing species can be
claimed to be unable to produce mycotoxins at all.
Evidence that at least not all strains of toxinogenic
species isolated from foods produce toxic metabo-
lites is given in different studies. Schroeder and
Boller (1971) examined a large number of
Aspergillus flavus strains isolated from peanuts,
cottonseed, rice and sorghum for the pro duc-
tion of aflatoxins and found that 98, 81, 20 and
24%, respectively, could produce aflatoxins. The
same can be said for the ochratoxin production by
Aspergillus niger strains. Varga et al. (2000)
observed that in only 6% of the strains represent-
ing the A. niger species complex could ochratoxin
production be detected.
VI. Conclusions
Increased amounts of food can be made available
by effective protection of crops (after harvesting)
49
and food products against spoilage fungi. The rela-
tionship between crops and fungi is plant parasitic
in nature and infection is dependent on accessi-
bility of the crop (e.g. by wounds) for the fungus.
The relationship between processed food and
fungi can be described in ecological terms (e.g.
Frisvad and Samson 1991; Filtenborg et al. 2001)
where specific food products relate to specific
fungal species. This specificity may be defined by
varying stress situations that fungi meet before
and/or during colonisation of food. In addition,
knowledge of the distribution of fungi around and
inside food during its preparation is of importance
to predict losses of food products due to spoilage.
Acknowledgements. The authors thank Mr.
Massimiliano Silvestri for providing Fig. 3.1.
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Mycotoxins and Detoxification
4 Genetics and Biosynthesis of Aßatoxins and Sterigmatocystin
JULIE K. HICKS, KIMINORI SHIMIZU, and NANCY P. KELLER
CONTENTS
I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . .
11. Afiatoxin and Sterigmatocystin
Biosynthesis . . . . . . . . . . . . . . . . . . . . . . . . . . .
A. Biochemistry..........................
B. Molecular Genetics . . . . . . . . . . . . . . . . . . . . .
1. Biosynthetic Genes . . . . . . . . . . . . . . . . . . .
2. Regulatory Genes . . . . . . . . . . . . . . . . . . . .
111. Regulation of Afiatoxin and Sterigmatocystin
Production . . . . . . . . . . . . . . . . . . . . . . . . . . . .
A. Endogenous Factors ....................
1. Signal Transduction . . . . . . . . . . . . . . . . . . .
2. RcoA-Mediated Regulation . . . . . . . . . . . .
B. Exogenous Factors .....................
1. Nutritional Components . . . . . . . . . . . . . . .
2. Host Factors ........................
IV Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . .
I. Introduction
Many microorganisms produce low molecular
weight compounds called secondary metabolites
whose benefit to the producing organism is often
obscure (Bu'Lock 1961). Fungi are among the
most prodigious producers of secondary metabo-
lites, many of which are either beneficial (e.g.
antibiotics) or detrimental (e.g., mycotoxins) to
humans. Two chemically related fungal secondary
metabolites that have been studied extensively
are the mycotoxins sterigmatocystin (ST) and
aftatoxin (AF), produced by several Aspergillus
species. AF was discovered in the 1960s when
thousands of fowl died of a mysterious disease
called "Turkey-X" disease (Lancaster et al. 1961).
Present address: IK. HICKS, Duke University Medical
Center, 322 CARL Building, Research Drive, Box 3546,
Durharn, NC 27710, USA
Present address: K. SHIMIZU, Research Center for Patho-
genic Fungi and Microbiol Toxicoses, Chiba University,
1-8-1 Jnohana, Chuo-ku, Chiba 260-8673, Japan
Present address: N.P. KELLER, Department of Plant Pathol-
ogy, University of Wisconsin-Madison, 1630 Linden Drive,
Madison, Wisconsin 53706, USA
Later, it was found that Turkey-X disease was not
55 actually caused by a pathogen but rather was a
poisoning stemming from contaminated peanut
57 meal in the feed that had been colonized by a
57
59 fungus identified as A. fiavus (Lancaster et al.
59 1961). The causal agent of Turkey-X disease was
59 found to be a polyketide mycotoxin produced by
A. fiavus, subsequently named "aflatoxin" for "A.
61
61 flgyus toxin". Since then, re-examination of early
61 records suggests that outbreaks of aflatoxicosis
62 (the name given to the disease caused by AF poi-
62 soning) may have been documented as early as the
62
64 1940s (CulIen and Newberne 1994).
65 Following the initial discovery of AF, further
65 studies revealed that aflatoxins exist in several
forms that differ in their abundancy in nature as
weIl as their toxicity. The four most abundant afla-
toxins are distinguished based on their relative
chromatographie mobility and characteristic blue
(AFB j ; AFB z) or green (AFG j ; AFGz) fluores-
cence (Sweeney and Dobson 1999). As the
primary aflatoxin species produced by A. fiavus
and A. parasiticus, AFB j is the best-studied of
these four, as it is the most toxic and the most
prevalent in field situations (Bhatnagar et al.
1994). Un1ess otherwise noted, "AF" refers to
"AFB j " in the remainder of this chapter.
Aspergillus nomius and Aspergillus tamari
also produce AF (Kurtzman et al. 1987; Goto et al.
1996) but these species are not as common as A.
fiavus and A. parasiticus. While isolates of A. par-
asiticus commonly produce both the Band G afla-
toxins (Pitt 1989), most North American A. fiavus
isolates produce only B aflatoxins. Recently, Cotty
and Cardwell (1999) have compared North
American and West African A. fiavus isolates and
found that none of the North American isolates
produced G aflatoxins, while 40 and 100% of the
West African isolates produced AFG 1 in ammo-
nium and urea media, respectively.
• AFB 1 and AFG1 contain dihydrobisfuran rings
that distinguish these compounds from AFB z
The Mycota XI
Agricultural Applications
Kempken (Ed.)
© Springer-Verlag Berlin Heidelberg 2002
56 IK. Hicks et al.
and AFGz, which contain tetrabisfuran rings
(Sweeney and Dobson 1999).
• The B aflatoxins have a cyclopentenone ring
that is replaced by a lactone ring in the G afla-
toxins (Dutton 1988).
• A third group of aflatoxins that was first dis-
covered in 1963 is the M aflatoxins, AFM1 and
AFM z (Allcroft and Carnaghan 1963). The "M"
designation indicates that these aflatoxins are
present in the milk from cows that have con-
sumed AFB1-contaminated feed. AFM1 and
AFMz are 4-hydroxy derivatives of AFB 1 and
AFB z, respectively (van Egmond 1994).
• A closely related molecule, sterigmatocystin
(ST), is produced by A. nidulans as well as other
fungal species including A. versicolor and some
species of Chaetomium and Bipolaris (Cole
and Cox 1981). As the penultimate precursor to
AFB 1, ST has similar toxie and carcinogenic
properties to AFB 1. The structure of ST includes
a xanthone moiety joined to a dihydrofuran or
tetrahydrofuran ring (Sweeney and Dobson
1999).
The LD so values of AF range as low as
0.34mg/kg for some mammals (Cullen and New-
berne 1994), making it the most potent of the
genotoxic agents produced by biological organ-
isms (Wang and Groopman 1999). Acute dos es of
AF are associated with a variety of severe con-
ditions including vomiting, abdominal pain,
pulmonary edema, fatty infiltration, immunosup-
pression and necrosis ofthe liver (Coulombe 1994;
Cullen and Newberne 1994). Among the geno-
toxic effects are the inhibition of DNA and RNA
synthesis, micronuclei production, sister chro-
matid exchange, and unscheduled DNA synthesis
and chromosomal strand breaks (Wang and
Groopman 1999). The most characteristic effect of
AF, as well as some of the other aflatoxins and ST,
is that of a mutagen (Groopman et al. 1980; Sab-
bioni, 1990; Raisuddin et al. 1993). Once in the
liver, the bisfuran moiety of the AF molecule is
oxidized by a cytochrome P-450 to an active, AF-
8,9-epoxide form (Koser et al. 1988). The epoxide
forms adducts with DNA by binding to guanine
residues in regions with alternating G-C
sequences (Yu et al. 1990). Ultimately, intercala-
tion of the activated AF in the DNA can result in
G:C to T:A transversions. It has been shown that
hepatocarcinomas caused by AF gene rally have a
unique "signature" transversion at the third base
(a guanine) of codon 249 in the p53 tumor-
suppressor gene resulting in an amino acid substi-
tution (Hsu et al. 1991; Aguilar et al. 1993). The
resulting defective p53 protein is associated with
tumorgenesis in the liver.
Due to the serious health threat presented by
AF, the United States government has imposed
stringent regulations on acceptable levels of AF in
agricultural crops. If AF levels exceed 20 parts per
billion (ppb), the crop must be decontaminated or
destroyed. Other countries have much lower
levels of tolerance, varying from a "no tolerance"
policy (Singapore) and 5ppb limit (regions of
Eastern Europe) to 10ppb (Japan) (Cardona et al.
2000). These lower acceptable levels of AF result
in the refusal of imported contaminated U.S. grain
and concomitant financial loss in export dollars
(Schumann 1998; Cardona et al. 2000). The most
extreme crop losses from AF contamination are
experienced when crops are subjected to high
temperatures and drought conditions, as these
conditions are optimal for Aspergillus invasion
and toxin production (Diener 1976). The southern
regions of the US, particularly Texas, and other
regions of the world, including the Afriean and
Asian continents, are frequently subject to envi-
ronmental conditions optimal for AF production.
In the past 30 years, Texas and the southeastern US
have experienced several major droughts that
have led to widespread AF contamination in corn
crops. In 1977, the southeastern region of the US
lost over 60% (approximately $200 million) of the
corn crop to AF contamination (Schumann 1998).
A 30% loss was sustained in Texas in 1996
(Houston Chronicle 1996; Bryan Eagle 1996), and,
in 1990 and 1998, over 50% of the Texas corn crop
was lost due to AF contamination. Indirect costs
include loss of livestock due to aflatoxicosis and
funds used to implement prevention and clean up
measures (Shane 1994). In Africa and Asia, AF
contamination of subsistence crops results in
deleterious health consequences due to lack of
adequate government monitoring and food-pro-
cessing technologies sophisticated enough to elim-
inate AF from food products. In some parts of
Africa, over one half of human hepatocarcinomas
show the signature G to T transversion at co don
249 of the p53 tumor-suppressor gene that is char-
acteristie of AF mutation (Hsu et al. 1991; Aguilar
et al. 1993). Other studies have revealed a corre-
lation between high levels of AF exposure and
liver cancer in Mexico, mainland China, and other
parts of southeast Asia (Yeh et al. 1989; Soini et
al. 1996; Sylla et al. 1999).
 Genetics and Biosynthesis of Aflatoxins and Sterigmatocystin
11. Aflatoxin and Sterigmatocystin
Biosynthesis
The discovery of AF as a serious disease agent for
livestock and poultry stimulated aseries of studies
investigating the AF biochemical pathway. Initial
efforts focused on identifying enzyme activities
and isolating A. flavus and A. parasiticus mutants
that were blocked at various stages of AF biosyn-
thesis. Feeding studies using these mutants were
used as primary means of establishing pathway
intermediate epistasis. This early work has been
reviewed extensively (see Dutton 1988). Conse-
quently, a model for the AF biosynthetic pathway
and accompanying prediction of enzymes likely to
be associated with the formation of AF was devel-
oped (Fig.4.1; Keller et al. 1992). In the course of
these studies, several groups determined that
sterigmatocystin, ST, was a late intermediate in the
AF pathway and the end metabolite of several
Aspergillus species (Hamasaki et al. 1973; Barnes
et al. 1994).
A. Biochemistry
The biochemistry of the AF pathway has been
thoroughly reviewed in the last few years
(Bhatnagar et al. 1992; Minto and Townsend 1997;
Payne and Brown 1998) and only the salient
points will be summarized here. A total of 16 enzy-
matic steps are believed to be involved in the for-
mation of AF from the initial polyketide precursor
(Fig.4.l). In the proposed pathway, acetate units
are synthesized into a fatty acid precursor mole-
cule by a fatty acid synthase (FAS, Brown et al.
1996a; Watanabe et al. 1996) and then converted
into an unstable intermediate, noranthrone, by
a polyketide synthase (PKS, Chang et al. 1995a;
Feng and Leonard 1995; Yu and Leonard 1995).
Noranthrone is immediately converted into the
anthraquinone, norsolorinic acid (NOR), through
a mechanism that has been hypothesized to
include a noranthrone oxidase (Vederas and
Nakashima 1980), a monooxygenase (Bhatnagar
et al. 1992) or possibly a spontaneous conversion
event (Dutton 1988). Because NOR is an orange-
colored intermediate that is easily visible to the
naked eye, mutants that accumulate NOR have
proven to be useful visual tools to determine
whether the AF/ST biosynthetic pathway is acti-
57
vated under various conditions (Chang et al. 1992;
Butchko et al. 1999).
The means by which NOR is converted into
averautin (AVN), the next step in AF biosynthe-
sis, is somewhat contro"ersial. Mutants of A. flavus
(Papa 1982) andA. parasiticus (Bennett 1981) that
accumulate NOR are not completely blocked for
AF production, but accumulate substantially
reduced levels of AF (Bhatnagar et al. 1992). This
suggests that the conversion of NOR to AVN
involves at least two enzymes, though the nature
of the specific enzymes has not been clearly estab-
lished (Bhatnagar et al. 1992). Chang et al. (1992)
have isolated nor-l, a gene encoding a dehydro-
genase that complements the NOR accumulation
defect in A. parasiticus. However, NorA, an un-
related enzyme with similarity to an aryl alcohol
dehydrogenase, has also been shown to have
the capacity to convert NOR into AVN (Cary et
al. 1996). The complexity of the conversion of
NOR to sub se quent intermediates in A. parasiti-
cus is reviewed in Bennett et al. (1997). InA. nidu-
lans, a single dehydrogenase enzyme, StcE, is
sufficient for the conversion of NOR to AVN
(Brown et al. 1996b; Butchko et al. 1999). The
ST gene cluster (see next section) also contains
stcV that appears to be a homologue of norA.
However, disruption of this gene has no effect on
ST production (Kelkar, Adams, and Keller,
unpubl. data).
The next step of the pathway utilizes the first
of several oxidases required for AF formation.
The conversion of averantin (AVN) to averufin
(AVF) requires at least two enzymatic steps
with one requiring incorporation of an oxygen
atom to form an intermediate described as
5'-hydroxyaverautin (HAVN, Yabe et al. 1991
1997). HAVN is then transformed into averufin
(AVF) by adehydrogenase activity (Chang et al.
2000b). Following AVF formation, sequential
oxidation, esterification, and cyclization steps
result in versiconal hemiacetal acetate (VHA; Yu
et al. 2000b), versiconal (VAL), and versicolorin B
(VERB), respectively.
The production ofVERB designates a branch
point in the pathway of the AF- and ST-producing
fungi. Another oxidation step converts VERB into
versicolorin A (VERA; Branch A; Fig. 4.1). VERB
and VERA are then metabolized by the same
enzymes to produce the branch Band branch A
end products: demethylsterigmatocystin (DMST)
and sterigmatocystin (ST) in A. nidulans (Kelkar
et al. 1996, 1997),AFB2 and AFB 1 in most A. flavus
 Mt
~~
OH
Ac.tyI CoA
MalonylCoA

~ stcA H
o No... olorinic Acid (NOR)

ll~/
Polykatida Precu ...or

P
steG?
HO

steB orstew~
OH

;c:y=, I/stcn
H

'0
~ steBorsteW

V....lcon.1 hernlacelal Acelala (VHA)

Hydroxyve""colorone (HVN)

HO H

lQ6 /O HO
O~OH ~steN?
V....icon.1 (VAL)
~MC
0 H

#
. I 4CO
'~"""l
<

ll&<
HO BranchA
o V....lcolorin A (VERA)

steS,steU >Branch B II
/ H
" OH

.(-0-).,.
. -0 ... O~ C). . .o O~
Dlhyd~methylsterigmatocystin (DHDMST)
Damethylslarigmatocystln (DMST)

< steP > II 9~OH

~0~H3
H

H3 Dlhydrosterigmatocystln lOHST)
Sterigmatocystln (ST)

An.toxln ~ (AF~) Aftetoxln Bz (AFBz)

Fig. 4.1. Aflatoxin/sterigmatocystin biosynthetic pathway.
Malonyl CoA and acetyl CoA are converted into a polyke-
tide precursor by the combined activities of a fatty acid syn-
thase and polyketide synthase. The polyketide precursor
undergoes aseries of conversions to result in sterig-
matocystin (A. nidulans), AFBj/AFB 2 (A. flavus) or
AFBj/AFBz/AFGj/AFGz (A. parasiticus). The A. nidulans
stc (gerigmatocystin ~luster) genes responsible far produc-
ing various enzymes in the pathway are indicated. A ques-
tion mark following the gene indicates that function has not
been demonstrated and activity is only postulated
 Genetics and Biosynthesis of Afiatoxins and Sterigmatocystin
strains, and AFB 2/AFG 2 and AFBj/AFG j in
A. parasiticus strains (Yu et al. 1998; Yabe et al.
1999).
B. Molecular Genetics
1. Biosynthetic Genes
In order to begin to develop strategies to control
the production of AF/ST by the producing
Aspergilli, an understanding of the genes and reg-
ulatory mechanisms underlying the AF/ST biosyn-
thetic pathway was deemed necessary. In 1992, the
first AF biosynthetic gene, nor-l, was isolated from
A. parasiticus (Chang et al. 1992). The product of
this gene functions in converting norsolorinic acid
(NOR) into averantin (AVN). The nor-l gene was
cloned by complementing a mutant that accumu-
lated NOR with anA. parasiticus wild-type cosmid
library. The complemented transformant no
longer accumulated NOR but instead produced
AF. Since this initial success, a large number of
genes encoding enzymes for 16 steps in the AF/ST
biosynthetic pathway have been isolated from A.
flavus, A. parasiticus and A. nidulans (e.g. Keller
et al. 1994b, 1995,2000; Trail et al. 1994; Kelkar et
al. 1996, 1997; Prieto et al. 1996; Silva et al. 1996;
Prieto and Woloshuk 1997; Yu et al. 1997,
1998; Payne and Brown 1998; Motomura et al.
1999; Chang et al. 2000b; Yu et al. 2000b). Figure
4.1 shows the placement of A. nidulans genes to
their respective steps in the pathway.
The AF/ST regulatory gene, aflR, and the
biosynthetic genes are clustered in regions of
DNA spanning approximately 60-75 kb (Trail
et al. 1995; Yu et al. 1995; Brown et al. 1996b).
Analysis of the three clusters reveals that, while
the AF/ST biosynthetic clusters of A. flavus, A.
parasiticus and A. nidulans contain essentially the
same genes (Bhatnagar et al. 2001), there are
re arrangements of gene order and direction of
the ST genes of A. nidulans in comparison to the
AF genes of A. flavus and A. parasiticus (Trail et
al. 1995; Keller and Hohn 1997; Cary et al. 2000).
Subsequently, genes involved in the biosynthesis
of other fungal secondary metabolites have been
found to be clustered (Hohn et al. 1995; Young et
al. 1998).
Although many of the enzymatic and regula-
tory functions of the AF/ST biosynthetic pathway
have been assigned to the products of specific
genes (Payne and Brown 1998), the role of some
59
of the cluster genes is still elusive. One of these
genes, aflI, is particularly intriguing. The gene
encoding AflJ was first discovered in the AF
cluster of A. flavus and has now been identified in
the clusters of A. nidulans (Genbank Accession #:
U34740) and A. parasiticus (Ehrlich et al. 1999a).
Of the three species, the A. flavus afli has been the
most extensively characterized (Meyers et al.
1998). Meyers et al. (1998) have shown that aflJ is
approximately 1.8 kb, contains two introns of less
than 100 bp, and encodes a predicted protein of
483 amino acids. The protein sequence does not
match any pro tein in Genbank. Recent results
suggest that AflJ is important in the transcription
of genes required for norsolorinic acid formation
but not for genes involved in later steps (Payne
2000).
In both the ST and AF clusters, there are a
number of genes that appear to have no obvious
role in the biosynthesis of these metabolites. For
example, disruption of the A. nidulans stcQ and
stc V had no affect on ST production under the
conditions examined (Kelkar, Adams, and Keller,
unpubl. data). It is possible that genes such as stcQ
and stcV encode proteins with redundant func-
tions. It is also possible that, even though these
genes are in the AF/ST clusters, they do not
encode for enzymes that are necessary for AF/ST
production. Finally, it is possible that these genes
do have a role in AF/ST production but that
the phenotype caused by the disruption of these
genes was not observed under the conditions
tested.
2. Regulatory Genes
In addition to the biosynthetic genes, all of the
AF/ST-producing Aspergillus species that have
been identified to date contain the regulatory
gene, aflR (aflatoxin regulation). AflR is necessary
for the positive co-regulation of AF/ST biosyn-
thetic gene expression (Yu et al. 1996a; Payne and
Brown 1998). Evidence for this conclusion
includes the observation that deletion or mutation
of aflR in A. flavus, A. nidulans and A. parasiticus
results in atoxigenic strains that do not express
AF/ST biosynthetic genes. These mutants can be
complemented by a wild-type aflR gene (Chang et
al. 1993; Payne et al. 1993; Yu et al. 1996a). In all
three species, additional copies of aflR or overex-
pression of aflR results in increased and/or altered
transcription of the AF/ST biosynthetic genes and
AF/ST production that par allels the transcript
60 IK. Hicks et al.
levels of aflR (Chang et al. 1995a; Flaherty and
Payne 1997; Yu J-H 1996a).
The aflR genes encode proteins belonging to
the fungus-specific Zn(II)2Cys6 (zinc-binuclear
cluster) transcription factor family (Chang et al.
1993; Woloshuk et al. 1994; Yu et al. 1996a; Todd
and Adrianopoulos 1997). Although the AflR pro-
teins from the three species are functionally
homologous (Chang et al. 1993; Yu et al. 1996a),
similarity between the aflR DNA sequences varies
greatly. The A. flavus aflR shares 95% sequence
identity with the A. parasiticus aflR (Payne and
Brown 1998) but only 33% identity with the A.
nidulans aflR. The highest region of identity
(71 %) between the A. flavus and A. nidulans aflR
genes occurs in the bases encoding the zinc-
binuclear cluster-binding domain (Yu et al.
1996a). Most Zn(II)2Cys6 proteins recognize and
bind to a palindromic sequence in the promoters
of target genes. The primary AflR binding
sequence was first identified in A. nidulans as [5'-
TCGN(5)CGA-3'] (Fernandes et al. 1998). This
motif is found one or more times in most of the
promoters in the ST cluster and is conserved in the
promoters of AF genes (Payne and Brown 1998;
Erhlich et al. 1999b). A second AflR consensus
binding sequence, [5'-TTAGGCCTAA-3'], has
also been reported for A. flavus and A. parasiticus
(Chang et al. 1995b; Payne and Brown 1998),
although the [5'-TCGN(5)CGA-3'] sequence has
been shown to be the major binding site in A. par-
asiticus (Ehrlich et al. 1999b).
The combined efforts of several researchers
have revealed specific regions of AflR that appear
to be important for correct functioning. Chang et
al. (1999) showed that the carboxy terminus of
AflR appears to be particularly important for acti-
vation and regulation of the pro tein. Ehrlich et al.
(1999b) found a similar result when site-directed
mutagenesis of specific acidic residues in a 23 a.a.
acidic region at the carboxy terminus caused a 50-
fold decrease in AflR activation. Other important
domains in AflR are a putative dimerization
domain (Ehrlich et al. 1998; Ehrlich et al. 1999a),
a classic zinc-binuclear cluster-binding domain
(Chang et al. 1993; Woloshuk et al. 1994; Yu et al.
1996a) and a nuclear localization signal. Mutation
of the A. parasiticus AflR nuclear localization
signal results in an 80% reduction in AF accumu-
lation compared to that of a wild-type strain
(Ehrlich et al. 1998) and elimination of ST pro-
duction in A. nidulans (Hicks, Shimizu, and Keller,
unpublished data).
The aflR promoter of A. paras!tlcus con-
tains both positive and negative regulatory
domains (Ehrlich et al. 1999a). An AflR binding
site (TTAGGCCTAA) has been found in the aflR
promoter of A. parasiticus (Chang et al. 1995b).
When this site was mutated, a 40% decrease in AF
production was observed (Ehrlich et al. 1998).
These data lend support to the hypo thesis that
AflR is involved in autoregulation (Ehrlich et al.
1998; Chang et al. 1999). The PacC binding site
has also been found in the aflR promoter (Ehrlich
et al. 1999a) as well as in the promoter of several
AF/ST biosynthetic genes (Genbank Accession #:
U34740). PacC is a zine-finger transeription factor
that is known to stimulate expression of alkaline-
expressed genes and repress transcription of acid-
expressed genes (Denison 2000). The presence of
this site in the aflR promoter is particularly inter-
esting in light of the role that pH has been shown
to have in regulation of AF/ST production (see
below).
In 1997, Prieto and Woloshuk showed that an
A. flavus strain lacking all of the AF cluster except
aflR and the AF biosynthetic gene, ord-l, was able
to convert the O-methylsterigmatoeystin (OMST)
substrate into AF. This showed that aflR is the only
regulatory gene within the AF cluster that is nec-
essary for expression of the biosynthetic genes or
at least ord-l. However, severallines of evidence
suggest that AflR is not sufficient for activating
AF/ST biosynthetic gene expression. In A. flavus,
constitutively expressed aflR did not result in con-
stitutive AF accumulation; instead, AF accumula-
tion patterns and levels fluctuated over time,
closely resembling those of a wild-type aflR strain
(Flaherty and Payne 1997). This result suggests
that presence of the aflR transcript is not sufficient
to cause biosynthetic gene expression during
primary growth and that other factors could be
acting as AF inhibitors at the early stages of fungal
growth. At least two lines of evidence exist to
suggest that AflR is not sufficient for stc gene acti-
vation in A. nidulans. First, Butchko et al. (1999)
identified 16 mutations that were not linked to the
ST cluster and resulted in a phenotype of no to
minimal ST production even though aflR was
expressed. Secondly, Hicks, Shimizu, and Keller
(unpub!. data) have found that strains with muta-
tions in the flbA or pkaA genes (encoding signal
transduction components, see below) do not
produce stc transcripts or ST even when aflR is
overexpressed. These data suggest a post-
transeriptional or post-translational regulation of
 Genetics and Biosynthesis of Aftatoxins and Sterigmatocystin
AflR by other protein(s). Erhlich et al. (1998) and
Chang et al. (1999) have suggested that AflR con-
tains domains for putative repressor and activator
functions. It is possible that the factors responsi-
ble for the observations in the studies noted above
serve these functions.
111. Regulation of Aflatoxin
and Sterigmatocystin Production
Raving achieved a good understanding of the
nature of the AF biosynthetic pathway and the
physical structure of the AF/ST biosynthetic gene
clusters, a major research effort is now focused
on und erst an ding the endogenous and exogenous
factors that regulate AF/ST production. This
section details the current understanding of
factors that are involved in AF/ST production.
A. Endogenous Factors
Progress in elucidating Aspergillus non-cluster
genes involved in AF/ST regulation has been
born out of the many observations linking loss of
AF/ST production to morphological abnormali-
ties, especially to decreases in asexual spore pro-
duction (KaIe et al. 1996; Bennett et al. 1997).
Below we describe progress in identifying a signal
transduction pathway and a WD pro tein both of
which genetically link AF/ST production to mor-
phological development in Aspergillus species.
1. Signal Transduction
One way in which AF/ST production is regulated
in Aspergillus spp. is through a signal transduction
I post-transcriptional regulation
I I
I
, I
I I
colony \, __ { transcription factor?
growth '----------'
transcriptional regulation
61
pathway that also regulates asexual reproduction
(Yu et al. 1996b; Ricks et al. 1997). Proteins that
have been identified as belonging to this signal
transduction pathway include FlbA, a RGS
(Regulator of G-protein Signaling; Dohlman and
Thorner 1997) pro tein and FadA, the a subunit of
a heterotrimeric G-protein (Yu et al. 1996b).
A third component of this signaling pathway
is PkaA, encoding the catalytic subunit of protein
kinase A (Shimizu and Keller 2001). Protein
kinase A (PKA) regulates expression of target
pro teins through phosphorylation of serine or
threonine residues in the consensus sequence, X-
R/K-R/K-X-SIT-X (Kemp and Pe ars on 1990). The
catalytic subunits of PKA are activated when
cAMP is synthesized by adenylyl cyclase and
induces the dissociation of the catalytic and
regulatory subunits. Adenylyl cyclase activity is
regulated by the a subunits of heterotrimeric
G-proteins, which either stimulate (Ga.) or inhibit
(GlXj) adenylyl cyclase (Kronstad et al. 1998).
Shimizu and Keller (2001) have identified a gene
(pkaA) in A. nidulans that encodes a PKA cat-
alytic subunit. PkaA functions in the FadA-
mediated signaling pathway (Fig. 4.2). Deletion of
pkaA results in hyperconidiation and altered
timing of stc gene expression and ST production
while overexpression of pkaA results in hypoconi-
diation and loss of aflR and stc gene expression
and subsequent ST production. The role of PkaA
in regulating ST production and conidiation is
likely more complex than merely acting through
the FadA-mediated signaling pathway. Shimizu
and Keller (2001) have shown that, when aflR and
pkaA are both overexpressed simultaneously, no
stc gene expression is observed despite the pres-
ence of abundant afiR transcript. This suggests a
post-transcriptional or post-translational regula-
Fig. 4.2. Proposed signal transduction pathway
.,.. affecting both fungal development and sterig-
I
matocystin production. PkaA regulates AftR activ-
ity both transcriptionally and post-transcriptionally.
) ____ ,/ Dashed arrows represent predicted steps. Shimizu
and Keller 2001; copyright 2001, with kind permis-
sion from the Genetics Society of America)
62 J.K. Hicks et al.
tion of AflR by PkaA in addition to the transcrip-
tional regulation of aflR by PkaA. Three PkaA
consensus phosphorylation sequences exist in the
carboxy terminus of the A. nidulans AflR, and two
in the A. parasiticus and A. flavus AflR. Current
research is focused on determining which, if any,
of these sites are necessary for regulation of AflR
activity by PkaA (Hicks, Shimizu, and Keller,
unpubl. data).
Although the initial characterization of the
FadA-mediated signaling pathway that regulates
ST production and conidiation was established in
A. nidulans, evidence shows that a similar signal-
ing pathway exists in both A. parasiticus and A.
flavus. Hicks et al. (1997) transformed the A. nidu-
lans dominant activating fadA allele (fadA d+) into
a NOR-accumulating strain of A. parasiticus and
showed that the resultant transformant did not
accumulate NOR and also had the same colony
phenotype as an A. nidulans fadA d+ mutant. Sim
and Keller (unpublished data) have performed a
similar experiment with A. flavus and achieved
comparable results. Recent work has also shown
that in Fusarium sporotrichioides the fadA d+ allele
increases the production of the trichothecene
mycotoxin. This suggests that regulation of sec-
ondary metabolism through a FadA-mediated sig-
naling pathway may be operational in a variety of
fungal species (Tag et al. 2000).
2. RcoA-Mediated Regulation
In addition to signal transduction effects on
Aspergillus, rcoA, a gene encoding a likely tran-
scriptional regulator, has recently been shown to
affect both conidiation and ST production. RcoA
is a me mb er of the WD [Tryptophan (W)/Aspar-
tate (D)] repeat family of proteins which are
implicated in global regulation of a variety of
functions in several organisms (Hicks et al. 2001;
Neer et al. 1994). Other members of this family
indude the Saccharomyces cereviseae Tup1p and
Neurospora crassa RC01 proteins (Keleher et al.
1992; Yamashiro et al. 1996; Mukai et al. 1999).
Deletion of both rco-l in N crassa and rcoA
(McoA) in A. nidulans results in significant re duc-
tion in asexual reproduction with concomitant
reduction in expression of brlA, a major
Aspergillus conidiation gene (Hicks et al. 2001;
Adams et al. 1988). Additionally, McoA strains
have gross vegetative growth defects and are
unable to produce ST or express the associated
aflR and stc genes (Hicks, Lockington, Strauss,
Dieringer, Kubicek, KeHy and Keller, unpubl.
results). While the role of RcoA in ST production
has not been fully elucidated, it is possible that
RcoA either positively regulates aflR or has a neg-
ative regulatory function in pathways that affect
aflR. Alternatively, it is possible that loss of RcoA
results in such gross defects in biomass accumula-
tion that the other processes of conidiation and ST
production are indirectly affected. It is interesting
to note that, when aflR is overexpressed in a
McoA background, the stc gene transcript, though
present, is not accumulated to the levels seen
when aflR is overexpressed in a wild-type
background. This suggests RcoA has an AflR-
independent effect on stc gene expression.
B. Exogenous Factors
One of the most challenging aspects in studying
regulation of AF/ST production is in elucidating
the complex nature of the interactions between
various environmental stimuli such as nitrogen
and carbon sources and ambient pH and host
influences. The following sections will detail
progress in identifying the possible molecular
factors that playa role in this inter action.
1. Nutritional Components
Studies have shown that simple carbon sources
such as glucose, sucrose and maltose are optimal
ror AF/ST production, as opposed to the more
complex carbohydrates such as peptone, lactose,
sorbose and others (e.g. Davis and Diener 1968;
Abdollahi and Buchanan 1981; Buchanan and
Stahl 1984; Luchese and Harrigan 1993). The
reason that simple carbon sources are more
optimal than complex sources for AF/ST produc-
tion is controversial. One hypo thesis is that simple
carbon sources are easily metabolized into the
acetate molecules that can be used as the raw
material from which the AF/ST-specific fatty acid
synthase enzymes generate substrate molecules
for the polyketide synthase (Hsieh and Mateles
1970). A second hypothesis suggests a role for gly-
colysis in AF/ST biosynthesis. Buchanan and
Lewis (1984) showed that activity of at least three
glycolytic enzymes was lowered in the presence of
glucose as opposed to peptone. They hypothesized
that this may be caused by carbon catabolite
repression that results in decreased activity of the
glycolytic cyde with consequent shunting of acetyl
 Genetics and Biosynthesis of Afiatoxins and Sterigmatocystin
coenzyme A to alternative pathways such as that
for AF production. The discovery of a cluster of
sugar metabolism genes located adjacent to the
AF biosynthetic cluster in A. parasiticus mayaiso
suggest a possible connection between carbon
metabolism and AF production (Yu et al. 2000a).
This cluster contains four genes, one of which,
hxtA, encoding a hexose transporter protein, is co-
regulated with the AF biosynthetic genes.
Nitrogen source has also been implicated in
the environmental regulation of AF/ST pro duc-
tion. The most defined results involve studies
where Aspergillus is grown in media containing
either nitrate or ammonium as the sole nitrogen
source. Several studies have shown that nitrate
inhibits AF production while ammonium pro-
motes biosynthesis. Kachholz and Demain (1983)
demonstrated that a nitrate medium reduced AF
production in A. parasiticus by 75%. When they
grew A. parasiticus in an ammonium-containing
medium and then transferred the cultures to a
nitrate medium, the cultures produced as much
AF as the cultures that had been transferred to
either an ammonium-containing medium or a
medium containing no nitrogen. This suggests that
nitrate does not inhibit the activity of AF biosyn-
thetic enzymes that have already been formed.
When Flaherty and Payne (1997) overexpressed
aflR in a medium containing nitrate and the same
medium containing no nitrate,AF gene transcripts
were present under both conditions but AF pro-
duction was reduced by 90% in the nitrate-
containing medium. The data from these two
studies suggest that nitrate inhibition may occur
post-transcriptionally. However, Chang et al.
(1995b) observed that when A. parasitcus was
grown in a nitrate medium, the aflR transcript was
only detected in strains with extra copies of aflR,
suggesting a transcriptional regulation of aflR by
nitrate. Interestingly, in the A. parasiticus aflR-afll
intergenic region, there are several putative
binding sites for AreA, a protein that regulates
genes involved in nitrogen metabolism (Wilson
and Arst 1998; Chang et al. 2000a).
Niehaus and Jiang (1989) observed that
nitrate causes increased activity of the mannitol
cycle resulting in enhanced levels of NADPH.
Based on these results they proposed an indirect
regulatory role for nitrate in AF production
through redox controls that limit the availability
of acetyl CoA, aprecursor necessary for AF pro-
duction. This hypothesis is further supported by
evidence showing that high NADPH/NADP
63
ratios favor incorporation of acetyl CoA into
lipids while low NADPH/NADP ratios favor
polyketide production (Kiser and Niehaus 1981;
Niehaus and Dilts 1982).
Nitrate mayaiso play an indirect role in AF
production through its affect on ambient pH.
Accumulating evidence supports a significant role
of pH in AF/ST production. The extent to which
this role overlaps with the carbon and nitrogen
effects has not yet been determined. Until 1988,
convention held that the effect of nitrogen on AF
production in A. flavus was directly related to
nitrogen source. However, Cotty (1988) buffered
nitrate (AF-repressive) medium at an acidic pH
and ammonium (AF-conducive) medium at a
neutral pH and demonstrated that AF was pref-
erentially produced in the acidified nitrate
medium, suggesting that the source of the nitro-
gen was only indirectly important in that it affects
the pH of the culture grown over time. In an
attempt to clarify the significance of pH in AF/ST
biosynthesis, Keller et al. (1997) examined the role
of pH on the expression of the functionally homol-
ogous AF and ST biosynthetic genes ver-l and
stc U in A. parasiticus and A. nidulans, respectively.
They found that transcription of these genes
occurred at higher levels in media buffered to
acidic pHs of 4-6 than in media buffered to pH 7
and 8. Consequently, AF/ST levels were also
high er in lower pHs. This trend was observed in
both A. parasiticus and A. nidulans regardless of
the nitrogen or carbon source used in the buffered
medium, supporting the hypothesis that pH is the
overriding determinant when compared to nitro-
gen or carbon source. Additionally, Keller et al.
(1997) reported that accumulation of norsolorinic
acid was less in nitrate media at pR 5 than in
ammonium medium at pH 5, suggesting a pH-
independent repression by nitrate on AF/ST pro-
duction as well as a strong role for pH regulation.
In contrast to the work of Keller et al. (1997)
and Cotty (1988), several other studies have
shown that, under certain conditions, pH may have
very little influence on AF production (Kachholz
and Demain 1983). It is possible that these con-
trary data are the result of using different strains,
media and/or growth conditions. Whatever the
reason, Flaherty and Payne (1997) demonstrated
that A. flavus grown in media differing only in the
absence or presence of nitrate produced approxi-
mately 90% less AF in the nitrate-containing
medium even though the final pH values of the
two media were comparable. Similarly, Kachholz
64 IK. Hicks et al.
and Demain (1983) buffered media differing
only in the presence or absence of nitrate to pH
4.5 and still observed reduced AF production in
the nitrate-containing cultures. These studies
demonstrate the complexities and uncertainties
involved in elucidating the relationships be-
tween carbon source, nitrogen source and pH as
weIl as the obvious impartance that these factars
have in influencing AF/ST production, at least
in vitro.
2. Host Factors
A relatively new field of study in AF research is
the examination at the molecular level of the role
that plant host factors have in regulating AF/ST
production. Investigations of host faetors involved
in Aspergillus infection, proliferation and AF
production in planta may help to identify new
targets for potential control measures. For
example, Fakhoury and Woloshuk (1999) have
recently demonstrated that A. flavus a-amylase
activity is necessary for normal infection and AF
production in com and that a trypsin inhibitor
from com inhibits A. flavus a-amylase activity.
They propose that the trypsin inhibitor may func-
tion in limiting A. flavus proliferation in the kemel
endosperm tissue by inhibiting the ability of the
fungus to degrade starch.
The role of fatty acids in pathogenesis is
another area of interest. It is weIl known that
Aspergillus species preferentially invade lipid-
rich seeds and, within those seeds, the lipid-rich
portions such as the embryo (Brown et al. 1993;
Keller et al. 1994a). Harbored within the seeds are
lipid-metabolizing enzymes called lipoxygenases
(LOXs), that are responsible for metabolizing
linoleic and linolenic polyunsaturated fatty acids
into either 9S- or 13S-hydroperoxylinoleic acid
(9S- or 13S-HPODE) or 9S- or 13S-hydroperox-
ylinolenie acid (9S- or 13S-HPOTE) (Gardner
1991). In the plant, 13S-HPODE and 13S-HPOTE
are converted into a wide array of compounds
with functions that range from elicitation of plant
defenses to fungal toxicity (Hildebrand 1989;
Farmer and Ryan 1992).
The first studies demonstrating a putative role
of plant-derived linoleic and linolenic acid deriv-
atives in AF production occurred as early as 20
years ago (Fabbri et al. 1983). Evidence for either
the inhibition (Doehlert et al. 1993; Goodrich-
Tanrikulu et al. 1995; Zeringue et al. 1996) or stim-
ulation (Passi et al. 1984; De Luca et al. 1995) of
AF production by LOX pathway intermediates
has been accumulating during the ensuing period.
In 1997, Burow et al. examined the differential
roles of 9S- and 13S-HPODE and 13S-HPOTE in
regulating AF/ST production in A. parasiticus and
A. nidulans. They found that both the 13S-
hydroperoxy fatty acids repressed AF/ST biosyn-
thetic gene expression and AF/ST accumulation.
In contrast, 9S-HPODE extended the period
during which the AF/ST biosynthetic genes were
expressed but did not have any apparent effect on
levels of AF/ST accumulation under the condi-
tions tested. This suggests a role for 9S-HPODE
producing LOX as AF stimulatory factors.
Burow et al. (2000) have demonstrated that
expression of a peanut LOX gene, pnloxl, was
induced during infection of the seed by A. para-
siticus. Concomitant with increased pnloxl tran-
script, the concentration of 9S-HPODE in the
seed increased during infection in contrast to
uninfected control seed, which still retained a high
level of 13S-HPODE. PnLOX1 is postulated to
contribute to the increase in 9S-HPODE during
Aspergillus infections; however, as it is a mixed
function LOX (i.e., produces both 13- and 9S-
HPODE), it is thought that other peanut seed
LOXs are active during the Aspergillus/peanut
seed interaction. In com, re cent studies have sug-
gested a role for the com LOX gene, CSSAP92,
in the Aspergillus-com inter action (Wilson et al.
2001). Expression of CSSAP92, which produces
96% 9S-HPODE, was highest in the susceptible
com lines in comparison to the resistant lines or
mock-inoculated control plants. This study sug-
gests that CSSAP92 may be a biomarker in com
indicating resistance or susceptibility of a com line
to AF contamination.
Linoleic acid derivatives, called psi faetors,
are also produced by Aspergillus species (Champe
et al. 1987; Wilson et al. 2001). Psi factors are
involved in fungal asexual and sexual develop-
ment. Calvo et al. (1999) have shown that addition
of exogenous linoleic acid, or the plant LOX
products, 9S- and 13S-HPODE, to cultures of A.
nidulans significantly increased asexual (conidial)
development and, when added at low concentra-
tions, sexual (cleistothecial) development. Addi-
tion of 13S-HPODE, but not 9S-HPODE, to an
A. flavus isolate resulted in a significant reduction
in sclerotial production. Additionally, conidial
production was enhanced in both A. parasiticus
and A. flavus when linoleic acid was added
exogenously.
 Genetics and Biosynthesis of Aflatoxins and Sterigmatocystin
In addition to linoleic and linolenic acid deriv-
atives, other host plant factors have been impli-
cated in affecting AF production and development
in Aspergillus spp. Green-McDowelle et al. (1999)
reported that exposure of A. flavus and A. para-
siticus to the cotton-Ieaf volatile, 3-methyl-l-
butanol (3-MB), resulted in both decreased AF
production and conidiation. Similarly, Zeringue
(1991) reported that exposure to specific C6 and
C9 cotton leaf derived volatiles resulted in
decreased AF levels in corn, cottonseed and
peanut infected with A. flavus. Finally, Wright et
al. (2000) demonstrated that exposure of A. para-
siticus to the corn-derived volatile n-decyl alde-
hyde resulted in reduced colony growth, reduced
conidiation and reduced AF production.
The identification of host compounds and host
gene products affecting either Aspergillus repro-
duction processes or AF production suggests that
control measures can be obtained through con-
ventional breeding and genetic engineering.
IV. Conclusions
Since the initial discovery of aflatoxin almost 40
years ago, marked progress has been made in
every aspect of AF research including the eluci-
dation of the intermediates in the biosynthetic
pathway and the characterization of a AF/ST gene
cluster.
Recent efforts have been focused on under-
standing the regulatory networks involved in
AF/ST biosynthesis, the majority, if not all, of
which ultimately work through regulating the
activity of the regulatory gene, aflR, at both the
transcriptional and post-transcriptional level. At
least one of these networks involves a G-protein
signaling pathway that also regulates asexual
development. As each component in this signaling
pathway is identified, it is becoming clear that
several of the components, such as FlbA and
PkaA, influence, and are influenced by, other reg-
ulatory networks. It is possible that some of these
networks may involve regulation of co-factors that
repress or stimulate AflR activity.
In addition to regulation by the G-protein
signaling pathway, exogenous factors impact
AF/ST production. The role of pR, nitrogen
source, carbon source and host factors in regulat-
ing AF/ST production is complex. The individual
factors, through mechanisms not yet clearly under-
65
stood, not only influence AF/ST production indi-
vidually, but also interact with the other factors in
relationships that are as yet largely undefined. It
is apparent that, even though substantial inroads
have been made with regard to our understanding
of the intricacies of AF/ST production, there is still
much work to do before the global problem of AF
contamination can easily be controlled.
Acknowledgements. We wish to express our grat-
itude to the members in our laboratory for their
help throughout this work. We thank Jeff Batten
and Deepak Bhatnagar for their critical review of
the manuscript.
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Yu J-H, Wieser J,Adams TH (1996b) The Aspergillus FlbA
RGS domain protein antagonizes G-protein signaling
to block proliferation and allow development. EMBO
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Yu J, Chang PK, Cary JW, Bhatnagar D, Cleveland
TE (1997) avnA, a gene encoding a cytochrome
P-450 monooxygenase, is involved in the conversion of
averantin to averufin in afiatoxin biosynthesis in
Aspergillus parasitieus. Appl Environ Microbiol
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Yu J, Chang PK, Ehrlich KC, Cary JW, Montalbano B, Dyer
JM, Bhatnagar D, Cleveland TE (1998) Characteriza-
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asitieus cytochrome P-450 monooxygenase encoded
by ordA that is involved in the biosynthesis of afia-
toxins BI, GI, B 2, and G 2• Appl Environ Microbiol
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Yu J, Chang PK, Bhatnagar D, Cleveland TE (2000a)
Cloning of a sugar utilization gene cluster in Asper-
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Yu J, Woloshuk Cp, Bhatnagar D, Cleveland TE (2000b)
Cloning and characterization of avfA and omtB genes
involved in afiatoxin biosynthesis in three Aspergillus
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Zeringue H, Brown R, Neucere J, Cleveland T (1996) Rela-
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Appl Environ Microbiol 57:2433-2434
5 Molecular Genetics of Lignin-Degrading Fungi
and Their Applications in Organopollutant Degradation
DANIEL CULLEN
CONTENTS
I. Introduction .......................... .
11. Physiology of Wood-Decaying Fungi ...... .
A. Peroxidases .......................... .
1. Lignin Peroxidases .................. .
2. Manganese Peroxidases .............. .
B. Laccases ............................. .
C. Other Enzyme Systems ................. .
111. Molecular Genetics .................... .
A. Peroxidases .......................... .
1. Gene Structure ..................... .
2. Genomic Organization ............... .
3. Regulation ........................ .
4. Heterologous Expression ............. .
B. Laccases ............................. .
C. Peroxide-Generating Enzymes ........... .
D. Cellobiose Dehydrogenase .............. .
IV. Conclusions and Future Prospects ........ .
References ........................... .
I. Introduction
This chapter provides an overview of the physiol-
ogy and associated molecular genetics of wood-
decaying fungi as they relate to organopoUutant
degradation. White-rot fungi are characterized by
an ability to fragment all major structural poly-
mers of wood including lignin. More poorly und er-
stood are the brown-rot fungi, which rapidly
depolymerize cellulosic materials while only
modifying lignin. Collectively, these wood- and
litter-degrading fungi play a pivot al role in the
carbon cycle. In fact, white-rot fungi are the only
microbes known to efficiently mineralize fully
lignified tissue.
The wood-rotting fungi are obligate aerobes,
deriving their nourishment from the biological
USDA, Forest Service, Forest Products Laboratory, One
Gifford Pinchot Dr., Madison, Wisconsin 53705, USA
combustion of wood and associated materials,
71 using molecular oxygen as a terminal electron
71 acceptor. The focus here is on the non-specific
72 extracellular enzyme systems of white-rot fungi.
72
These oxidative enzymes have been shown to
74
75 transform and degrade an array of organopollu-
75 tants in addition to depolymerizing lignin. Intra-
77 cellular metabolism of small molecular weight
77
77
products is also covered, although relatively
78 little is known of these processes. This chapter
78 does not comprehensively review the voluminous
79 literature on fungal degradation of lignin and
79
79
related organopollutants. Recent developments
80 are emphasized, and the reader is referred to
80 previous review articles (Kirk and Farrell 1987;
81 Eriksson et al. 1990; Gold and Alic 1993; Hammel
1995b; Cullen and Kersten 1996; Cullen 1997; Kirk
and Cullen 1998; Cameron et al. 2000; Pointing
2001) for more detailed information. Areas of
uncertainty are highlighted.
11. Physiology of Wood-Decaying Fungi
Lignin is a formidable substrate. The polymer is
large and initial depolymerization must be ex-
tracellular. The structure is comprised of inter-
unit carbon-carbon and ether bonds that
demand oxidative rather than hydrolytic degrada-
tive mechanisms. In addition, the polymer's lack
of stereoregularity requires ligninolytic agents
that are less specific relative to the hydrolytic
enzymes attacking cellulose. White-rot fungi have
met these challenges through the evolution of
extracellular peroxidases and oxidases that act
non-specifically via the generation of unstable
lignin free radicals, which undergo a variety of
spontaneous cleavage reactions. The major
enzymes acting directly or indirectly on lignin are
lignin peroxidase (LiP), manganese peroxidase
(MnP), and laccase (Fig. 5.1). All three enzymes
can act with low molecular weight mediators to
The Mycota XI
Agricultural Applications
Kempken (Ed.)
© Springer-Verlag Berlin Heidelberg 2002
72 D. Cullen

OCH 3
0, Cation radical
L

H2~r
many
0yproducts

o~
OH
"-
/L

Mnp+Mn:~i~
° °
+ unsaturate!."
OCH 3
0, 0·
L Phenoxy radical
OH Benzylic radical

/L
o
8 c
OH

Fig. 5.1. Extracellular oxidative enzyme systems of P. peroxidation oxidize ~O-4 models as shown in reactions A
chrysosporium and related white-rot fungi. Lignin peroxi- and B, respectively. Laccases and Mn(III) oxidize phenols
dase (LiP) and manganese peroxidase (MnP) coupled lipid (C)

bring about oxidation of lignin as weH as an A. Peroxidases

impressive array of xenobiotics (Table 5.1). The
microbiology and physiology of ligninolytic fungi
have been reviewed (Kirk and FarreH1987; Eriks-
son et al. 1990; Gold and Alic 1993; Higuchi 1993;
Tuor et al. 1995; Cullen and Kersten 1996).
1. Lignin Peroxidases
LiP was first discovered based on HzOz-dependent
Ca-Cß cleavage of lignin model compounds
and subsequently shown to catalyze the partial
Table 5.1. Examples of organopollutants degraded by wood-decaying fungi. PCBs polychlorinated biphenyls; PAHs poly-
cyclic aromatic hydrocarbons
Species
Bjerkandera adusta
Ceriporiopsis subvermispora
Coriolus versicolor
Corolopsis gallica
Corolopsis polyzona
Gloeophyllum striatum a
Gloeophyllum trabeum a
Irpex lacteus
Kuehneromyces mutabilis
Laetiporus sulphureus a
Nematoloma frowardii
Phanerochaete chrysosporium
Phanerochaete laevis
Phanerochaete sordida
Phlebia radiata
Phlebia tremellosa
Pleurotus eryngii
Pleurotus ostreatus
Pleurotus pulmonarius
Pycnoporus cinnabarinus
Trametes hirsutus
Trametes hispida
Trametes versicolor
aBrown-rot fungi. All others are classified as white-rot fungi.
Organopollutant References( s)
Azo dyes Heinfling et al. (1998)
Phenyl urea herbicides Khadrani et al. (1999)
PCBs Beaudette et al. (1998)
PAHs Field et al. (1992); Kotterman et al. (1998a,b)
PCBs Ferrey et al. (1994)
Anthracene Krivobok et al. (1998)
Fluorene Garon et al. (2000)
4-Methyldibenzothiophene Ichinose et al. (1999)
Textile dyes Kapdan et al. (2000)
PAHs Pickard et al. (1999)
PCBs Novotny et al. (1997)
2,4,-Dichlorophenol Schlosser et al. (2000)
Pentachlorophenol Fahr et al. (1999)
Pentachlorophenol Fahr et al. (1999)
Polyethylene glycol Kerem et al. (1998); Kerem and Hammel (1999)
2,4,6-Trinitrotoluene Kim and Song (2000)
Phenanthrene and pyrene Sack and Fritsche (1997); Sack et al. (1997a)
Phenanthrene and pyrene Sack and Gunther (1993); Sack et al. (1997a)
PAHs Sack and Gunther (1993); Sack et al. (1997a-c)
2,4,6-Trinitrotoluene Scheibner and Hofrichter (1998)
Alkyl halide insecticides Kennedy et al. (1990)
Chloroanilines Sandermann et a1. (1998)
DDT Bumpus and Aust (1987)
2,4-Dichlorophenol Valli and Gold (1991)
Heterocyclic dyes Cripps et al. (1990)
Endosulfan (cyclodiene) Kullman and Matsumura (1996)
Various PAHs Bumpus 1989; Hammel et al. (1991); Vazquez-
Duhalt et al. (1994); Bogan et al.
(1996a); May et al. (1997)
PCBs Bumpus et al. (1985); Eaton (1985); Thomas
et al. (1992); Dietrich et al. (1995); Yadav et al.
(1995); Novotny et al. (1997)
Pentachlorophenol Hammel and Tardone (1988); Mileski et al.
(1988); Lamar et al. (1990b); Aiken and Logan
(1996); Reddy and Gold (1999,2000)
Trichloroethylene Yadav et al. (2000)
2,4,5-Trichlorophenol Joshi and Gold (1993)
2,4,6-Trichlorophenol Armenante et al. (1994); Reddy et al. (1998)
2,4,6-Trinitrotoluene Fernando et al. (1990); Sublette et al. (1992);
Hodgson et al. (2000)
PAHs Bogan and Lamar (1996)
Creosote Davis et al. (1993); Lamar et al. (1994)
Pentachlorophenol Lamar and Dietrich (1990); Lamar et al.
(1990a,b, 1993)
Dioxins Takada et al. (1996)
2,4,6-Trinitrotoluene Van Aken et al. (1999)
PCBs Ferrey et al. (1994)
Azo dyes Heinfling et al. (1998)
Industrial dyes Vyas and Molitoris (1995); Rodriguez et al.
(1999)
PCBs Beaudette et al. (1998)
Phenanthrene Bezalel et al. (1996)
Phosphorothiolates Amitai et a1. (1998)
Various PAHs Martens and Zadrazil (1998); Novotny et a1.
(1999); Baldrian et a1. (2000)
Benzo[ a]pyrene Maspahy et al. (1999)
Triclosan Hundt et a1. (2000)
Lindane Singh and Kuhad (1999)
Industrial dyes Rodriguez et a1. (1999)
PAHs Majcherczyk et a1. (1988); Collins et al. (1996);
Johannes et al. (1996); Johannes and
Majcherczyk (2000)
PCBs Novotny et a1. (1997); Beaudette et al. (1998)
Triclosan Hundt et al. (2000)
74 D. Cullen
depolymerization of methylated lignin in vitro
(Glenn et al. 1983; Tien and Kirk 1983, 1984; Gold
et al. 1984; Fig. 5.1A). Lignin peroxidase catalyzes
a variety of oxidations, all of which are dependent
on HzO z. Simply stated, LiP oxidizes aromatie
nuclei ofsubstrates by one electron and the result-
ing aryl cation radicals degrade spontaneously via
many reactions dependent on substrate structure
and on the presence of reactants (Harvey et al.
1985; Kersten et al. 1985; Shoemaker et al. 1985;
Hammel et al. 1986b). Reactions subsequent to
cation radical formation are complex. The chem-
istry of LiP-catalyzed reactions has been reviewed
(Higuchi 1990).
Direct involvement of LiP has been demon-
strated in the degradation of a wide range of
organopollutants. Polyeyclie aromatie hydroear-
bons (PAHs) with ionization potentials below
approximately 7.6eV, including benzo[a]pyrene,
anthracene, and pyrene, are substrates for LiP
(Hammel et al. 1986a). Similarly, free radical
mechanisms involving purified LiP have been
shown for chlorinated phenols (Hammel and
Tardone 1988; Mileski et al. 1988; Valli and Gold
1991; Reddy and Gold 2000), tetrahydrofurans
(Vazquez-Duhalt et al. 1994), dioxins (Hammel et
al. 1986a; Valli et al. 1992b), methoxybenzenes
(Kersten et al. 1985), and various chloro- and
nitromethoxybenzenes (Valli and Gold 1991;
Valli et al. 1992a,b; Teunissen et al. 1998). The
biochemistry of organopollutant degradation has
been reviewed (Higson 1991; Hammel 1995a,b).
Isozymic forms of Phanerochaete chrysospo-
rium LiPs are commonly distinguished by their
pI and order of elution from a MonoQ anion-
exchange column. Ten peroxidases have been
separated by this method and are designated Hl
through HlO. Six of these, Hl (pI 4.7), H2 (pI 4.4),
H6 (pI 3.7), H7 (pI 3.6), H8 (pI 3.5), and Hl0
(pI 3.3), have veratryl alcohol oxidation activity
(Renganathan et al. 1985; Kirk et al. 1986; Leisola
et al. 1987; Farrell et al. 1989). Analytical isoelec-
tric focusing resolved 15 proteins with LiP activity
(Leisola et al. 1987). The number of isozymes
observed depends on the growth conditions (e.g.
nitrogen- versus carbon-limited), the me ans of
purification, and storage conditions. Proteolysis,
dephosphorylation and other post-translation al
modifications contribute to isozyme multiplicity
(Eriksson and Pettersson 1982; Kuan and Tien
1989; Dosoretz et al. 1990a,b; Datta 1992; Dass et
al. 1995; Feijoo et al. 1995; Rothschild et al. 1997,
1999). Although the various LiPs have similar sub-
strate specificities towards aromatic substrates,
their kinetic behavior varies more clearly when
tert-butyl hydroperoxide is substituted for HzO z
(Farrell et al. 1989; Glumoff et al. 1990). The pro-
cessing, biological roles and interactions among
isozymes are poorly understood.
The precise role of LiPs in complex substrates
such as wood and organopollutant-contaminated
soils remains uncertain. As mentioned above,
extracellular peroxidases are physically too large
to enter the pores of the cell wall (reviewed by
Blanchette et al. 1997). Thus, LiP has been pro-
posed to act indirectly by oxidizing low molecular
weight substrates, which in turn penetrate the wall
and oxidize the polymer (reviewed by Hammel
1996). Specifically, the secondary metabolite vera-
tryl aleohol, which is synthesized and secreted by
P. chrysosporium and which is oxidized by LiP to
a cation radical (Khindaria et al. 1995), was
proposed to play this role (Harvey et al. 1986).
However, the veratryl alcohol cation radical is too
short-lived to function as a diffusible mediator
(Khindaria et al. 1995). Recent studies show LiP
is capable of oxidizing ferroeytoehrome e and syn-
thetic lignin at the protein surface by a long-range
electron transfer mechanism (Wariishi et al. 1994;
Johjima et al. 1999).
2. Manganese Peroxidases
Low molecular weight diffusible oxidants could be
provided by manganese peroxidases (MnPs;
Glenn et al. 1986; Paszczynski et al. 1986). Like
LiPs, MnPs are glycosylated and have acidic pIs
and pH optima. Also like LiPs, they have a con-
ventional peroxidase catalytic cycle, but with
Mn(H) as the substrate. The Mn(H) must be
chelated by bidentate organic acid chelators such
as glycolate or oxalate, which stabilize the product
Mn(HI) and promote its release from the enzyme.
The resulting Mn(IH) chelates are diffusible oxi-
dants that can act at some distance from the
enzyme. However, they are not strong oxidants
and cannot attack the non-phenolic units of lignin.
Instead, they oxidize phenolic structures (Fig.
5.1e), which make up ab out 10% of the units in
lignin. The phenoxy radicals resulting from the
one-electron oxidation und ergo a variety of reac-
tions, some of which result in polymer cleavage
within certain units, e.g., between the aromatic
rings and Ca (Wariishi et al. 1991; Tuor et al.
1992; Fig. 5.1e). Purified MnPs from cultures of
P. chrysosporium, Nematoloma frowardi, and
 Molecular Genetics of Lignin-Degrading Fungi and Their Applications in Organopollutant Degradation
Phlebia radiata oxidize xenobiotics such as pen-
tachlorophenol and 2,4,6-trinitrotoluene (TNT)
in an Mn-dependent manner (Scheibner and
Hofrichter 1998; Van Aken et al. 1999; Reddy and
Gold 2000). In contrast, azo dye decolorization by
Pleurotus eryngii and Bjerkandera adusta MnP
isozymes is Mn-independent (Heinfling et al.
1998).
It seems unlikely that the MnP-catalyzed
reaction should result in the extensive depoly-
merization of lignin or the efficient oxidation
of organopollutants of relatively high ionization
potential. However, certain efficient lignin
degraders, including Ceriporiopsis subvermispora,
Phanerochaete sordida, and Dichomitus squalens,
lack detectable LiP but produce MnP (Perie and
Gold 1991; Ruttimann et al. 1992, Ruttimann-
Johnson et al. 1993, 1994; Hatakka 1994; Orth et
al. 1994). Also, phenanthrene and fluorene are not
LiP or MnP substrates and yet both are efficiently
degraded by P chrysosporium cultures (George
and Neufield 1989; Hammel et al. 1992; Vazquez-
Duhalt et al. 1994; Bogan et al. 1996a). These
studies point to alternative mechanisms, and
recent research shows that the production of dif-
fusible oxyradicals by MnP can occur by a mecha-
nism other than via chelates of Mn(HI). In the
presence of Mn(H), MnP promotes the peroxida-
tion of unsaturated lipids. Transient lipoxyradical
intermediates are generated and these have been
shown to oxidize non-phenolic lignin model com-
pounds (Fig. 5.1B). The MnPllipid peroxidation
system depolymerizes phenolic- and phenol-
blocked (methylated) synthetic lignins (Bao et al.
1994), and oxidizes fluorene (Bogan et al. 1996a),
phenanthrene (Moen and Hammel 1994), and ß-
0-4linkages within lignin model compounds (Bao
et al. 1994; Kapich et al. 1999). Taken together,
these data suggest that lipid peroxidation may be
a component of the lignin-degrading system of
certain fungi. The identity of the substrate lipides)
is under investigation (Enoki et al. 1999), but cur-
rently unknown.
MnP/lipid peroxidation models aside, LiP
remains the only fungal oxidant known that can
efficiently mimic, in vitro, the Ca-Cß cleavage
and extracellular cleavage of aromatic rings that is
characteristic of ligninolysis by white-rot fungi.
Recently identified peroxidases defy simple clas-
sification as MnP or LiP, and can oxidize Mn(H)
as weIl as non-phenolic substrates (e.g., veratryl
alcohol) in the absence of mangane se (Mester and
Field 1998; Camarero ct al. 1999).
75
B. Laccases
Laccases are blue copper oxidases that catalyze
the one-electron oxidation of phenolics, aromatic
amines, and other electron-rich substrates. Like
Mn(IH) chelates, they oxidize the phenolic units
in lignin to phenoxy radicals, which can lead to
aryl-Ca cleavage (Kawai et al. 1988; Fig. 5.1e).
Most white-rot fungi produce laccase but some
do not, indicating that laccase is not absolutely
required in lignin degradation. Isozyme multiplic-
ity is common among basidiomycetes including C.
subvermispora (Fukushima and Kirk 1995; Salas et
al. 1995), P chrysosporium (Srinivasan et al. 1995;
Dittmer et al. 1997; Rodriguez et al. 1997), and D.
squalens (Perie et al. 1998).
Laccase can oxidize non-phenolic lignin-
related substrates in the presence of certain auxil-
iary substrates such as ABTS (2,2'-azino-bis-3-
ethylthiazoline-6-sulfonate; Collins et al. 1996;
Bourbonnais et al. 1997, 1998). For example,
purified P ostreatus laccase efficiently degrades
organophosphorus insecticides and related nerve
agents in the presence of ABTS (Amitai et al.
1998). High ionization potential PAHs are also
oxidized by C. gallica and T. versicolor laccases in
the presence of synthetic mediators (Johannes
et al. 1996; Pickard et al. 1999). Pycnoporus
cinnabarinus and Trametes versicolor cultures
produce small molecular weight compounds
that may act as natural intermediaries for
oxidation of non-phenolic lignin substructures
(Eggert et al. 1996) and PAHs (Johannes and
Majcherczyk 2000). The structure and function of
fungal laccases have been reviewed (Thurston
1994; Youn et al. 1995).
C. Other Enzyme Systems
Although numerous laboratory studies show
extracellular peroxidases and laccases capable of
oxidizing various xenobiotics, it is clear that a
multitude of additional extracellular and intracel-
lular processes must be involved in the transfor-
mation and/or mineralization of these compounds.
For examples, O-methylation of PCP and triclosan
has been observed in P chrysosporium (Lamar
and Dietrich 1990) and P cinnabarinus (Hundt
et al. 2000) cultures, respectively. Glycosyl
conjugation of triclosan has also been shown
in T. versicolor cultures (Hundt et al. 2000).
Membrane-bound and intracellular enzymes have
76 D. Cullen
received relatively little attention to date, but
cytochrome P-450 monooxygenases have been
implicated in bioconversions of phenanthrene by
P. ostreatus (Bezalel et al. 1996), benzo[a]pyrene
by Pleurotus pulmunoarius (Maspahy et al. 1999),
endosulfan by P. chrysosporium (Kullman and
Matsumura 1996), and 4-methyldibenzothiophene
by Coriolus versicolor (Ichinose et al. 1999). In the
case of 2,4,6-trichlorophenol (Reddy et al. 1998)
and PCP (Hammel and Tardone 1988; Mileski et
al. 1988; Reddy and Gold 1999,2000) degradation
by P. chrysosporium, extracellular LiP or MnP
may intitially catalyze oxidative dechlorination
and the products subsequently undergo various
intracellular reductive dechlorination and hydrox-
ylation reactions.
Organopollutant degradation by brown-rot
fungi may involve extracellular oxidative mecha-
nisms that are fundamentally different from
white-rot species. Gloeophyllum spp. and related
brown-rot fungi lack detectable LiP, MnP, or
laccase activities but nevertheless efficiently
degrade 2,4 dichlorophenol (Fahr et al. 1999;
Schlosser et al. 2000), PCP (Fahr et al. 1999), and
polyethylene glycol (PEG; Kerem et al. 1998;
Kerem and Hammel 1999). Hydroxyl radical,
formed by a Fenton-type re action of Fe(II) and
H 2 0 2 , has long been suspected as the powerful
oxidizing agent needed to rapidly depolymerize
cellulose during brown rot (Koenigs 1974a,b).
Analysis of metabolites and the identification of
Fe(II) and H 20 2 in Gleophyllum cultures support
a Fenton-type degradation (Hyde and Wood 1997;
Kerem et al. 1998; Kerem and Hammel 1999;
Paszczynski et al. 1999; Schlosser et al. 2000).
Several systems have been suggested as
potential sources of extracellular H 20 2 for peroxi-
dase activity and hydroxyl generation (Fig. 5.1A).
One likely source of H 20 2 for P. chrysosporium
LiPs is glyoxal oxidase (GLOX; Kersten and
Kirk 1987). GLOX is a radical-copper oxidase
(Whittaker et al. 1996) that utilitizes a wide
variety of small aldehydes such as glyoxal and
methylglyoxal (extracellular metabolites of P.
chrysosporium) and transfers the electrons to O 2 ,
gene rating H 20 2 • Another substrate, glycolalde-
hyde, is produced by the action of LiP on ß-0-4
lignin substructures. Perhaps the most interesting
property of GLOX, and of considerable physio-
logical significance, is that in the absence of a
peroxidase system, the oxidase is reversibly inac-
tivated (Kersten 1990). The enzyme is reactivated,
however, by reconstituting the complete peroxi-
dase system, including both LiP and substrate.
This suggests that the supply of H 20 2 by GLOX is
responsive to the demand of the peroxidases,
thereby providing an extracellular regulatory
mechanism for control of the coupled enzyme
systems. GLOX is also produced by other white-
rot fungi, although apparently not by all (Hatakka
1994; Orth et al. 1994).
Another likely candidate for gene rating H 20 2
in some white-rot fungi, including P. chrysospo-
rium and B. adusta, is aryl alcohol oxidase (AAO;
Muheim et al. 1990; Asada et al. 1995b). This
enzyme oxidizes benzyl alcohols to aldehydes,
transferring the electrons to O 2, producing H 20 2•
Interestingly, B. adusta secretes chlorinated benzyl
alcohols that are not substrates for LiP but are for
the AAo. Because B. adusta also secretes LiP, this
strategy circumvents the non-productive oxida-
tion of the AAO substrate by LiP. P. ostreatus
secretes a mixture of benzyl alcohols, including
anisyl alcohol, and an AAO that oxidizes them
(Sannia et al. 1991).
Various sugar oxidases have also been sug-
gested to be producers of the required extra-
cellular H 20 2 • Most are intracellular enzymes.
Pyranose oxidase, however, which oxidizes various
monosaccharid es at C-2, with transfer of electrons
to O 2 to produce H 20 2, has been located in the
extracellular polysaccharide matrix that coats the
lumens of cells during white-rot. The enzyme has
been studied in P. chrysosporium, T. versicolor,
Oudemansiella mucida, and Agaricus bisporus
(Daniel et al. 1994; Artolozaga et al. 1997; Volc et
al. 1997). Glucose I-oxidase activity has been
reported in P. chrysosporium mycelium (Kelley
and Reddy 1986, 1988) but appears to be less
common than pyranose 2-oxidase (reviewed by
Ander and Marzullo 1997).
In addition to the oxidases, extracellular H 20 2
mayaiso be genera ted by the oxidation of organic
acids that are secreted by many white-rot fungi.
Specifically, Mn(II)-dependent oxidation of gly-
oxylic and oxalic acids in C. subvermispora cul-
tures generates H 20 2 (Urzua et al. 1995, 1998a,b;
Aguilar et al. 1999).
Cellobiose dehydrogenase (CDH) is widely
distributed among white-rot and brown-rot fungi,
and mayaiso play an important role in organopol-
lutant degradation (Westermark and Eriksson
1974a,b, 1975; Henriksson et al. 2000a). The
enzyme has two domains containing FAD or he me
prosthetic groups, and these can be cleaved by P.
chrysosporium proteases. CDH binds to cellulose
 Molecular Genetics of Lignin-Degrading Fungi and Their Applications in Organopollutant Degradation
and oxidizes cello dextrins, mannodextrins, and
lactose. Suitable electron acceptors include
quinones, phenoxy radicals, and Fe 3+. (CDH can
use Oz as a poor electron acceptor, but it is still
classified as adehydrogenase, not an oxidase.)
The biological function of CDH is uncertain
(Eriksson et al. 1990). One model suggests that
CDH generates hydroxyl radicals by Fenton-type
reactions [Fe(II) and HzO z reactants] as described
above (Kremer and Wood 1992; Mansfield et al.
1997; Henriksson et al. 2000b). The model is con-
sistent with many of the properties of CDH and
entails the concerted efforts of additional enzymes
for efficient lignin depolymerization. In addition
to Fe(II), the Fenton mechanism requires a system
for generating extracellular HzO z, and enzymes
such as GLOX may be involved. Possibly, the
function(s) of CDH differ among species. This
issue has been recently reviewed (Henriksson et
al. 2000a).
III. Molecular Genetics
The molecular genetics of lignin-degrading fungi
has advanced considerably over the past 15 years.
Phanerochaete chrysosporium has emerged as the
model system, in large part due to the develop-
ment of experimental procedures. Methodologies
for auxotroph production, recombination analysis,
rapid DNA and RNA purification, pulsed-field
electrophoretic separation of chromosomes, and
genetic transformation by auxotroph complemen-
tation and by drug resistance markers have been
described (Alic et al. 1989, 1990, 1991, 1993; Alie
1990; reviewed by Cullen and Kersten 1996;
Cullen 1997). Another auxotroph complementa-
tion system was recently reported (Zapanta et al.
1998). No other white-rot fungi provide a range of
experimental tools comparable to P chrysospo-
rium, aIthough genetic recombination analysis
(Eichlerova and Homolka 1999; Larraya et al.
1999a), pulsed field electrophoretic karyotyping
(Larraya et al. 1999b), and genetic transformation
(Yanai et al. 1996; Kim et al. 1999; Honda et al.
2000) are now available for Pleuotus ostreatus.
Most recently, transformation of Trametes
versicolor has been achieved using phleomycin
resistance as a dominant selectable marker
(Bartholomew et al. 2001). Virtually nothing is
known of the molecular genetics of brown-rot
fungi. The molecular biology of P chrysosporium
77
has been reviewed (Alic and Gold 1991; Pease and
Tien 1991; Gold andAlic 1993; Cullen and Kersten
1996; Cullen 1997; Kirk and Cullen 1998).
A. Peroxidases
1. Gene Structure
Tien and Tu (1987) were first to clone and
sequence a P. chrysosporium cDNA encoding a
LiP. It is now clear that P. chrysosporium LiPs are
encoded by a family of at least ten c10sely related
genes that have been designated lipA through Zipf
(reviewed by Cullen and Kersten 1996). One
allele, ZipI2, is transcriptionally inactivated by an
unusual class II non-autonomous transposon
(Gaskell et al. 1995). Residues believed essential
to peroxidase activity are conserved (Tien and Tu
1987; Schalch et al. 1989). The gene-encoding
isozyme H8, ZipA, also features a putative propep-
tide (Schalch et al. 1989). A similar sequence was
shown in the LiP2 gene of strain OGC101, and the
proenzyme was identified as an in vitro translation
product (Ritch et al. 1991). The P chrysosporium
clones contain 8-9 short introns, and the positions
of six of these are highly conserved (Brown et al.
1988; Schalch et al. 1989; Ritch and Gold 1992;
Gold and Alie 1993; Stewart and Cullen 1999). The
crystal structure of LiP is similar to the overall
three-dimensional structure of cytochrome c per-
oxidase (CCP), even though sequence identity is
only approximately 20% (Edwards et al. 1993;
Poulos et al. 1993).
Several LiP genes have been characterized
from other fungal species, including Trametes
versicolor genes LPGIILPGIV (Jonsson and
Nyman 1992, 1994; Johansson and Nyman 1996),
LPGIIILPGIII (Jonsson and Nyman 1994;
Johansson and Nyman 1996), VLGI (Black and
Reddy 1991), LPGVI (Johansson and Nyman
1995), Bjerkandera adusta clone LPO-1 (Asada et
al. 1992), and Phlebia radiata Ipg3 (Saloheimo
et al. 1989). Further, PCR amplification of LiP-
like sequences also suggests the existence of LiP
genes in Ceriporiopsis subvermispora and Phane-
rochaete sordida (Rajakumar et al. 1996). These
results appear to contradict numerous biochemi-
cal investigations that failed to detect LiP activity
in C. subvermispora (Ruttimann et al. 1992; Orth
et al. 1993) and P. sordida (Ruttimann-Johnson et
al. 1994) cultures.
Multiple MnP genes have been sequenced
from several white-rot fungi including P.
78 D. Cullen
chrysosporium (Pe ase et al. 1989; Pribnow et al.
1989; Orth et al. 1994; Alic et al. 1997), T vers i-
color MPGI (Johansson 1994), T versicolor
PGVII (Johansson and Nyman 1996), C. subver-
mispora (Lobos et al. 1998; Tello et al. 2000), and
p. ostreatus (Asada et al. 1995a). The actual
number of MnP genes in these or other species
remains to be established. The N-terminal amino
acid sequences of P. chrysosporium isozymes
indicate the existence of at least two more genes
(Datta et al. 1991; Pease and Tien 1992).
Multiple sequence alignments reveal features
that help to distinguish MnP and LiP genes
(reviewed by Cullen 1997). The crystal structure of
MnP shows similarities with LiP. Experimental
support for Asp203 in Mn z+ bin ding has been pro-
vided by site-directed mutagenesis of the residue
(Kusters-van Someren et al. 1995). Consistent with
its ability to oxidize Mnz+ and its Mn-independent
activity on aromatic substrates, the gene encoding
Pleurotus eryngii "Mn-independent" peroxidase
features putative Mn-binding ligands (Ruiz-
Duenas et al. 1999b). Analogous genes have not yet
been identified in P. chrysosporium.
2. Genomic Organization
Genetic linkage in P. chrysosporium has been
demonstrated by restriction fragment length poly-
morphisms (RFLPs; Raeder et al. 1989a,b) and
later refined by PCR-based segregation analysis
(Gaskell et al. 1994). Recombination frequencies
are based on analyses of single basidiospore cul-
tures, which are the homokaryotic products of
meiosis. Genetic maps are entirely consistent with
physical distances established by Southern blot-
ting of CHEF gels (Gaskell et al. 1991; Covert et
al. 1992a; Stewart et al. 1992; Kersten et al. 1995)
and by walking in genOInic libraries (Huoponen et
al. 1990; Gaskell et al. 1991).
Gene clustering and chromosome length poly-
morphisms are common in P. chrysosporium. For
example, ZipA, ZipB, ZipC, ZipE, ZipG, lipH, ZipI, and
Zipf are clustered within 3 % recombination and
hybridize to a 3.5/3.7mb chromosome pair on
CHEF gel Southern blots (see Stewart and Cullen
1999 and refs. cited therein). In contrast, lipD and
lipF are unlinked and hybridize to 4.8/4.4mb and
1.8/2.0mb pairs, respectively (Stewart et al. 1992;
D'Souza et al. 1993). The gene encoding glyoxal
oxidase, glx, hybridizes to a 3.7/3.9mb pair
(Kersten et al. 1995). These length dimorphisms
are mitotically stable in strain BKM-F-1767, but
the number and sizes of bands rearrange substan-
tially following meiosis, presumably due to recom-
bination. Simultaneous resolution of all bands
has not been achieved under a single set of
electrophoretic parameters (Gaskell et al. 1991;
Covert et al. 1992b; D'Souza et al. 1993). Beyond
P. chrysosporium, little is known of the organiza-
tion of peroxidase genes, although a cluster con-
taining two LiP- and one MnP-encoding genes has
been described for T versicolor (Johansson and
Nyman 1996).
Homologous chromosomes differing in elec-
trophoretic mobility have also been observed in
numerous eukaryotes such as Plasmodium falci-
parum (Corcoran et al. 1988), dozens of fungi
(reviewed in Zolan 1995), and, most recently, in
the related white-rot fungus P. ostreatus (Larraya
et al. 1999a,b). Mechanism(s) giving rise to these
chromosome length polymorphisms (CLPs) and
their possible role in genetic variability are not
well understood. Intrachromosomal recombina-
tion between repetitive sequences or unequal
exchange between homologues could generate
shortened chromosomes (Zolan 1995; Gray
2000). The presence of repetitive elements in P.
chrysosporium (Gaskell et al. 1995), and their
apparent restriction to a single allelic homologue,
has some impact on length differences. However,
re cent sequence analyses showed no recombina-
tion between elements (Stewart et al. 2000).
3. Regulation
LiP genes are differentially transcribed and their
expression is dramatically modulated by culture
conditions (Holzbaur and Tien 1988; Stewart et al.
1992; Reiser et al. 1993; Janse et al. 1998; Vallim et
al. 1998; Stewart and Cullen 1999). Although the
P. chrysosporium LiP genes are tightly clustered
and structurally similar, no clear relationship
between genomic organization and transcriptional
regulation has been observed (Stewart et al. 1992;
Stewart and Cullen 1999). Areport suggesting that
nutrient nitrogen limitation regulates LiP ex-
pression post-translationally by heme processing
(Johnston and Aust 1994) has been directly con-
tradicted (Li et al. 1994).
Quantitative transcript analyses by competi-
tive RT-PCR support a role for P. chrysosporium
peroxidases in organopollutant-contaminated
soils. Consistent with an MnP-dependent lipid
peroxidation mechanism, depletion of high ion-
ization potential PAHs generally coincided with
increased transcripts of mnpl, mnp2, and mnp3 as
well as MnP activity (Bogan et al. 1996c). In con-
 Molecular Genetics of Lignin-Degrading Fungi and Their Applications in Organopollutant Degradation
trast to mnps, the transcript patterns among LiP
genes varied dramatically during anthracene
transformation in soils (Bogan et al. 1996b). Com-
parison of anthracene and PCP-contaminated
soils suggests differential regulation of LiP genes
in response to the particular organopollutant
(Lamar et al. 1995; Bogan et al. 1996b).
Manganese peroxidase production in P.
chrysosporium is generally dependent on Mn con-
centration (Bonnarme and Jeffries 1990; Brown
et al. 1990, 1991). Differential regulation of
MnP isozymes has been observed in response to
media composition for C. subvermispora and P.
chrysosporium (Pease and Tien 1992; Lobos et al.
1994). Transcriptional control of MnP expres-
sion is complex. Possible transcriptional control
elements, particularly metal response elements
(MREs), have been identified in 5' -untranslated
regions (Godfrey et al. 1990,1994; Alic and Gold
1991; Brown et al. 1993; Alic et al. 1997). Consis-
tent with the occurrence of upstream MREs,
Gettemy et al. (1998) observed upregulation of P.
chrysosporium mnpl and mnp2 in response to
Mn2+ concentration in defined media, but tran-
script levels of mnp3 were unaffected. In contrast,
T. versicolor mnp2 does not contain any MREs but
is dramatically upregulated in response to Mn (T.
Johansson and D. Cullen, unpubl.). Differential
display analysis of Coriolus versicolor cultures
exposed to PCP identified upregulated genes
encoding a heat shock protein, but not MnP genes
(Iimura and Tatsumi 1997).
4. Heterologous Expression
Basic biochemical investigations have been ham-
pered by low yields and purification difficulties
associated with LiP/Mnp isozymes. As a result,
considerable research effort has been expended
on the development of heterologous expression
systems. Baculovirus systems have been used to
produce active recombinant MnP isozyme H4
(Pease et al. 1991) and LiP isozyme H8 (Johnson
and Li 1991; Johnson et al. 1992; Lin et al. 1997).
Yields are relatively low, but baculovirus pro duc-
tion may be useful for experiments requiring
limited quantities of recombinant protein, e.g.,
site-specific mutagenesis. Techniques have also
been developed for recovering active P.
chrysosporium isozymes MnP H4 (Whitwam et al.
1995; Whitwam and Tien 1996), LiP H8 (Doyle and
Smith 1996), and LiP H2 (Nie et al. 1998) from E.
coli inclusion bodies. P. chrysosporium MnP and
LiP can also be produced in a "homologous
79
expression" system in which mnp transcriptional
control is placed under the glyceraldehyde-3-
phosphate dehydrogenase promoter (Mayfield et
al. 1994; Sollewijn Gelpke et al. 1999). The system
temporally separates the recombinant protein
from other peroxidases, and it has been suc-
cessfully used in site-directed mutagenesis
(Kusters-van Someren et al. 1995; Kishi et al.
1997). Effident secretion of fully active MnP
isozyme H4 has been achieved in A. oryzae
(Stewart et al. 1996) and A. niger (Conesa et al.
2000). The recombinant enzyme has served mech-
anistic studies (Jensen et al. 1996; Kapich et al.
1999), and Aspergillus expression systems have
been extended to peroxidases from C. subvermis-
pora [Larrondo et al. 2001, #1418], P. eryngii
(Ruiz-Duenas et al. 1999a), and Geotrichym
candidum (Sugano et al. 2000). Aifa et al. (1999)
has recently reported low-level secretion of P.
chrysosporium LiP by Aspergillus niger.
B. Laccases
Laccases of white-rot fungi are also encoded by
complex families of structurally related genes
(Kojima et al. 1990; Saloheimo et al. 1991; Coll et
al. 1993; Yaver and Golightly 1996; Yaver et al.
1996; Karahanian et al. 1998; Giardina et al. 1999;
Temp et al. 1999). Although laccase activity has
been demonstrated in P. chrysosporium cultures
(Srinivasan et al. 1995; Dittmer et al. 1997;
Rodriguez et al. 1997), the corresponding genes
have not yet been isolated (D'Souza et al. 1996).
Laccase genes are often differentially regulated
and the patterns of regulation differ substantially
between species (Wahleithmer et al. 1995; Yaver
and Golightly 1996; Yaver et al. 1996; Smith et al.
1998; Palmieri et al. 2000). Little information is
available on the genomic organization of laccases,
but Trametes villosa pulsed field gels suggest the
possible linkage of certain laccase genes (Yaver
and Golightly 1996). Basidiomycete laccases have
been expressed in several systems including A.
oryzae (Yaver et al. 1999) and Pichia pastoris
(Otterbein et al. 2000).
c. Peroxide-Generating Enzymes
Glyoxal oxidase (GLOX) of P. chrysosporium is
encoded by a single gene with two alleles (Kersten
and Cullen 1993; Kersten et al. 1995). The deduced
amino acid sequence lacks clear homology with
80 D. Cullen
any other known proteins (Kersten and Cullen
1993), although Bork and Doolittle (1994) have
identified a 50-residue 'kelch' motif in a variety
of database sequences including glyoxal oxidase
and galactose oxidase. On the basis of catalytic
similarities with Dactylium dendroides galactose
oxidase, potential copper ligands were tentatively
identified at Tyr135, Tyr377, His378, and His
471(Whittaker et al. 1996). The deduced amino
acid sequences of allelic variants differ by a single
residue (Lys308«Thr308), which may explain the
two isozyme forms observed on isoelectric focus-
ing gels (Kersten and Kirk 1987; Kersten 1990).
Southern blot analysis of CHEF gels and segrega-
tion analysis show that glx is unlinked to MnP or
LiP genes (Gaskell et al. 1994; Orth et al. 1994;
Kersten et al. 1995). The GLOX gene (glx) is
transcriptionally regulated in P. chrysosporium
(Kersten and Cullen 1993). Consistent with a close
physiological relationship between GLOX and
LiP, glx transcript appearance in defined media
(Stewart et al. 1992; Kersten and Cullen 1993), soil
(Bogan et al. 1996b) and in wood chips (Janse et
al. 1998) is coincident with lip and mnp. Pro duc-
tion of mutant GLOX in Aspergillus nidulans and
Pichia pastoris has confirmed the identity of
catalytic residues (Kersten et al. 1995; Whittaker
et al. 1999).
Beyond GLOX, relatively little is known of
the molecular genetics of white-rot oxidases.
Although numerous studies implicate pyranose 2-
oxidase in lignin degradation, the corresponding
gene has only been isolated from Coriolus versi-
color (Nishimura et al. 1996). The sequence shows
weak similarity to a short region within the FAD
domain of several cellobiose dehydrogenase
genes. Glucose I-oxidase genes have not been
isolated from any basidiomycete. Martinez and
coworkers have cloned and sequenced aryl
alcohol oxidase (AAO) encoding genes from
Pleurotus pulmunarius (Varela et al. 2000) and
Pleurotus eryngii (GenBank accession No.
AF064069). The predicted proteins are over 90%
identical, and show substantial sequence similarity
toA. niger glucose I-oxidase (35% identity). Blast
searches clearly show that the P. pulmunarius
sequence is related to an array of fungal "oxi-
dases" including P. pastoris alcohol oxidase (24 %),
and the FAD domain of cellobiose dehydroge-
nases from P. chrysosporium (25%), Trametes
versicolor (25%), and Pyconporus cinnabarinus
(24 %). We have recently cloned and partially
sequenced cDNAs from P. chrysosporium colo-
nized wood that exhibit striking similarity to these
FAD oxidases and to GLOX (T. Johansson and D.
Cullen, unpubl.). Taken together, these results
suggest involvement of a wide range of sugar oxi-
doreductases, aryl alcohol oxidases, and glyoxal
oxidase.
D. Cellobiose Dehydrogenase
Genes encoding CDH have been cloned from
several fungi including the white-rot fungi P.
chrysosporium (Raices et al. 1995; Li et al. 1996),
T. versicolor (Dumonceaux et al. 1998), and P.
cinnabarinus (Moukha et al. 1999). Sequences are
highly conserved. All share a common architec-
ture with separate FAD, heme, and cellulose-
binding domains (CBD), although the latter
domain has no obvious structural similarity to
functionally similar bacterial or fungal CBDs.
Li et al. (1997) characterized both allelic variants
of P. chrysosporium cdh. Northern blots show
upregulation of cdh in cellulose-containing media
(Li et al. 1996; Moukha et al. 1999), and com-
petitive RT-PCR revealed transcripts in P.
chrysosporium colonized wood (Vallim et al.
1998). Renganathan and coworkers (1985) tem-
porally altered expression of P. chrysosporium
CDH by fusing cdh with the promoter of the
highly expressed glyceraldehyde-3-phosphate
dehydrogenase gene (Li et al. 2000). No evi-
dence for CDH gene multiplicity has been
reported.
IV. Conclusions and Future Prospects
Experiments with purified enzymes, analysis of
metabolites in whole cultures, and field trials
demonstrate efficient degradation of a wide range
of organopollutants by wood-decaying fungi.
Oxidative mechanisms involving extracellular
peroxidases and laccases have been repeatedly
shown, although their relative importance in
complex substrates such as organopollutant-
contaminated soils remains uncertain. Fenton-
type reactions and an array of membrane-bound
and intracellular enzyme systems probably
augment or supersede peroxidases and laccases in
certain circumstances.
Establishing the roIe of specific genes and
enzymes in degradation pathways requires ex-
 Molecular Genetics of Lignin-Degrading Fungi and Their Applications in Organopollutant Degradation
perimental tools that have only recently become
available. Biochemical analysis in cell-free
systems is greatly facilitated by the increasing
availability of pure recombinant enzymes. Quan-
titative analysis of transcripts and organOPollutant
transformations in complex substrates provides
further evidence for the involvement of specific
genes. A more direct experimental approach
might be disruption or replacement of individual
genes, and transformation systems are now avail-
able for Phanerochaete chrysosporium, Pleurotus
ostreatus, and Trametes versicolor. Gene replace-
ment through homologous recombination occurs
at low frequency in P chrysosporium (Alic et al.
1993), but the technique has not yet been brought
to bear on questions of organopollutant degra-
dation. Agrobacterium-mediated gene transfer
systems may facilitate gene disruption experi-
ments in related wood-decaying fungi (Covert et
al. 2001 #1538; Chen et al. 2000 #531; de Groot
et al. 1998 #1537).
Current genomics research offers significant
opportunities for elucidating the roles and inter ac-
tions of known genes and for identifying new and
novel genes involved in degrading lignin and
organopollutants. The US Department of Energy's
Joint Genome Institute (JGI) has completed wh oie
genome shotgun sequencing of P chrysosporium.
A draft assembly is currently available for Blast
searching at <url>http://www.jgi.doe.gov/programs/
whiterot/whiterocmainpage.htmk/url>. Our la-
boratory and the JGI are collaborating on sequenc-
ing ESTs from P chrysosporium colonized wood,
and all sequences should be publicly available
by 2002. Clearly, these efforts will reveal an enor-
mous number of genes of potential relevance to
organopollutant degradation. When coupled to
transcript profiling, heterologous expression, and
gene disruptions, these investigations will elucidate
major degradation pathways, and answer funda-
mental questions concerning lower eukaryotic
genome organization.
Acknowledgements. The research of the author
was supported by U.S. Department of Energy
grant DE-FG02-87ER13712.
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Disease Control, Diagnostic, and Management
6 Biological Control of Fungal Plant Pathogens
YIGAL ELAD and STANLEY FREEMAN
CONTENTS
I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . .
11. Biocontrol Modes of Action ..............
A. Induced Resistance .....................
B. Competition...........................
C. Cell Wall Degrading Enzymes (CWDEs)
and Parasitism . . . . . . . . . . . . . . . . . . . . . . . . .
D. Inhibitory Compounds ..................
E. Restraining Pathogenicity of Pathogens .....
F. Suppression of Inoculum Production
by the Pathogen .......................
G. Combination of Modes of Action .... . . . . . .
111. Factors Affecting Efficacy of Biocontrol . . . . .
A. Plant Host Effect . . . . . . . . . . . . . . . . . . . . . . .
B. Microclimate, Abiotic Factors .............
C. Physical and Chemical Nature of Plant
Surfaces and the Environment ............
D. Indigenous Microbial Populations . . . . . . . . . .
IV. Implementation of Biocontrol Agents
Under Field Conditions . . . . . . . . . . . . . . . . . .
A. Barriers That Limit Implementation . . . . . . . .
V. Improved Control ......................
A. Integration with Other Control Agents . . . . . .
B. Application Timing .....................
C. Genetically Manipulated Agents . . . . . . . . . . .
VI. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . .
I. Introduction
Vast experience has been gained in the biological
control of soilborne and foliar diseases of fruits,
grain, fiber and wood products at pre-planting,
during cultivation or at post-harvest. Infection by
pathogens can be reduced under controlled or
field conditions by pre-inoculation of the plant
surfaces with filamentous fungi, bacteria, yeasts or
viruses (Blakeman and Fokkema 1982; Andrews
1992; Tronsmo 1992; Blakeman 1993; Brasier 1998;
Funck Jensen and Lumsden 1999). Biocontrol
offers attractive alternatives or supplements to the
use of conventional methods for disease control.
Department of Plant Pathology, ARO, The Volcani Center,
Bet Dagan 50250, Israel
Microbial biocontrol agents (BCAs) are perceived
93 as being less demanding to the environment and
94 their generally complex mode of action makes it
95 unlikely that resistance will develop. Tbe interest
96
in biocontrol is reflected in the number of scien-
96 tific publications that relate to this subject. Figure
97 6.1 compares the amount of publications that
97 relate to some fungal plant pathogens with the
98 amount related to biocontrol in the last three
98 decades. Tbe number of publications that deal
98 with plant pathogens increased during the 1980s
98
99
and 1990s by approximately almost 200% as com-
pared with the amount of publications during the
99 1970s (Fig.6.1A). On the other hand, the amount
99 of publications that relate to the biocontrol of
100 these pathogens and to Trichoderma spp. that are
101 popular biocontrol agents increased in the same
101 period by approximately up to 1170% (more than
101 11-fold; Fig. 6.1B). Tbe portion of biocontrol pub-
103
103 lications of the total amount of publications on the
104 respective pathogens increased from less than
104 10% during the 1970s to between 10 and 30%,
depending on the pathogen in question (Fig. 6.1 C).
However, enthusiasm for the use of biocontrol
needs to be balanced by the realization that few
microbial agents have been registered as com-
mercial products. Tbere are numerous examples
of successful biocontrol but in this chapter we will
be able to mention only some of them. Tbe reader
is encouraged to refer to various reviews that are
cited here for more detailed information.
Some examples of biocontrol of foliar
pathogens with common species of microorgan-
isms are from the works of Newhook (1951) and
Wood (1951) who inoculated senescent lettuce
leaves with antagonists such as Fusarium spp. and
Penicillium claviforme that were isolated from the
same crop in order to prevent primary establish-
ment of Botrytis cinerea; Cladosporium herbarum
suppressed strawberry gray mold by protecting
the flowers under field conditions (Bhatt and
Vaughan 1962). Several fungi controlled S. sclero-
tiorum on various crops (Boland and Hunter 1988;
The Mycota XI
Agricultural Applications
Kempken (Ed.)
© Springer-Verlag Berlin Heidelberg 2002
94 y. Elad and S. Freeman

Whipps et al. 1993). Control of Botrytis cinerea by simultaneously with the pathogen (Nelson and
Trichoderma spp. has been reported for grape Powelson 1988). Some yeast species and bacteria
(Dubos 1992; O'Neill et al. 1996) and strawberry have also been reported effective in controlling
(Tronsmo and Dennis 1977). Trichoderma Botrytis cinerea in various crops (Redmond et al.
hamatum reduced pod infection by 94 % in snap 1987; Elad et al. 1994a). For example, Bacillus
beans when applied to the blossom before or brevis reduced disease on protected Chinese
cabbage by 64-71 % by decreasing the period
of leaf wetness (Edwards and Seddon 1992).
500 - , - - - - - - - - - - - - - - - - - - - - , Research on biocontrol of soilborne fungal
A pathogens has concentrated on antagonistic
+ POIvdery milde",
~
fnngi belonging to the genera Trichoderma and
400 -. - BOfrylis cillerea
A- Sc/erofill;a spp.
Gliocladium, mycoparasitic Pythium spp., non-
+ Pythi/llll spp. pathogenic Fusarium spp., binucleate Rhizoctonia
.... 300 -B-- Sclerotilllll rolfsii spp., Laestisaria spp., Bacillus spp., ftuorescent
......= --.- Rhhoctollia spp. Pseudomonads and Streptomycetes (Funck
'"' Trichoderma spp. Jensen and Lumsden 1999). Some of the biocon-
•
<öl
Q.
200 trol isolates originate from suppressive soils where
development of the pathogens is inhibited. In
some fungal plant pathogens, deleterious RNA
100
virus es are involved in biological control. For
--j

example, in the chestnut blight (Cryphonectria

o ---,----,--,----,-~ parasitica), take-all disease (Gaeumannomyces
Year: 1982 1985 1988 1991 1994 1997 2000 graminis) , Sclerotinia molds (Sclerotinia spp.)
and Dutch elm disease (Ophiostoma novo-ulmi)
1200 pathosystems, viruses are responsible for natural
disease suppression under field conditions and
1000 l studies are being conducted to utilize this hypo-
virulence phenomenon (Duffy and Weller 1996;
800
... -1
Brasier 1998; Zhou and Boland 1998). Therefore,
'"=
't;"' 600 • a diverse range of microbial agents including
Q.
viruses, bacteria, fungi and other organisms (such
400 as nematodes) possess antagonistic activities
against as wide a range of fungal pathogens.

o ------------------~------~--~ 11. Biocontrol Modes of Action

Year: 1982 1985 1988 1991 1994 1997 2000

Prevention of infection by BCAs or suppression

30
c of disease is based on induced resistance in the
host plant, competition for nutrients and space,

... 20

...s-=
Fig. 6.1. Three decades of research work on biological
'c:.;"' control of fungal plant pathogens as reflected in the
Q.
amount of publications cited in the CABI database. A Per-
centage of the number of publications on selected fungal
10 pathogens during the 1980s and 1990s as compared with the
number of publications during the 1970s. B Percentage of
the number of publications on biocontrol of selected fungal
pathogens and on the biocontrol agents belonging to Tri-
choderma spp. during the 1980s and 1990s as compared
o +-~---r--~~--~--~~--~--~- with the number of publications during the 1970s. C Per-
centage of publications on biocontrol of total publications
Year: 1973197619791982 1985 1988 1991 19941997 2000 relating to the respective fungal plant pathogens
 Biological Control of Fungal Plant Pathogens
antibiosis, hyperparasitism, reduction of the sapro-
phytic ability and reducing spore dissemination
and/or restraining of pathogenicity factors of the
pathogens. The behavior of pathogens during the
pre-penetration phase of their development on a
healthy plant surface and the relative importance
of necrotic tissues within the crop as an inoculum
source determine the mechanism that may be
effective in biocontrol. Unlike the necrotrophs
that are dependent on exogenous nutrients
and are susceptible to competition, biotrophic
pathogens such as powdery mildews and rusts,
which are independent of exogenous nutrients
during germination and penetration, can still
establish infection on a nutrient-depleted plant
surface. However, on the plant surface, the conidia
or germ tubes of the biotrophs are exposed to
antibiotics and lytic enzymes produced by micro-
organisms (mainly bacteria such as Bacillus spp.
and Pseudomonas spp.) that can inhibit germina-
tion and Iyse germ tubes (Doherty and Preece
1978).
A. Induced Resistance
Induced resistance (IR) is recognized as an impor-
tant mode of biocontrol in vegetative tissues
(Sequeira 1983; Kua: 1987; Kloepper et al. 1992).
Resistance may be induced locally or may be sys-
temic. Induced systemic resistance (ISR) caused
by various microorganisms can protect plants
against soil or foliar pathogens (Paulitz and Matta
1999). ISR is attributed to a variety of micro-
organisms (nonpathogenic microorganisms such
as saprophytes, plant growth promoting, and avir-
ulent races of the pathogen) and can contral mul-
tiple pathogens. In addition to biotic elicitors of
resistance, toxic and nontoxic chemicals, UV,
compost and other agents have also been shown
to induce resistance. A pathogen such as Col-
letotrichum orbiculare can be applied to the
cotyledon or first true leaf and induce resistance
in upper leaves of the same plant (Tuzun and Kua:
1985). IR results in enhanced and more rapid
secretion of defense re action enzymes such as
proteases, peroxidases and chitinases, and finally
in lignification of the cell walls limiting pathogen
development and spread (Hammerschmidt and
Kua: 1982; Hammerschmidt et al. 1982; Metraux
and Boller 1986). Raupach and Kloepper (1998)
reported that mixtures of plant-growth-promoting
rhizobacteria (PGPR) caused ISR against three
95
foliar and soil-borne pathogens of cucumber. IR
to wilt diseases with avirulent fungi has been
repeatedly demonstrated under laboratory condi-
tions but under field conditions it was rarely effec-
tive, as in the case of protection of sweet potato
from Fusarium oxysporum f. sp. batatas by a non-
pathogenic Fusarium oxysporum strain (Ogawa
and Komada 1986).
Salicylic acid (a signal molecule in systemic
acquired resistance, SAR) produced by
Pseudomonas aeruginosa induced resistance to
Botrytis cinerea in bean (De Meyer and Höfte
1997), and isolate T39 of Trichoderma harzianum
had a similar ISR effect to that of Pseudomonas
aeruginosa on bean, challenged by Botrytis cinerea
(De Meyer et al. 1998). Root inoculation of Tri-
choderma harzianum caused increased peroxidase
and chitinase activities in leaves of cucumber
seedlings (Yedidia et al. 1999). Wilson (1989) sug-
gested that part of the mode of action of yeasts
against certain citrus fruit rots might involve IR in
the fruit, a fact later confirmed by Droby et al.
(1991). ISR is demonstrated by applying a BCA at
a location separated from the plant organ that is
challenged by a pathogen, whereas the suppres-
sion of disease by dead cells of the inducer may
demonstrate a local IR. For instance, dead cells of
T39 were capable of controlling Botrytis cinerea
infection on bean, tobacco and pepper (Elad and
Kapat 1999) and powdery mildew on cucumber.
Given the spatial separation of the T39 root appli-
cation from the foliar pathogens, this effect could
be attributed to ISR imparted by Trichoderma
harzianum T39 (De Meyer et al. 1998; Elad 2000).
Few, if any, plants in natural ecosystems exist
independent of symbiotic associations with myc-
orrhizal and/or endophytic fungi (Petrini 1986).
Mutualistic symbioses have been shown to confer
disease resistance (Freeman and Rodriguez 1993;
Redman et al. 1999). A pathogenic isolate of Col-
letotrichum magna (pathogen of cucurbits) was
converted to a nonpathogenic endophytic mutual-
ist by UV mutagenesis and gene disruption
enabling the symbiont to confer disease resistance
against pathogenic isolates of Colletotrichum,
Fusarium, and Phytophthora. Disease resistance
was correlated to a decrease in the time of activa-
tion of host defense systems after exposure to
the pathogens (Freeman and Rodriguez 1993;
Redman et al. 1999). The phenomenon was
defined as "endophyte associated resistance"
(EAR) since it did not induce SAR or hypersen-
sitive reactions (Redman et al. 1999). Mycorrhizal
96 y. Elad and S. Freeman
fungi are associated with defending plants against
soil-borne diseases and have even been implicated
in some induced-resistance-like reactions (Dehne
1982; St. Arnaud et al. 1994). However, mycor-
rhized plants may be more susceptible to foliar
pathogens, due to delayed accumulation of
pathogenesis-related (PR) proteins in the foliage
(Shaul et al. 1999).
B. Competition
Species of bacteria, yeasts and filamentous fungi
can inhibit fungal pathogens by competing for
nutrients such as nitrogen, carbon, macro-
elements and micro-elements. In such cases,
reduction of the concentration of nutrients gener-
ally results in a reduced rate of pathogen spore
germination and in slower germ tube growth,
thereby reducing the number of infection courts
and the extent of subsequent necrosis incited by
the pathogen (Blakeman and Fokkema 1982;
Loper and Buyer 1991; Blakeman 1993; Elad et al.
1995; Funck Jensen and Lumsden 1999).
Competition for nutrients or space was
demonstrated when post-harvest biocontrol was
tested with various yeasts on apple fruits, e.g., the
yeast strain 87 of Candida spp. (McLaughlin et al.
1990), the basidiomycetous yeast Cryptococcus
laurentii (Roberts 1990), Sporobolomyces roseus
(Janisiewicz et al. 1994) and Candida oleophila
(Mercier and Wilson 1994). Several bacterial and
fungal isolates were able to reduce germination of
conidia of Botrytis cinerea and the severity of rot
symptoms on detached leaves, and to control
disease on whole plants. Since the nutrient
concentration on leaves had an effect on control
efficacy, activity was partially attributed to com-
petition for nutrients (Elad et al. 1994a; Zimand
et al. 1996). Competition for nutrients such as
nitrogen, carbon and iron and for sites on root
surfaces is important in the biocontrol of soil-
borne fungal pathogens. Induced rhizosphere
competence in Trichoderma harzianum is an
example of an antagonist having an improved bio-
control effect, probably because of the ability to
compete for cellulose in the mucilage layer at the
root surface (Ahmad and Baker 1987). Similarly,
the ability to effectively compete for iron and
carbon enabled the control of Fusarium by
Pseudomonads (Elad and Baker 1985; Loper and
Buyer 1991).
C. Cell Wall Degrading Enzymes (CWDEs)
and Parasitism
Parasitism of fungi by various microorganisms has
been recorded in many systems (Kranz 1981;
Adams 1990; Wisniewski et a1.1991; Elad 1995),
and relies on the production of fungal CWDEs
(Elad 1995). Trichoderma, Gliocladium and
Pythium spp. are among the known mycoparasites
(Elad 1995). Tronsmo and Dennis (1977) claimed
that Trichoderma controls Botrytis rot of fruits,
presumably by direct parasitism and antibiotic
production. Pythium spp. are nonspecific myco-
parasites that interact with many soil-borne host
fungi (Lifshitz et al. 1984).
Most of the microorganisms reported as
BCAs ofpowdery mildew fungi are hyperparasites
or antibiotic producers (Elad et al. 1995, 1999;
Belanger et al. 1997). The most common hyper-
parasite of powdery mildews is the coelomycete,
Ampelomyces quisqualis (Sztejnberg et al. 1989).
The species does not appear to be confined to one
host; for instance, an isolate from an Oidium sp.
infecting Catha edulis in Israel was able to antag-
onize several powdery mildew fungi belonging
to the genera Oidium, Erysiphe, Sphaerotheca,
Podosphaera, Uncinula and Leveillula (Hashioka
and Nakai 1980; Sztejnberg et al. 1989). Other iso-
lates parasitized Sphaerotheca fuliginea in cucum-
bers (Jarvis and Slingsby 1977; Philipp and Crüger
1979; Sundheim and Amundsen 1982; Sztejnberg
et al. 1989). The hyperparasite Verticillium lecanii
of several pathogenic fungi reduced the incidence
of Sphaerotheca fusca in the greenhouse when
certain conditions were respected, namely, low
vapor pressure deficit (VPD) (Spencer and Ebben
1983; Verhaar et al. 1996). Considered for a long
time to be a strict hyperparasite, re cent findings
have suggested that early degradation of powdery
mildew cells in interaction with Verticillium lecanii
was mediated by the production of antibiotic sub-
stances (Askary et al. 1997). Among other fungi
reported as parasites are Acremonium alternatum
and Cladosporium cladosporioides that antago-
nized and destroyed the thallus of Sphaerotheca
fusca (Malathrakis and Klironomou 1992).
CWDEs produced by antagonistic microor-
ganisms can affect pathogenic fungi (Chernin et al.
1995). Combination of endochitinase and chito-
biosidase of Trichoderma harzianum resulted in a
synergistic increase in antifungal activity, which by
itself was more pronounced than the activity of
 Biological Control of Fungal Plant Pathogens
Enterobacter cloacae chitinases (Lorito et al.
1993). Labudova and Gogorova (1988) found
isolates of Trichoderma reesei and Trichoderma
harzianum capable of producing proteinase, man-
anase, laminarinase and chitinase and claimed that
this implied that the nature of the antagonism of
the two isolates was based on mycoparasitism.
One member of the group of hydrolytic enzymes
that is believed to be involved in this mechanism
of biocontrol was purified and characterized as a
serine-protease (Geremia et al. 1993). Autoclaved
mycelium, fungal cell wall preparations or chitin
induce this enzyme; however, it is not induced in
the presence of glucose. It has been suggested
(Wisniewski et al. 1991) that the mode of action
of the post-harvest biocontrol yeast, Pichia guil-
liermondii, combines tenacious attachment with
secretion of CWDEs. On the contrary, there was
no correlation between ability to degrade Botrytis
cinerea cell-wall polymers and biocontrol activity
in correlations between chitinases and ß-1,3-
glucanase and the control achieved by five isolates
of Trichoderma (Zimand et al. 1991) or by 74
isolates of Trichoderma (Elad et al. 2000). There-
fore, the fact that Trichoderma isolates have the
potential to produce CWDEs does not necessar-
ily guarantee effective biocontrol.
D. Inhibitory Compounds
Antibiosis, as a means of biocontrol, has been
associated with many microorganisms (Fravel
1988; Andrews 1992; Whipps 1997; Funck Jensen
and Lumsden 1999). It is sometimes difficult to
predict the importance in natural ecosystems of
inhibitory compounds that are produced in vitro
by antagonists. The ultimate proof for the role of
these compounds in biocontrol is the loss of activ-
ity in nonproducing mutants of the antagonist.
This was the case with gliotoxin-negative mutants
of Gliocladium virens that lost 50% of the disease
suppressive effect compared to the wild type
(Wilhite et al. 1994).
Trichoderma and Gliocladium spp. are
common producers of antibiotics. Trichoderma
virens (syn. Gliocladium virens) produces two
major antifungal antibiotics - gliotoxin (toxic
to Rhizoctonia solani more than to Pythium
ultimum) and gliovirin (toxic to Pythium spp. but
not to Rhizoctonia solani) (Howell et al. 1993).
Tronsmo and Dennis (1977) reported that Tricho-
97
derma pseudokoningii and Trichoderma viride
produce substances that inhibit Botrytis cinerea
on strawberry fruits. Likewise, Trichoderma
hamatum, which pro duces inhibitory volatiles,
reduced grey mould of snap bean pods and
blossom by 77-97% (Nelson and Powelson 1988).
Janisiewicz and Roitman (1988) found an isolate
of Pseudomonas cepacia to be effective against
Botrytis, Penicillium and Mucor rots of apples and
pe ars, by production of the powerful antibiotic
pyrrolnitrin. In addition, Botrytis cinerea has
been controlled by antibiotic producers of certain
Bacillus spp. in various hosts (Edwards and
Seddon 1992; Leifert et al. 1995).
The antagonistic epiphytic yeast-like fungus
Sporothrix flocculosa colonized powdery mildew
of rose and cucumber more rapidly than Sporothrix
rugulosa; Sporothrix flocculosa did not penetrate
its host but rather induced a rapid plasmolysis of
the powdery mildew cells (Hajlaoui et al. 1992;
Hailaoui et al. 1994), presumably the result of the
production of fatty acids by the antagonist
(Benyagoub et al. 1996). The fatty acids appear to
interfere with the integrity of the plasmalemma,
the composition ofwhich determines the specificity
of the antagonist. Hoch and Providenti (1979)
found strong antagonism between the common
phyllosphere yeasts, Tilletiopsis spp. and the
cucumber powdery mildew pathogen. Inoculation
with a Tilletiopsis cell suspension on detached,
mildew-infected cucumber leaves destroyed the
superficial thallus of the powdery mildew. Similar
to Sporothrix flocculosa, Tilletiopsis spp. do not
seem to penetrate the powdery mildew fungus sug-
gesting that antibiosis is the main mode of action
(Klecan et al. 1990).
E. Restraining Pathogenicity of Pathogens
Synthesis of hydrolytic enzymes by some
pathogens during the first phase of host-pathogen
interactions is crucial for the infection process.
These enzymes include cutinase, which hydrolyzes
ester linkages of the cutin polymer, and CWDEs,
i.e., pectolytic enzymes exo- and endo-
polygalacturomnase (PG), pectate lyase (PL), and
pectin methyl esterase (PME) and cellulase
(Alghisi and Favaron 1995; Kapat et al. 1998b).
Interference with pathogenicity processes has only
recently been studied in Trichoderma harzianum
T39 on germination and subsequent lesion pro-
98 Y. Elad and S. Freeman
duction by Botrytis cinerea on bean leaves. No
difference in the amount of pathogen mycelium
could be observed 24--48h after inoculation. Nev-
ertheless, penetration of the host tissue and disease
were significantly reduced. The activities of PG,
PME and PL (Zimand et al. 1996) and chitinase, ß-
1,3-glucanase and cutinase (but not that of cellu-
lase) produced by Botrytis cinerea was reduced in
the presence ofT39 (Kapat et al. 1998b). Cervone
et al. (1989) suggested that the accumulated pectic
enzyme products, i.e., oligogalacturonides with a
degree of polymerization higher than nine, act as
elicitors of defense mechanisms of plants. It has
been suggested that, since the intensity of the pec-
tolytic enzyme activity is reduced in the presence
of Trichoderma harzianum T39, the larger oli-
gogalacturonides, which do not undergo further
breakdown, may accumulate and elicit the host
plant's defense mechanism (Zimand et al. 1996).
Proteases are involved in biocontrol as suggested
by Pietr et al. (1994), i.e., secretion of proteolytic
enzymes that deactivate pathogenicity related
hydrolytic enzymes is common for microorganisms
including fungi (Haab et al. 1990). Proteolytic
activity of Trichoderma viride was claimed to be
involved in biocontrol of Sclerotium rolfsii in
autoclaved soil (Rodriguez-Kabana et al. 1978).
Endoproteinase of Bacillus megaterium can inacti-
vate extracellular enzymes of Rhizoctonia solani
(Bertagnolli et al. 1996). Trichoderma harzianum
T39 produces protease on leaves that reduces
Botrytis cinerea germination, pathogenicity
enzymes and subsequent disease development,
whereas protease inhibitors reduce efficacy of
biocontrol (Elad and Kapat 1999).
F. Suppression of Inoculum Production
by the Pathogen
Antagonistic interactions can also be exploited
during the saprophytic stage of necrotrophs such
as Botrytis cinerea. Reduction in inoculum pro-
duction followed by a suppression of its ability to
infect would create an accumulative effect over
several disease cycles in pathosystems where
inoculum produced within the field is the major
contribution to the development of epidemics.
Köhl et al. (1995a,b) reported that Ulocladium
atrum consistently reduced sporulation of Botrytis
cinerea on dead lily and onion leaves exposed to
field conditions for more than 90% in aseries of
experiments und er different microclimatic condi-
tions. Naturally senescing leaves within the dense
canopy of a crop may constitute the initial sub-
strate for Botrytis cinerea. Therefore, microbial
suppression of sporulation from these necrotic
tissues will result in a lower spore load in the crop
leading to a slower progression of disease epi-
demics (Köhl and Fokkema 1993).
G. Combination of Modes of Action
Although not shown, in many cases it can be
assumed that biocontrol is a result of mnlti-
mechanism action of the antagonist (Elad 1996).
Synergism between different forms of antagonism
may occur, as in the case of the in vitro inhibition
of conidial germination of Botrytis cinerea by
antibiotics combined with the action of CWDEs
(Lorito et al. 1993). Nonpathogenic Fusarium
oxysporum re duces fusarium wilt diseases by
me ans of saprophytic competition with the
pathogen for the nutrients in the soil (Alabouvette
et al. 1996) as weIl as by parasitic competition
for infection sites (Schneider 1984). Trichoderma
virens, the producer of the antibiotics gliovirin and
gliotoxin, is also a mycoparasite that pro duces
CWDEs. As described above, the mode of action
of Trichoderma harzianum T39 is competition for
nutrients and interference with the production of
pectolytic enzymes of the pathogen; thus, in addi-
tion to slowing the germination of the pathogen's
conidia, T39 also prevents the penetration of the
host tissue and the maceration process, and has
been shown to induce resistance (De Meyer et al.
1998; Kapat et al. 1998b).
111. Factors Affecting Efficacy
of Biocontrol
A. Plant Host Effect
Mechanisms and attributes of biocontrol other
than those described above are of importance
under field conditions. Among these is the ability
of the BCA to survive and proliferate on the plant
surface under varying nutrition al and micro-
climatic conditions, and to colonize the plant in
such a way as to prevent the establishment of
the pathogen. This complicates the use of BCAs
because of the fact that an isolate, although effec-
tive against a certain pathogen on one crop, may
 Biological Control of Fungal Plant Pathogens 99

be ineffective against the same pathogen on ated with reduced efficacy of Botrytis cinerea
another. The host plant may directly infiuence the suppression in cucumber and tomato by T39,
BCA by stimulating its establishment and survival Aureobasidium pullulans and Cryptococcus
and by stimulating antagonism towards the alb idus (Dik and Elad 1999). Tilletiopsis minor
pathogens (Smith et al. 1997; Paulitz and Matta was very effective in controlling powdery mildew
1999). The host can provide favorable environ- on cucumber plants under controlled conditions,
mental niches and nutrients that infiuence BCA- but, under greenhouse conditions, the effect was
pathogen interaction by supplying plant exudates disappointing due to elevated VPD (Hijwegen
needed for the production of antibiotics or for 1992).
competition. These factors are determined by the
genetic background of the host plant and by its
C. Physical and Chemical Nature of Plant
physiological status as manipulated by the growth
Surfaces and the Environment
conditions.
Soluble and volatile nutrients that arise from
Chemical exudates on the plant surface contain
the root, seed, fiower and leaf exudates and
macro- and micro-elements, sugars, sugar alcohols,
mucigal of the root cap can support introduced
pectic substances, amino acids, and organic acids.
BCAs. The plant can infiuence microbial popula-
The quality and quantity of leachates from plants
tions associated with its surfaces, as shown for fiax
are affected by plant age and factors such as tem-
and tomato that were grown in the same soil and
perature, VPD and surface moisture, light, fertil-
found to select for different bacteria (Lemanceau
ization and pollen that change constantly. These
et al. 1995). Strains of Pseudomonas fluorescens
changes may affect plant surface microfiora
CHAO that were engineered to overproduce
directly or have an indirect impact by modifying
diacetylphloroglucinol and pyoluteorin increased
leaf characteristics, e.g., metabolic state, morphol-
in biocontrol efficacy much more on roots of
ogy and surface chemistry (Cutter 1976). As nutri-
cucumber than on wheat, suggesting that the
ents fiuctuate, there are community changes in
plant species may affect antibiotic production
colonization by bacteria, yeasts and filamentous
(Maurhofer et al. 1995). Likewise, different plant
fungi (Blakeman 1985). Plant surfaces are covered
cultivars may accommodate BCAs to a different
with a hydrophobic wax layer on which water is
degree (Smith et al. 1997); for example, different
distributed as discrete droplets. Fertilization of
cultivars of wheat responded differently to PGPR
plants affected the survival of Trichoderma on the
(Chanway et al. 1988) and biocontrol (Weller
foliage (Elad and Kirshner 1993). Similar to the
1986) strains.
phyllosphere, the rhizosphere is subject to dra-
matic changes on a short temporal scale. Rain
events and daytime drought can result in fiuctua-
B. Microclimate, Abiotic Factors
tion in salt concentration, pH, osmotic potential,
water potential and soil particle structure. The
Suppression of plant diseases by BCAs is largely
rhizosphere can also change due to root growth,
affected by environmental conditions (Burpee
interaction with other soil biota and weathering
1989; Hannusch and Boland 1996; Shtienberg and
processes. These changes affect the establishment
Elad 1997). Under field conditions the plant
and activity of the introduced BCAs. Duffy et al.
surface is subjected to fiuctuating temperature,
(1997) calculated that the chemical and physical
VPD, surface wetness, gases and air movement
properties of soil that are associated with sup-
(Burrage 1971). These factors affect the indige-
pression of take-all disease of wheat by Tricho-
nous microfiora and BCAs directly, or may have
derma koningii are nitrate-nitrogen, pH, copper,
an indirect effect by modifying the disposition of
and soluble magnesium and suggested amending
the host plant, e.g., the metabolic state and the
the inoculants with beneficial factors that would
plant surface chemistry. For example, Trichoderma
enable biocontrol activity.
harzianum T39 was less effective in suppression of
cucumber fruit and stern grey mould under wet
conditions and temperatures below 20°C than D. Indigenous Microbial Populations
under dry conditions at elevated temperatures
(Elad et al. 1993). High temperature during the On the plant surface, the introduced BCA faces
day and high VPD during the night were associ- not only the pathogen, but also a variety of other
100 y. Elad and S. Freeman
microorganisms with which it constantly interacts.
For example: cucumbers inoculated with PGPR
strains interacted with the population of endo-
phytes within the plant (Press et al. 1995) and the
BCA Bcillus cereus interacted with the bacterial
communities in the rhizosphere of soybean
(Gilbert et al. 1996). The colonization of apple
wounds by natural microorganisms and an intro-
duced yeast, Candida oleophila, was monitored
by Mercier and Wilson (1994) who found that the
presence of naturally occurring microftora was in
some cases beneficial to biocontrol of storage rot.
The effect of introduced Trichoderma harzianum
T39 on other phylloplane microorganisms (Elad
and Kirshner 1992) was found to either promote
or inhibit bacteria, yeasts or filamentous fungi,
depending on plant nutrition and the microcli-
matic conditions under which the plants were
incubated.
IV. Implementation of Biocontrol
Agents Under Field Conditions
Biocontrol preparations can serve as an alterna-
tive means of control if they reduce disease to
acceptable levels. They can be used when no effec-
tive manageable solution is available, especially in
cases of fungicide failure. Several microorganisms
have been fully commercialized for the control of
plant pathogens and their number has increased
in re cent years. Of the 35 products listed by Fravel
et al. (1999) only five were not aimed at fungal
pathogens. A few examples will be described here.
Control of fruit post-harvest diseases caused
by common pathogens is usually achieved by the
application of yeast or bacterial isolates that are
applied directly to the harvested fruits or prior to
harvest. These BCAs are effective protectors of
wound pathogens, such as the product "Aspire "
(Droby et al. 1991). The ability to control the
atmosphere of BCA-treated stored and shipped
fruits results in high efficacy of the BCAs (Droby
et al. 1991; Jijakly et al. 1999). An isolate of Strep-
tomyces griseovirides is the core of apreparation
called Mycostop that has been developed com-
mercially and claimed to control several diseases
(White et al. 1990). For instance, it is aimed at
lettuce gray mold and applied by drenching the
plantlets with the suspension. The control of
several cereal seed-borne diseases that is achieved
by astrain of Pseudomonas cholororaphis is as
good as the control achieved by standard
fungicides reported in over 100 field trials in
Sweden (Gerhardson et al. 1998). One isolate of
Ampelomyces quisqualis (Sztejnberg et al. 1989)
has been developed into a commercial product
named AQ10 (Ecogen, Jerusalem, Israel). This
product is currently available for the control of
powdery mildew on grape leaves and is being eval-
uated under greenhouse conditions for the control
of powdery mildew on cucurbits (Rofstein 1994)
and roses (Pasini et al. 1997).
Isolate T39 of Trichoderma harzianum
(Trichodex, 20P, Makhteshim Ud., Be'er Sheva,
Israel), registered for agricultural use in many
countries worldwide, has effectively controlled
Botrytis diseases in greenhouse crops and grape
vineyards, and other greenhouse diseases such as
white mold (Sclerotinia sclerotiorum), leaf mold
(Cladosporium fulvum) and powdery mildew
(Sphaerotheca fusca) in various countries (Elad et
al. 1994b, 1995,1998a, 1999; O'Neill et al. 1996;
Elad 2000). Dik and Elad (1999) reported that
in cucumber, the most consistent control of gray
mold was achieved by Aureobasidium pullulans
and Trichoderma harzianum which maintained
sufficiently high population densities and con-
trolled disease more significantly than the broad-
spectrum fungicide tolylftuanid under all tested
climate regimes.
Establishment of the BCA on plants is impor-
tant for field application. For instance, recovery
of the antagonist on nonsprayed control plots is
indicative of the spread of airborne Ampelomyces
quisqualis inoculum (Falk et al. 1995). Similarly,
Trichoderma harzianum T39 established itself on
sprayed plants as weIl as on nontreated plants in
the greenhouse; a drawback in the experiment.
Rowever, the secondary dispers al of the BCA may
enable the protection of new plant parts, advanta-
geous for practical use (Elad et al. 1993). The
system by which the BCA is delivered to the
appropriate plant site may inftuence its success. A
conidial preparation of Gliocladium roseum was
transferred to strawberry ftowers by bees resulting
in suppression of Botrytis cinerea on its target -
ftowers and fruits (Peng et al. 1992).
In efficacy trials, Sporothrix flocculosa was
tested on a commercial scale against powdery
mildew in roses and was found as effective as
a fungicide, provided that the VPD was favorable
to the antagonist (Belanger et al. 1994). In com-
parative large-scale trials on cucumber, Sporo-
thrix flocculosa was found more efficient at
 Biological Control of Fungal Plant Pathogens
controlling Sphaerotheca fuliginea than other
BCAs of powdery mildew including Ampelomyces
quisqualis, Verticillium lecanii and Tilletiopsis
washingtonensis (Hajlaoui and Belanger 1991; Dik
et al. 1998). The fungus is currently being targeted
for commercial exploitation under the trade name
Sporodex.
Forest trees, stumps of cut logs and wood
products pose achallenge for biocontrol. Basid-
iomycete fungi such as those belonging to the
genera Heterobasidium, Chondostereum, Phelli-
nus, Ophiostoma, Pellinus, Armilaria and Lentinus
that infect these trees and stumps cause rot of the
wood before or after logging and several staining
fungi such as Ophiostoma and Ceratocystis spp.
cause discoloration of the wood. Trichoderma spp.
and Scytalidium have received the greatest atten-
tion as control agents for this purpose (Bruce
1999; Schoeman et al. 1999). The biocontrol activ-
ity is based on depletion of resources and com-
petition for nutrients, inhibitory volatile and
nonvolatile compounds, mycoparasitism and
production of extracellular enzymes. However,
biocontrol in the wood industry is not yet com-
mercially implernented.
A. Barriers That Limit Implementation
Most of the studies reporting high efficacy of
BCAs were conducted under controlled environ-
ments. Occasionally, introduction of antagonists
that were highly effective under controlled envi-
ronments on commercially grown plants were only
moderately effective and sometimes ineffective
under field conditions. Problems of biocontrol effi-
cacy may stern from lack of establishment and/or
persistence of the introduced microorganisms.
They may adversely be affected by the fluctuating
environmental conditions or may be affected by
other practices of the farmer.
A survey of 60 greenhouse experiments
conducted worldwide revealed that, in ca. 70%
of them, Trichoderma harzianum T39 suppressed
Botrytis cinerea infections in tomatoes and cucum-
ber proving as effective as the chemical fungicide
applied for comparison. Discrepancy in the effi-
cacy of biological control between controlled and
field conditions may be due to several reasons. It
has been suggested that the environment al condi-
tions which are unpredictable in commercial pro-
duction may influence the survival, establishment
in the phyllosphere and activity of the biocontrol.
101
Inconsistent efficacy hinders the large-scale imple-
mentation of biological control, especially in high-
value cash crops.
Powdery mildew antagonists, for instance,
are dependent on conditions of low VPD for
maximum efficiency. Some hyperparasites colo-
nize powdery mildews only when there is free
water on plant surfaces; others need VPD below
2.5 mbar if a rapid and complete colonization
is to be achieved (Hajlaoui and Belanger 1991;
Belanger et al. 1997; Dik et al. 1998). VPD remains
the most critical factor affecting colonization and
destruction of powdery mildew structures on
plant surfaces and can be manipulated under a
controlled environment.
V. Improved Control
A. Integration with Other Control Agents
BCAs may be combined together for the control
of more than one disease that occurs in a crop,
they may be combined with additives to improve
their efficacy or survivaI, or they may be combined
with other me ans of control in order to achieve
acceptable levels of disease suppression. To
reduce the dependence of hyperparasites on low
VPD, researchers have used several additives.
Spencer and Ebben (1983) mixed spores of Verti-
cillium lecanii with 2% glycerol and 1% gelatin
and increased their longevity on cucumber leaves.
Malathrakis and Klironomou (1992) found in
laboratory experiments that glycerol increased
the effectiveness of Acremonium alternatum when
applied 3 or 4 d after inoculation of cucumber
plants with Sphaerotheca [uliginea. On the other
hand, Jarvis (1992) was unable to increase natural
populations of Ampelomyces quisqualis using
sugars, peptone, gelatin or glycerol. Trichoderma
harzianum T39 and Ampelomyces quisqualis
AQ10 have been applied with oils to ensure activ-
ity against powdery mildews (Elad et al. 1998).
Finally, paraffin oil improved the effectiveness of
Sporothrix fiocculosa against rose and cucumber
powdery mildews (Belanger et al. 1994; Dik et al.
1998).
A mixture of Trichodex with iprodione was
superior to either component alone for the control
of grey mould (Elad et al. 1994b). Trichodex mixed
with Phyton 27 (a copper-based pesticide) was
most effective (97% control) on greenhouse
102 Y. Elad and S. Freeman
tomato against grey mould (Bourbos and
Skoudridakis 1994). However, in cucumber grown
in heated ggreemhouses, alternation of TRI-
CHODEX with iprodione did not improve control
efficacy of Botrytis cinerea compared with weekly
applications with TRICHODEX. It did improve
control compared with iprodione. Apparently, in
heated glasshouses, adverse conditions for the
BCA do not prevail and biocontrol as a stand-
alone treatment is as good or better than chemical
control or alternation (Dik and Elad 1999).
Knowledge of the compatibility between fungi-
cides and BCAs is essential for their successful
combination. Some strains of Ampelomyces
quisqualis are compatible not only with fungicides,
but also with pesticides used on crops in which the
antagonist might be used (Sundheim 1986).
Sztjenberg et al. (1989) studied the effects of
Ampelomyces quisqualis and the fungicide pyra-
zophos and found that pyrazophos alone and in
alternation withAmpelomyces quisqualis achieved
better results than Ampelomyces quisqualis alone.
Integration of biological control with genetic
resistance will usually enhance disease control.
Verhaar et al. (1996) and Dik et al. (1998)
improved biocontrol of powdery mildew when
they applied Verticillium lecanii on tolerant,
as opposed to susceptible, cucumber cultivars.
Control was further improved by the addition of
silicon to the nutrient solution, which renders the
plants less susceptible to powdery mildew (Dik et
al. 1998). Falk et al. (1995) suggested that Fusar-
ium proliferatum, a mycoparasite that re duces
sporangial production of the grape downy mildew
Plasmopara viticola, might be practical in con-
junction with moderately resistant cultivars or
with a management program in which chemical
agents are used.
During post-harvest and storage there are
opportunities to improve disease suppression by
combining BCAs with various me ans that have
been found to be effective on their own. IR was
obtained by successful integration with sugar and
sugar analogs such as 2 deoxY-D-glucose, calcium
and magnesium salts, chitosan and chitin deriva-
tives, coating materials, modified atmosphere and
UV-c irradiation (Janisiewicz 1998). Similarly,
control of Bipolaris sorokiniana on tall fescue by
Strenotrophomonas maltophilia strain C3 was
improved by the addition of colloidal chitin
(Zhang and Yuen 1999). Low water activity toler-
ant cells of Candida sake that were produced on
glycerol-amended growth medium improved
control of blue mold in apples as compared with
the unmodified yeast (Teixido et al. 1998).
Efforts to enhance BCAs of soil-borne dis-
eases have been directed primarily at enhancing
activity of BCAs immediately after seeding to
improve suppression of preemergence damping-
off, enhancing rhizosphere competence of BCAs
to improve suppression of root rot and enhancing
compatibility of BCAs with other agricultural
practices. Antifungal metabolite production
alte red in situ by genetic manipulation has been
used to enhance establishment of the BCA in
target environments. The physiological status or
differentiation state of BCA cells harvested from
fermentation can have an effect on survival and
activities when applied. Furthermore, the sub-
strates on which the microorganisms are delivered
and their formulation can inftuence efficacy of the
BCA and enhance desirable characteristics of
biocontrol.
Attempts to apply more than one BCA were
made for several reasons. Elad et al. (1994a) sug-
gested that application of more than one isolate
could be needed for targeting two life cycle stages
of Botrytis cinerea, i.e., prevention of infection and
reduction of sporulation. Isolates with different
nutrition al profiles had different niche require-
ments and could coexist in wounds of apple allow-
ing better control of post-harvest blue mold
(Janisiewicz 1996). Similarly, enhanced suppres-
sion of Fusarium wilt of radish was obtained with
strains of ftuorescent Pseudomonas spp. that had
different mechanisms and were compatible with
each other (De Boer et al. 1999). A combination
of BCAs can be used for the control of multiple
diseases in a crop, as was achieved with PGPR that
control multiple cucumber diseases (Raupach and
Kloepper 1998). This was also the case when the
post-harvest pathogens Penicillium expansum,
Botrytis cinerea and Pezicula malicorticis on apple
were controlled by a mixture of two yeast isolates
and one bacterium (Leibinger et al. 1997) and
when powdery mildewand grey mould on cucum-
ber were controlled by AQlO and TRICHODEX
(Elad et al. 1998). Guetsky et al. (2000) recently
suggested that application of more than one
antagonist, provided that the antagonists have dif-
ferent ecological requiremeuts, would increase the
reliability and decrease the variability of biologi-
cal control. Thus, combined application of a yeast
(Pichia sp.) and a bacterium (Bacillus sp.) resulted
in significant suppression of Botrytis cinerea in
strawberries under all regimes tested.
 Biological Contral of Fungal Plant Pathogens
B. Application Timing
In many trials, the BCAs were treated as biologi-
cal fungicides, i.e., applied at predetermined fixed
intervals, either on a weekly basis (in greenhouses)
or according to the developmental stage of the
crop, as is practiced in grapes (Elad et al. 1993;
O'Neill et al. 1996). Alternation of biocontrol
treatment with the chemical treatments on a cal-
endar basis was more effective than biocontrol
alone, and more reliable than the other treat-
ments since variations in the control results were
reduced (Elad et al. 1994b). However, disease sup-
pression achieved by the alternation treatment
was sometimes insufficient if the BCA was applied
in weather highly stimulatory to gray mold epi-
demics (Shtienberg and Elad 1997). The integra-
tion of biological and chemical controls aided by
the use of a forecaster to predict disease out breaks
in unheated greenhouses was examined with the
goal of developing integrated control pro grams.
"BOTMAN" (Botrytis manager) and "GREEN-
MAN" (greenhouse disease manager) were devel-
oped to also solve problems of other diseases, in
which the BCA is the most important tool and
chemical control is implemented on a need-basis
only (Elad et al. 1994b; Shtienberg and Elad 1997).
Decisions on whether to apply biological or chem-
ical measures are taken before each spray accord-
ing to the weather forecast, occurrence and
severity of foliar diseases, resistance of pathogens
towards fungicides and conduciveness of the
greenhouse and the crop to disease development.
When weather, crop and the greenhouse condi-
tions are extremely unfavorable for disease devel-
opment, then spraying is not to be recommended
at all; when conditions are extremely favorable,
then a chemical fungicide is suggested; in all other
cases, a BCA (TRICHODEX) is recommended.
In plots treated according to GREENMAN, four
chemical and six biological sprays were applied
and suppression of disease was equivalent to a full
chemical program.
c. Genetically Manipulated Agents
Manipulation of antagonistic microorganisms by
genetic modification may improve their biocontrol
activities. The molecular techniques required for
genetic manipulation include mutagenesis, proto-
plast fusion and molecular transformation, all of
which are well-established methods used in fungal
103
and bacterial microbiology. Therefore, introduced
genes pertaining to cell wall lysis, antibiotics and
hypovirulence, which are produced or overex-
pressed, have been shown to increase antifungal
activity of manipulated BCAs.
As mentioned in the previous sections, lytic
enzymes involved in mycoparasitism, interference
of pathogenicity enzymes and production of
antibiotics by antagonistic BCAs are major mech-
anisms of biocontrol. In recent years, cloning and
characterization of various genes encoding these
enzymes has been conducted for understanding
the mechanisms of biocontrol and for improving
antagonistic activity. These genes usually occur in
the fungal genome in single copies; however, by
transformation, multiple copies can be inserted
resulting in overexpression of a selected lytic
enzyme. U sing this approach, overexpression of
the EGLl ß-1,4-endoglucanase enzyme of Tricho-
derma longibrachiatum resulted in enhanced
disease suppression of Pythium ultimum in cucum-
ber over that of the single copy wild-type BCA
(Migheli et al. 1998). Likewise, increased antifun-
gal activity against Rhizoctonia solani was deter-
mined in Trichoderma harzianum transformants
overexpressing a 33-kDa chitinase (Lim6n et al.
1999). In this case, overexpression resulted in a
200-fold increase in chitinase activity compared
to that of the wild-type strain. Similarly, im-
proved biocontrol against Rhizoctonia solani
was achieved using transformed Trichoderma
harzianum overexpressing a proteinase-encoding
gene (Flores et al. 1997). Additional strategies
have relied on constitutive expression of a chiti-
nase gene from the bacteria Serratia marcescens in
Trichoderma harzianum producing high er levels
of the enzyme; however, proteolysis was observed
(Haran et al. 1993). Studies relying on expression
of chitinase genes in bacterial BCAs for control-
ling fungal pathogens have also been reported.
Examples of these include expression of chitinase
genes in Pseudomonas putida (Chet et al. 1993)
and Pseudomonas fluorescens (Sundheim et al.
1988) that controlled Rhizoctonia solani and
Fusarium oxysporum f. sp. redolens, respectively.
Toxins have been implemented in biocontrol
and constitute one of the major mechanisms of
biocontrol, namely antibiosis. Thus, regulatory
genes involved in toxin biosynthesis have been
used to enhance biocontrol activities of phy-
topathogenic fungi. For example, elevated levels
of toxins produced by the bacteria Erwinia her-
bicola have been shown to possess enhanced anti-
104 y. Elad and S. Freeman
fungal activity (Tenning et al. 1993). Overproduc-
tion of the toxin pyoluteorin in Pseudomonas fiu-
orescens resulted in improved control of various
fungal pathogens in cucumber (Maurhofer et al.
1992; Hill et al. 1994).
Hypovirulence can be achieved by transform-
ing the chestnut blight phytopathogen, Cry-
phonectria parasitica, with a double-stranded (ds)
RNA molecule (Nuss 1992). Furthermore, it was
shown that synthetic viral transcripts can reduce
virulence in additional pathogenic fungi indicating
the potential use of virus-mediated hypovirulence
in biocontrol (Chen et al. 1994).
VI. Conclusions
Significant progress has been achieved toward bio-
logical and integrated control of plant diseases
in various plant pathosystems over the last two
decades. Some biofungicides are already sold
commercially in a few countries and a wider avail-
ability of these products is expected, as registra-
tions will be obtained in more areas. Other BCAs
should reach the market soon and one can rea-
sonably assume that biological alternatives will be
available for most important greenhouse diseases
in the near future. These encouraging develop-
ments should not predude from reality that BCAs
are living organisms, the activity of which greatly
depends on biotic and abiotic conditions. Biocon-
trol preparations will rarely stand alone as a
complete measure for disease control under all
conditions. For this reason, scientists, and growers
alike, must accept the fact that BCAs are usually
not as effective as pesticides. Rather, biocontrol
should be viewed as an important if not essential
component of an integrated disease management
scheme if we are to achieve a significant and per-
manent reduction of pesticide use.
The research towards biocontrol improve-
ment and implementation is gaining momentum.
New modes of action have been elucidated and
the activity of some BCAs is becoming dearer.
However, more research into understanding the
behavior of BCAs on plant surfaces and during
interaction with pathogens, plants and indigenous
microflora is definitely needed. Genetic manipu-
lation of BCAs can be used as a tool for their
improvement in order to achieve better control
under variable conditions. However, the potential
risk associated with release of these organ-
isms into the environment should be further
studied to enable acceptable guidelines for their
implementation.
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7 Use of Entomogenous Fungi for the Control of Insect Pests
TARIQ M. BUTI
CONTENTS
I. Introduction...........................
11. Fungal Pathogenesis ....................
A. Adhesion and Germination . . . . . . . . . . . . . . .
B. Host Recognition . . . . . . . . . . . . . . . . . . . . . . .
C. Cuticle-Degrading Enzymes (CDEs) .......
D. Toxins ...............................
E. Colonisation of the Haemocoel . . . . . . . . . . . .
F. Attenuation of Virulence . . . . . . . . . . . . . . . . .
III. Epizootics and Factors Influencing
Fungal Efficacy ........................
A. Epizootics ............................
B. The Pathogen . . . . . . . . . . . . . . . . . . . . . . . . . .
C. The Insect Host . . . . . . . . . . . . . . . . . . . . . . . .
D. The Environment. . . . . . . . . . . . . . . . . . . . . . .
1. Solar Radiation . . . . . . . . . . . . . . . . . . . . . .
2. Temperature ........................
3. Relative Humidity . . . . . . . . . . . . . . . . . . . .
4. Rainfall ............................
5. Edaphic Factors . . . . . . . . . . . . . . . . . . . . . .
6. Host Plant . . . . . . . . . . . . . . . . . . . . . . . . . .
IV. Increasing the Efficacy of Mycoinsecticides ..
V. Potential Hazards and Safety Concerns .....
A. Allergenicity ..........................
B. Toxicity ..............................
C. Pathogenicity . . . . . . . . . . . . . . . . . . . . . . . . . .
D. Depletion of Hosts .....................
E. Competitive Displacement ...............
VI. Conclusions ...........................
References . . . . . . . . . . . . . . . . . . . . . . . . . . . .
J. Jntroduction
Insect-pathogenic (= entomogenous) fungi can be
divided into two broad categories: the speeialists
and generalists (for a summary of the attributes
of these two categories, see Table 7.1). The
most important group of specialist fungi belongs
to the order Entomophthorales which have been
recently reviewed by Pell et al. (2001). Because
of the difficulty in culturing entomophthoralean
School of Biological Sciences, University Wales Swansea,
Single ton Park, Swansea SA2 8Pp' UK
fungi, none have yet been commercialised. This
111 chapter focuses on the generalist "Mitosporic
112 fungi" belonging to the dass Hyphomycetes that
112 are or have been developed as biological control
113 agents (BCAs, Table 7.2).
114
115 The main reasons for developing fungal BCAs
117 are: (i) they offer an enviranmentally friendly
118 alternative to chemical pesticides, and (ii) they can
be used where pests have developed resistance to
118
118 conventional pesticides. Unfortunately, there is
118 comparatively little investment in the research
119 and development of these organisms compared
120
120
with that spent on the discovery of chemical pes-
121 ticides. Two reasons for this are that mycoinsecti-
121 eides usually have a narrow host range and that
122 they often give inconsistent and poor contral
122
123 in field trials (Butt et al. 1999). The factors re-
124 sponsible for the initiation and development
125 of epizootics in insect populations are extremely
126 complex, involving interactions among the
126
126 pathogen, insect host and environment (e.g. host
126 plant, soil, dimate). Understanding these dynamic
126 interactions and elucidation of the factors limiting
127 epizootics will ultimately lead to efficacious
127
control of insect pests. To date, particular attention
has focused on improving the production, formu-
lation and application technologies. Currently,
efforts are being made to optimise the impact of
fungal BCAs by integrating them with other novel
crop pratection strategies (see Sect. IV).
This chapter pravides a broad overview of the
factors infiuencing epizootics and the strategies to
improve the efficacy of mycoinsecticides. It also
examines the risks these organisms pose to human
health. However, the initial focus is on fongal
infection processes since an understanding of
these processes is axiomatic to their development
as mycoinsecticides. For example, knowledge of
infection pracesses can help in strain selection,
quality control, and the development of strategies
to increase the efficacy of these BCAs for pest
contral.
The Mycota XI
Agricultural Applications
Kempken (Ed.)
~ SnrinIYer- Vp:r):l!J Beflin Hpirip.lhPTO ?OO?
112 T.M. Butt

Table 7.1. General characteristics of specialist and generalist entomogenous fungi. (Adapted from Pell et al. 2001)

Characteristic Specialists Generalist

Example Taxa Entomophthorales Hyphomycetes

Conidia size and number >10 J.1m in length, relatively small <10 J.1m in length, relatively large numbers per
per cadaver numbers per cadaver cadaver
Conidia discharge Active, with the exception of Not active
Massospora spp.
Pre-formed mucous on Present Not usually present. Some exceptions e.g.
conidia Verticillium spp., Hirsutella spp., Aschersonia spp.
Protoplasts Produced by many species Not usually present
Rhizoids Present in many species Absent
Ability to modify host Can modify behaviour e.g. Do not usually modify behaviour
behaviour Entomophthora muscae,
Entomophaga grylli
Pre-death sporulation Present in some species e.g. Uncommon
Strongwellsea castrans
Epizootics Most common in foliar insects Most common in soil insects
Host range Generally narrow Generally wide, with exceptions, e.g. Nomuraea
rileyi infects noctuid lepidoptera, Aschersonia spp.
infect whitefly and scales, Metarhizium album
infects hemipteran insects
Toxin production Not known Observed in several species
Saprophytic Not known, with exceptions Known in some species
Conidiobolus spp.
Resting Spores Present in most species Not usually present
Virulence Few conidia required for infection Many conidia usually required for infection
Sporulation and Fast Slow
germination rate
Higher order conidium In most species Uncommon
production
Culture media Expensive, complex (very few Inexpensive, wide range of simple media can be
developed) used
Mass production Expensive Comparatively inexpensive

11. FungaI Pathogenesis (1) poor/good adhesion, (2) good/poor germina-

A. Adhesion and Germination
Fungi are unique among the insect pathogens in
that they infect their hosts primarily through the
external cuticle, although a few species do invade
through the alimentary canal (e.g. Culicinomyces).
Adhesion of propagules to the host surface is the
first step in the pathogenic process. Initial attach-
ment is usually passive but subsequent consolida-
tion of the attachment and germination are active
processes. Enzymes are secreted prior to germ
tube emergence and play an important role in the
consolidation stage, as illustrated by the imprints
propagules (conidia, hyphae) leave behind on the
host surface (Wraight et al. 1990). Cross-links
between components of the cell wall and/or
the extracellular matrix with the host's outer
surface are formed. Concomitantly, the pathogen
responds to the physical-chemical cues resulting in
one or a combination of the following scenarios:
tion, (3) slow/rapid germination, (4) germ tube
emergence, and (5) appressorium differentiation.
Hydrophobie conidia of many pathogens will
bind in a non-specific manner to the epicuticle
of both susceptible and resistant host insects
(Boucias et al. 1988, 1991). However, production
of penetrant germ tubes does not normally occur
on non-host organisms. The germination process
requires both an appropriate humidity regime and
available nutrients for germ tube production. In
addition to chemical requisites, the surface topog-
raphy of the cuticle has also been shown to playa
role in adhesion, germination and appressorium
differentiation (St. Leger et al. 1989a,b, 1991a,b;
Butt et al. 1995).
In many cases, various fungistatic activities
have been associated with the cuticle of in-
sects. For example, constitutive fungistatic fatty
acids, epicuticular lipids, cuticle phenolic com-
pounds, and even plant-derived allelochemicals
(sequestered in insect cuticles) have been sug-
gested to inhibit fungal growth (Smith and Grula
 Use of Entomogenous Fungi for the Control of Insect Pests 113

Table 7.2. Entomogenous fungi that are or have been developed for pest contro!. (Butt and Copping 2000)

Product Fungus Target Producer

Mycotal Verticillium lecanii Whitefly and thrips Koppert, The Netherlands

Vertalec Verticillium lecanii
Meta-Guard Metarhizium anisopliae
Green Guard Metarhizium anisopliae
vaL acridum
Biogreen Metarhizium anisopliae
Metaquino Metarhizium anisopliae
Bio-Path Metarhizium anisopliae
Bio-Blast Metarhizium anisopliae
Cobican Metarhizium anisopliae
Bio-Cane Metarhizium anisopliae
Conidia Beauveria bassiana
Green Muscle Metarhizium anisopliae
vaL acridum
Ostrinil Beauveria bassiana
CornGuard Beauveria bassiana
Mycotrol GH Beauveria bassiana
Mycotrol WP and Beauveria bassiana
BotaniGard
Naturalis-L Beauveria bassiana
Proecol Beauveria bassiana
Boverosil Beauveria bassiana
Betel Beauveria brongniartii
Engerlingspilz Beauveria brongniartii
Schweizer Beauveria Beauveria brongniartii
Melocont Beauveria brongniartii
PFR-97 Paecilomyces fumosoroseus
Pae-Sin Paecilomyces fumosoroseus
Laginex Lagenidium giganteum
Aphids Koppert, The Netherlands
Termites Ajay Bio-Tech Ud, India
Locusts CSIRO, Australia
Scarab larvae on pasture Bio-care Technology, Australia
Spittle bugs Brazil
Cockroaches EcoScience, USA
Termites EcoScience, USA
Sugarcane spittle bug Probioagro, Venezuela
Cane grubs Bio-Care Technology Pty Ud,
Australia
Coffee berry borer Live Systems Technology,
Colombia
Locusts, grasshoppers CABI - BioScience, UK
Corn borer Natural Plant Protection (NPP),
France
European corn borer Mycotech, USA (now Emerald
BioAgriculture)
Grasshoppers, locusts Mycotech, USA
Whitefly, aphids, thrips Mycotech, USA
Cotton pests e.g. bollworms Troy Biosciences, USA
Armyworm Probioagro, Venezuela
Colorado beetle Czechoslovakia
Cockchafers NPP,France
Cockchafers Andermatt, Switzerland
Cockchafers Eric Schweizer, Switzerland
Cockchafers K wizda, Austria
Whitefly ECO-tek, USA
Whitefly Agrobionsa, Mexico
Mosquito larvae AgraQuest, USA

1982; Lecuona et al. 1997; Sosa-G6mez et al. 1997;
Vega et al. 1997; Inyang et al. 1999a,b). It is pos-
sible that successful germination is also dependent
on the ability of the pathogen to produce enzymes
that destroy fuugistatic or toxic compounds in the
cuticle, irrespective of their origin. Some cuticular
components may be peculiar to a specific pest. For
example, (E)-2-decenal, a defensive secretion of
the Southern Green Stink Bug (Nezara viridula),
inhibits the germination of conidia of Metarhizium
anisopliae but not those of Beauveria bassiana
and Paecilomyces fumosoroseus (Sosa-G6mez
et al. 1997 and references therein). There is no
doubt that the cuticle infiuences spore adhesion,
germination and differentiation (Butt et al. 1995;
Lecuona et al. 1997).
On conducive surfaces (e.g. hard, hydropho-
bie, nutrient-poor substrates), conidia produce a
germ tube that terminates in an appressorium
(Butt et al. 1995). The size and shape of the
appressorium depends on the fungal species
and/or strain as weIl as cuticular cues (Butt et al.
1995). Nearly all strains of M. anisopliae readily
produce appressoria, whereas some strains of B.
bassiana and Verticillium lecanii produce appres-
soria and others do not. In contrast, Nomuraea
rileyi does not produce appressoria but hyphae
penetrate the cuticle directly. The appressoria
of most fungi secrete a copious mucilage that
helps anchor the fungus during penetration. The
mucilage is particularly pronounced around the
appressoria of Aschersonia spp. and V. lecanii
(Butt, unpublished observations; Schreiter et al.
1994).
B. Host Recognition
Exactly how the pathogen recognises its host
remains unclear. In the case of plant pathogens it
is proposed that the pathogen releases a specific
elicitor mole eule that is detected by a membrane-
bound receptor in the host cell (Fig. 7.1). This
probably triggers the formation of a specific
114 T.M. Butt

ignaling between host uc1eus - Gene

and pathoge n regulation
• Fungus possesses lnduction of same
signal transducin g
machinery (e.g.
Fungal response to bost
cuticle
• Induction of cuticle-
degrading enzymes
(proteases, eh itinases )
• Mucous secretion
• Differentiation of
appressorium and/or
penetration peg

Host cutic1e - Primary barrier to infectioD

• Fungistalic/fungiloxic components - retard or prevent spore germination
ignal moleeules - influence induction of euLicle-degrading enzymes and
differentiation of infection structures
• Prefomled urrients - timulale genninalion and growtb
• Injury 10 cuticle - activates host immnune system (e.g. melanisatioD of cuticle,
inducLion ofantirnierobial substaßees, migration ofhaemocytes 10 wound)

Fig.7.1. Hast-pathogen interactions

product that feeds back to the pathogen to elicit The relationship between some CDEs and viru-
formation of enzymes specific for attack of the lence is unclear. For example, Paris and Ferron
host cuticle. Such a system mayaiso be present in (1979) showed that lipase-negative isolates of
entomogenous fungi and is likely to involve spe- Beauveria brongniartii were avirulent. However,
cific virulence genes of the pathogen, resistance Da-Silva et al. (1989) found a better correlation
genes of the host, avirulence genes and suscep- between amylase activity and virulence than
tible genes. When the initial recognition signal is between lipase and virulence for M. anisopliae,
received by the pathogen it induces growth and no correlation at all between protease and vir-
and development, further release of virulence · ulence. Likewise, Jackson et al. (1985) reported
determinants leads to disease. Entomogenous that in V. lecanii the ability to degrade lipid
fungi possess a complex signaIling apparatus (G- and pro tein was not correlated with virulence or
proteins, receptors, kinases, secondary messen- avirulence because all isolates produced these
gers ) that plays a role in host recognition and enzymes. The role of chitinases also remains
induction of the appropriate cuticle-degrading unclear. EI-Sayed et al. (1989) showed that viru-
enzymes (Butt, Irving and Robson, unpublished lent and avirulent isolates of N rileyi had similar
results). The hydrolytic enzymes may release chitinolytic activity throughout their growth;
signal molecules and destroy fungistatic com- however, virulent isolates consistently had signifi-
pounds. The net result of such complex events cantly higher levels of activity at the time of
may influence fungal-insect compatibility and penetration. In contrast, Coudron et al. (1984)
the speed by which entomopathogens access the detected low levels of chitinase activity in
nutrient-rich haemolymph and kill their host. germlings of virulent strains of B. bassiana, M.
anisopliae and N rileyi.
Since proteins constitute a major component
C. Cuticle-Degrading Enzymes (CDEs) of the insect cuticle it follows that proteases must
play an important role in the penetration process.
All fungi use a combination of enzymes and Evidence for the particular importance of pro-
mechanical force to penetrate the host cuticle. A tease derives largely from studies of their produc-
wide range of enzymes are secreted including pro- tion in infected cuticles associated with cuticle
teases, esterases, lipases, and chitinases (Table 7.3). degradation, the effects of protease inhibitors on
 Use of Entomogenous Fungi for the Control of Insect Pests 115

Table 7.3. Extracellular enzymes of entomogenous fungi including cuticle-degrading enzymes

Enzyme Pathogen pH optimum pI MW (kDa) Reference( s)

Subtilisin Pr1 Serine Mal 8-9 Several 29-33 St. Leger et al. (1998);
protease isoforms Butt et al. (1998b)
Extracellular protease Bb 2 7.5-9.5 7.5 31.5 Urtz and Rice (2000)
BBP
Trypsin-like Pr2 serine Mal 8-9 4.4,4.9,5.2 27-30 St. Leger et al. (1994, 1996c);
protease Butt et al. (1998b)
Cysteine endoprotease Mal Not known 4.6 26.7 Cole et al. (1993)
Metalloproteinase Mal 7 7.3 42 St. Leger et al. (1994, 1997);
Af3 Markaryan et al. (1996)
Carboxypeptidase Mal 6.8 9.9 30 St. Leger (1994)
Esterase Mal 8 Not known Not known Segers et al. (1995)
Bb 2
Aminopeptidases Mal 7 4.5 45 St. Leger et al. (1993)
Prolyl- Mal 8 4 74 Segers et al. (1995);
dipeptidylpeptidase St. Leger (1993)
Lipase Bb 2 6 Not known Not known Varela and Morales (1996)
Chitinase Mal 5-5.5 Several 33-60 St. Leger et al. (1993, 1996a);
isoforms Bogo et al. (1998),
Kang et al. (1999)
Bb 2 5.4-8.6 45 Havukkala et al. (1993)

I Ma = Metarhizium anisopliae; 2 Bb = Beauveria bassiana; 3 Af = Aspergillus fumigatus.

pathogen behaviour, by the analysis of protease-
deficient mutants or transgenic strains over-
expressing the subtilisin Pd (e.g. St. Leger et al.
1996b; Butt et al. 1998b). More recently, studies
have included cloning, identification, and manipu-
lation of specific protease genes of M. anisopliae,
particularly those of Pr1, which is also produced
by many other entomogenous, hyphomycete fungi
(Joshi et al. 1995, 1997; Butt et al. 1998b). Studies
by Segers et al. (1995) suggest that immunologi-
cally related subtilisins are also important patho-
genicity determinants of nematophagous fungi
like Verticillium chlamydosporium. In addition to
subtilisins, a wide range of other proteases partic-
ipate in cuticle degradation (Table 7.3).
Many CDEs are induced by cuticular compo-
nents (Paters on et al. 1994; Butt et al. 1998b) and
it is known that several of these enzymes are
produced under nutrient-poor conditions and
repressed by excess nutrients (St. Leger et al.
1992). Ambient pR is also a major determinant
in the expression of CDEs by M. anisopliae with
each enzyme (Prla, Pr1b, Pr2, metalloproteases,
aspartyl proteases, aminopeptidase, and chiti-
nases ) being synthesized only at pRs at which they
function effectively (St. Leger et al. 1998). Several
subtilisin-coding genes are present, all of which
are homologous to those of a number of sapro-
phytic fungi (St. Leger et al. 1997). Butt et al.
(1998b) have reviewed the significance of the
inter- and intra-specific variation in subtilisins
while Bidochka et al. (1999), Segers et al. (1999)
and St. Leger et al. (1997) have discussed the
adaptation of proteases and carbohydrases of
saprophytic, phytopathogenic and entomogenous
fungi to the requirements of their ecological
niches.
Various factors in the cuticle may limit the
activity of fungal proteases including phenoloxi-
dases and protease inhibitors. Indeed, Samue1s
and Reynolds (2000) identified an ll-kD, heat
stable protease inhibitor in the moulting fluid of
pharate adult Manduca sexta that was highly spe-
cific for the M. anisopliae Prl.
D. Toxins
Entomogenous fungi secrete a wide array of
compounds with biological activity against other
organisms, mostly products of secondary metabo-
lism (Table 7.4). The metabolites serve different
116 T.M. Butt

Table 7.4. Selected metabolites of some important ento- al. 1999; Amiri-Besheli et al. 2000). Specialised
mogenous fungi species like M. album, which is restricted to
Pathogen Metabolite(s) hemipteran insects, produce very little destruxin,
while generalist species like M. anisopliae var.
Metarhizium anisopliae Destruxins (>27 types), anisopliae produce a wide range of destruxins
swainsinone, cytochalasin c (A-F) often in significant quantities. Amiri-
Beauveria bassiana Bassianin, beauvericin,
bassianolide, beauverolides, Besheli et al. (2000) suggest that destruxins
tenellin may be significant in determining host specificity
Beauveria brongniartii Oosporein and are possibly important pathogenicity deter-
Paecilomyces Beauvericin, beauverolides,
fumosoroseus pyridine-2,6-dicarboxylic
minants for some strains of Metarhizium.
acid Insects injected with low doses of destruxins
Verticillium lecanii Dipcolonic acid, exhibit tetanus within 3 min. and, at higher doses,
hydroxycarboxylic acid, tetanus paralysis is brief, or absent, and a flaccid
hyclosporin, vertilecanins,
bassianolide
paralysis occurs (Roberts 1981). Destruxins have
Tolypocladium spp. Cyclosporin, efrapeptins a spectrum of biological activities including dis-
(5 types) ruption of the calcium balance in cells (Dumas
Hirsutella thompsonii Hirsutellin A, hirsutellin B, et al. 1996) and inhibition of vacuolar ATPases
phomalactone
(Bandani et al. 2001). Some insects can remove
injected destruxins from circulation with the fat
body possessing a greater affinity for this cyclic
functions, depending on the ecological niche of peptide than any other tissue or organ (Vey et al.
the fungus. Some metabolites may be antibiotics 2001).
to protect the BCA against antagonistic microor- Destruxin E is probably the most toxic mole-
ganisms, others may prevent growth of sapro- cule to a wide range of insect species (Roberts
phytic microbes on the host after it is killed, and 1981; Proprawski et al. 1985; Fargues et al. 1986;
thus improve the survival of the BCA. Some Thomsen and Eilenberg 2000). However, insects
bioactive metabolites are important pathogenicity differ in their sensitivity to the different destruxin
determinants (Strasser et al. 2000b) while others molecules. For example, Galleria larvae are more
have antifeedantJrepellent properties that pre- sensitive to destruxins A and Ethan destruxin
sumably deter mycophagous organisms (e.g. Band D (Vey and Quiot 1989; Vey et al. 2001)
invertebrates which may feed on mycosed cadav- whereas housefly maggots are more sensitive to
ers). The fact that some entomogenous fungi over- destruxin Ethan destruxins A or B (Robert and
come their host before extensive invasion of Fargues 1986).
organs takes place suggests that toxins are respon- Efrapeptins are linear peptides isolated from
sible for host mortality. Tolypocladium species (Krasnoff et al. 1991). At
The most-studied species of entomogenous least five different congeners have been iden-
fungi are M. anisopliae and B. bassiana. Less tified (efrapeptins C-G); their production varies
studied, but equally important, species of between species and between strains of the same
commereial importance are M. jlavoviride, B. species (Bandani et al. 2000). Efrapeptins exhibit
brongniartii, P fumosoroseus, Aschersonia spp., insecticidal and miticidal activity against a wide
Tolypocladium spp., V. lecanii and Hirsutella range of arthropods including spider mites, potato
spp. These fungi secrete an array of secondary beetle, tobacco budworm and diamondback moth
metabolites, some of which are restricted to spe- (Matha et al. 1988; Krasnoff et al. 1991). The LD so
eific genera, while others are more widespread. values of the purified efrapeptin mixture for the
A major group of metabolites secreted by M. last instar larvae of G. mellonella and Manduca
anisopliae are cyclic peptides called destruxins. sexta in injection assays were approximately 30
These have a basic structural backbone that con- and 47 ng, respectively (Bandani and Butt 1999).
sists of five amino acids and a a-hydroxy acid Efrapeptins also have limited antimicrobial acitiv-
(Strasser et aL 2000b; Vey et al. 2001). Destruxin ity (Bandani et al. 2000).
congeners have also been isolated from Ascherso- Oosporein is a red-coloured dibenzoquinone
nia sp. (Krasnoff and Gibson 1996). Destruxin produced by a large number of soil fungi and
production varies between species as weIl as entomogenous fungi belonging to the genus Beau-
between strains of the same fungus (Kershaw et veria (Strasser et al. 2000a; Vey et al. 2001). The
 Use of Entomogenous Fungi for the Contral of Insect Pests
exact role of oosporein is unclear even though
it is found in insects infected with some strains
of Beauveria. The cytotoxicity properties of
oosporein have been reviewed by Strasser et al.
(2000a,b).
The hexade psi peptide, beauvericin, is pro-
duced by the entomopathogens Beauveria and
Paecilomyces spp. and the saprophyte Fusarium
spp. Beauveriein is structurally and functionally
similar to the membrane-damaging enniatin
antibiotics (Steinrauf 1985). It shows antibiotic
activity against several species of bacteria and
moderate insecticidal properties (Vey et al. 2001,
and references cited therein).
Beauveria species also produce bassianolide,
a cyclo-octadepsipeptide ionophore, and related
peptides called beauveriolides (Suzuki et al. 1977;
Namatame et al. 1999). Bassianolides, when fed
to silkworm larvae, are lethai at high doses but
induce atonical symptoms at low doses. Bassiano-
lides are also produced by V. lecanii (Kanaoka et
al. 1977).
Bassianin and tenellin are non-peptide toxins
produced by some strains of Beauveria that can
inhibit erythrocyte membrane ATPases (Jeffs and
Khachatourians 1997). There is very little infor-
mation on the effect of these toxins on target and
non-target organisms.
Hirsutellin A is an extracellular insecticidal
pro tein produced by the hyphomycete Hirsutella
thompsonii (Mazet 1992). This antigenic, ther-
mostable toxin does not appear to be glycosylated,
nor shows proteolytic activity. Mazet and Vey
(1995) have determined the amino acid composi-
tion and the N-terminal sequence of hirsutellin A
and have shown that the pure compound is highly
toxic to larvae of G. mellonella and Aedes aegypti.
Vertilecanins belong to the group of phenopi-
colinic acids produced by V. lecanii in solid state
fermentation systems. Very little is known ab out
these compounds except that they are supposed to
have insecticidal properties (Soman et al. 2001).
E. Colonisation of the Haemocoel
Once the fungus reaches the haemocoel, it grows
as thin-walled, single or multi-cell, hyphal bodies.
The spherical to ovoid budding cells are some-
times referred to as "blastospores". During the
colonisation phase the fungus must overcome the
insect's immune response (Gillespie et al. 2000).
Some of the fungal secondary metabolites have
117
been shown to interfere with the host's cellular
and humoral defences (Vilcinskas et al. 1997a,b;
Vey et al. 2001).
The insect's response to fungal infection may
involve a combination of humoral and/or cellular
(e.g. phagocytosis and/or encapsulation) defence
mechanisms (Butt et al. 1996; Bidochka et al. 1997;
Lamberty et al. 1999). Insects with few circulating
haemocytes are usually dependent upon humoral
defences which include phenoloxidases, lectins,
antimierobial peptides, reactive oxygen species
and protease inhibitors (Gillespie et al. 2000).
Ekengren and Hultmark (1999) showed that
cecropins were toxic to certain fungi; however,
astrain of B. bassiana was completely resistant
and caused 100% mortality of Drosophila
within 5 days of injection. Furthermore, minimum
inhibitory concentration (MIC) assays showed
that B. bassiana tolerated more Drosophila cer-
copin A than Hyalophora cecropin A (Ekengren
and Hultmark 1999). These studies suggest inter-
specific variation in the anti-fungal properties of
cecropins that, in turn, could influence the suscep-
tibility of insects to entomogenous fungi. In other
words, some insects may produce antimicrobial
peptides toxie to some fungal species (or fungal
strains) and not others. Conversely, some fungi
may have evolved strategies to overcome the toxic
effects of insect defence compounds.
Insect death may ultimately result from a
combination of toxicosis, invasion of vital organs
and depletion of nutrients. Indeed, fungi secrete
a wide range of enzymes in the host including
esterases, proteases and osidases (Butt, unpub-
lished observations; Joshi and St. Leger 1999).
Metarhizium anisopliae has been shown to secrete
acid phosphatases that degrade host sugar phos-
phates, thus providing phosphorus for fungal
growth at the expense of the insect (Xiu et al.
2001).
Soon after host death, and under favourable
environmental conditions, hyphae emerge from
the cadaver, usually through orifices and the inter-
segmental membrane. The hyphae differentiate
conidiophores and conidia. The latter are dis-
persed by the wind, however, other factors such as
arthropods and rain can playa role in dissemina-
tion. As mentioned previously, many hyphomyce-
tous fungi (e.g. Beauveria, Metarhizium, and
Paecilomyces) produce dry conidia possessing
hydrophobic properties due to cysteine-rich
proteins called hydrophobins within the rodlet
layer of the cell wall. In contrast, Aschersonia spp.
118 T.M. Butt
and V lecanii possess hydrophilic conidia often
immersed in mucilage. The hydrophobicity of the
conidial cell wall can influence the choice of for-
mulation and method of application against insect
pests. For example, hydrophilic conidia will readily
suspend in aqueous carriers, whereas hydrophobic
conidia are best suspended in oil carriers.
F. Attenuation of Virulence
Successful pathogenesis is influenced by the bio-
chemical and physiological processes during infec-
tion. The interaction between fungi and their
insect hosts is complex and dynamic and no single
trait or component of the invasion process is likely
to determine virulence. Two key attributes of
highly pathogenic (= hypervirulent) isolates are
their ability to kill in a relatively short period of
time (i.e. low LTso value) and causing high mor-
tality at relatively low doses (i.e. low LCso value).
It is important to select hypervirulent strains, with
low production costs, to ensure the end product is
commercially viable.
Most microorganisms will become attenuated
if maintained in artificial media for long periods of
time. Attenuation can be defined as areduction in
strength, density, force or virulence of a pathogen.
In the case of entomogenous fungi this can result
from a single subculture or repeated subculturing
on artificial media. Little is known about the
underlying mechanisms of attenuation. However,
certain attributes are clearly affected, such as
an increase in LTso and LCso values, changes in
phenotype, and sporulation. Attenuated isolates
readily produce sectors, and in the case of M.
anisapliae, the green-pigmented conidia usually
turn pale yellow or white (Butt, unpublished
observations). Attenuated cultures may lose
genetic material and ultimately become sterile
(Wang and Butt, unpublished observations).
The rate of attenuation c1early depends on
the isolate rather than species of pathogen. For
example, some isolates retained their virulence
even after repeated passage in vitra (e.g. Culici-
nomyces clavisporus, Sweeney 1981; B. bassiana,
Samsinakova and Kalalova 1983; B. bassiana,
Brownbridge et al. 2001; V lecanii, Hall 1980;
Paecilamyces farinosus, Hayden et al. 1992)
whereas others rapidly lost virulence after only a
few subcultures on artificial media (e.g. V lecanii,
Nagaich 1973; N rileyi, Morrow et al. 1989). Viru-
lence of attenuated strains could be regained by
passaging through an appropriate host (Wasti
and Hartmann 1975; Fargues and Robert 1983;
Perenerova 1994). Attenuation can be delayed
or stabilised by growth on nitrogen rich or
osmotic stress (high KCl content) media but
usually at the expense of spore yield (Shah and
Butt, unpublished observations).
III. Epizootics and Factors Inßuencing
Fungal Efficacy
A. Epizootics
Epizootics are the result of a complex interaction
among the host, the pathogen, and environment
over time (Fig. 7.2). Aspects of this "disease
tetrad" are discussed in more detail below. Assum-
ing c1imatic conditions are favourable, some of the
factors responsible for the induction of epizootics
in insect populations inc1ude fungal virulence,
spore density (because mortality is dose-related),
host density and behaviour (e.g. preening and
movement can influence spore acquisition), and
the host plant (allelochemicals can interfere with
the fungal pathogen). Host specificity is also an
important factor because strains with a wide host
range could potentially jump hosts thus increasing
their chances of dispersal with the concomitant
amplification of inoculum. Since the inoculum
threshold is not static and is influenced by all
aspects of the disease tetrad this contributes to
erratic suppression of pest populations. Consider-
able research is needed before we will be able to
adequately predict conditions under which fungal
epizootics are initiated and developed in pest pop-
ulations. Such an understanding is necessary if we
are to exploit the full potential of these pathogens
successfully in integrated pest management (IPM)
programmes (Inglis et al. 2001).
B. The Pathogen
Fungal pathogenicity is primarily dependent upon
host-pathogen compatibility. Virulent strains must
be compatible with the target host or they will fail
to cause infection. This compatibility is partly
dependent upon the ability of the pathogen to
recognise its host and to overcome the host's
defence barriers. The ability of a pathogen to
cause disease is also determined by the impact of
 Use of Entomogenous Fungi for the Contra! of Insect Pests 119

ENVlRONMENTALFACTORS
• Rainfall - can remove iooculum from plant surface
• Hurnidity - high RH essential ror spore gennination and conidiogenesis
• Temperature - affects germination .nd development rates - speed orkill
• uv - can inaetivateJlcili fungal spores
• 5oil- ean inOuence fuosaJ survivaI W1d distribution
• Biota - mycopabgollS orgWIisms. antagonists!competito". hOSl plant

~------------~.,
ARTHRQPOD PE T ~
o Proening will dislodge spores
o Basking in the SUD exposes conidia to Iwmful
UV radiation NGALPATHOG
o Ecdysis - many conidia shed witb old culicle ....~_ _. .__+l... • Pathogenieity detenninants- innuenee
o Cellular derences - encapsulation or fuogal 0'" ,';mlence (dose and speed ofkill) and
elements too large 1.0 be phagocytosed specifieity (host range)
o Humoral der.nees - W1timicrobial substances • Ecological fitness - dctennones shelr
and melanin inhibit fuogal development life/persistence in the fjeld or storage
• Nicbe - inocuJum may not reacb target ._g.
inside plant material or mJled up within leRf

HOST PLANT/CRQP
o Growth - leaf expansion dilutes inocuJum per unit area
o Epicuticular waxes - may trap inoculum
• Cbemistry - may produce fuogistaticlfungitoxie compounds
• Stomata and topograpby - couId influence spore survival and effieacy
•. 8- abaxial swface olfers more protection to spores againsl h.rmful
UV and a humid environment essential ror gennination and
conidiogcnesis

Fig.7.2. Disease "tetrad"

environment al especially c1imatic factors
(irradiation, temperature, precipitation, relative
humidity) - on host-pathogen interactions_
In the field, the spore density must be suffi-
ciently high to ensure that an insect will contact
sufficient infective propagules to exceed the
threshold to cause disease_ Two important con-
siderations in strain selection are virulence and
"ecological fitness". Virulent strains require less
inoculum to cause infection (low LD so value) and
kill their hosts quickly (low LTso value)_ Ecologi-
cally fit strains are those that persist well in
the field and can successfully infect a host under
sub-optimal conditions_ Bioassays are usually per-
formed und er conditions that favour fungal infec-
tion and are not necessarily indicative of field
environments. This may explain, in part, why field
trials often result in poor or inconsistent pest
control and emphasises the need to conduct bioas-
says using parameters that closely simulate field
conditions (Butt and Goettel 2000).
Better persistence in the environment also
improves the chances of a susceptible insect
coming into contact with sufficient levels of inocu-
lum to cause disease. Ecologically fit strains are
usually more tolerant of UV radiation, resist des-
iccation and microbial attack, and have sufficient
endogenous reserves to survive long periods in the
absence of a suitable host or other nutrient source.
The significance of ecological fitness becomes
clearer when the impact of environmental factors
on fungal survival is discussed (see below).
C. The Insect Host
A complex array of physiological, behavioural and
morphological factors inftuence the susceptibility
of insect pests to entomogenous fungi including:
insect density, developmental stage, diet, ecdysis,
basking, preening, and injury caused by mechani-
cal, chemical or macrobial agents. A detailed
review of the inftuence of host factors on disease
development is beyond the scope of this chapter.
This section focuses on selected factors that sig-
nificantly influence the efficacy of entomogenous
fungi in managing insect pests.
Stress has long been recognised as a factor
that may increase the susceptibility of insects
to entomopathogens (e.g. Steinhaus 1958; Vago
1963). Stress factors include crowding, nutrition,
chemical pesticides and the environment. Exactly
how these factors predispose insects to fungal
infection is poorly understood. Whereas some
120 T.M. Butt

stress factors will act synergistically with fungal importance is the horizontal transfer of conidia
BCAs others have little or no effect. For example, from live inoculated insects to healthy uninfected
exposure of larvae of Colorado potato beetle insects. Meadow et al. (2000) demonstrated that it
to acute or chronic sublethai doses of the Bt was possible to transmit fatal doses of B. bassiana
CrylIIA delta-endotoxin does not significantly conidia from adult (Delia radicum) fly-to-fly for
increase their susceptibility to B. bassiana (Costa aseries of at least six flies. Furthermore, when
et al. 2001). In contrast, some chemical pesticides female flies were exposed to the fungal BCA, then
enhance the efficacy of M. anisopliae by weaken- transferred to small cages containing males and
ing the host immune system (Hiromori and an oviposition substrate, no eggs were laid. Butt
Nishigaki 2001). Interestingly, starved Plutella and Ibrahim (unpublished observations) ob-
xylostella larvae were more susceptible than fed tained significant control of adult pollen beetles
larvae to Paecilomyces fumosoroseus isolate 1576 by exposing them to individuals inoculated with
but starved and fed larvae were similar in suscep- M. anisopliae. Several laboratories are currently
tibility to P. fumosoroseus isolate 4461 (Altre and developing systems whereby insect pests are
Vandenberg 2001). These observations illustrate attracted to a baited trap or trap crop where they
the complexity of host-pathogen interactions. A become inoculated with the fungus and then
better understanding of stress-induced host sus- spread it to a field population (see Sect. IV).
ceptibility could improve the efficacy of fungal Inseet behaviour (foraging, grooming) can
BCAs in pest control programmes. influence the development of epizootics and the
Insect nutrition is an important but much dispersal of fungal BCAs. For example, conidia
neglected factor regulating the susceptibility of of M. anisopliae are spread among individual
insects to entomopathogens and subsequent epi- termites by grooming (Kramm et al. 1982). The
zootics. The host plant not only influences nutri- termite, Reticulitermes jiavipes, resists infection
tion, but also persistence of inoculum and efficacy. due to a complex social behaviour including the
These parameters are dealt with in more detail in removal of infected individuals from the colony
Section III.D.6. (Boucias et al. 1996). Secretion of fungistatic com-
The development stage of an inseet is another pounds, often combined with complex social inter-
host faetor shown to play an important role in the actions, influence the susceptibility of termites
success of entomogenous fungi. Early instar larvae (e.g. Nasutitermes spp., Coptotermes formosanus)
or nymphs are usually more susceptible than older to fungal infection (Rosengaus et al. 2000; Wright
stages. For example, young larvae of the European et al. 2000). Foraging coccinellids will transfer
corn borer (Ostrinia nubilalis) were more suscep- conidia of P. fumosoroseus from sporulating
tible to B. bassiana than older larvae (Feng et al. cadavers to healthy aphids inducing significant
1985). Similarly, first and second instar larvae of mortalities in the aphid population (Pell and
mustard beetles and vine weevil were more sus- Vandenberg 2002).
ceptible to M. anisopliae than older larvae (Butt,
unpublished observations). In contrast, tsetse flies
(Glossina morsitans) <1 day old were more resis- D. The Environment
tant to infection by M. anisopliae than 20- or
40-day-old insects (Maniania and Odulaja 1998). Some of the salient climatic parameters influenc-
Furthermore, the female tsetse flies were generally ing the success of entomogenous fungi against
more susceptible than male flies. Insect develop- insects include solar radiation, temperature,
mental rates cannot be considered independent relative humidity, precipitation, and wind. These
of environment al factors (e.g. temperature ). High parameters interact with each other and
temperatures accelerate insect development and other environmental variables (e.g. soil and crop)
will reduce the time between moults that can sub- to impact on entomopathogens and should be
sequently reduce the prevalence of infection due addressed interactively (Inglis et al. 2001).
to loss of inoculum on exuviae (Vestergaard et al.
1995).
1. Solar Radiation
Insect density has a significant influence on
epizootics in insect populations. As the density of One of the most important parameters affecting
insects increases, there is a higher prob ability of an propagule persistence in epigeal habitats is inaeti-
insect coming into contact with the fungal BCA vation by solar radiation (Moore et al. 1993;
directly or with infected individuals. Of particular Fargues et al. 1996; Smits et al. 1996a,b). Fungal
 Use of Entomogenous Fungi for the Contra! of Insect Pests
propagules are highly susceptible to damage by
UVB (280-320nm) and to a lesser extent UVA
(320-400nm). Significant differences in suscepti-
bility to irradiation among genera and strains
within species have been observed. For example,
conidia of M. jlavoviride are most resistant to arti-
fici al sunlight followed by conidia of B. bassiana,
M. anisopliae, and P. fumosoroseus (Fargues et al.
1996). Braga et al. (200Ia) showed that 4 hr expo-
sures of conidia of M. anisopliae strains ARSEF
324, 23 and 2575 to solar UVA reduced their cul-
turability by 10, 40 and 60%, respectively. Fur-
thermore, an increase in UV irradiance from 920
to 1200mWm-2 significantly decreased the cultur-
ability of strains 2575 and 23 by 36% and 48%,
respectively (Braga et al. 2001b). Unprotected
conidia of B. bassiana are alm ost completely
inactivated by exposure to 1 h of direct sunlight or
20s of UVB (Edgington et al. 2000).
UV radiation is also harmful to fungal BCAs
used on protected crops in polythene tunnels.
Costa et al. (2001) conducted a field study to
assess the effect of various commercial polyethyl-
ene plastic greenhouse coverings on the persis-
tence of viable spores of B. bassiana. They showed
that the persistence of viable spores was signifi-
cantly longer under the plastic that blocked a
greater portion of the UV spectrum «380nm)
than the plastics that only blocked UV wave-
lengths below 360nm. One week after application,
the percentage spore germination was at least
twice as high under the <380nm blocking plastic
compared to <360 nm blocking plastics (Costa et
al. 2001).
Solar and UV blockers are often incorporated
into formulations to protect conidia from harmful
radiation (Burges 1998). Although several com-
pounds have demonstrated excellent protection of
conidia in laboratory-based environment studies
very few have been evaluated in the field. For
example, several oils or oil-soluble sunscreens sig-
nificantly increased the survival of B. bassiana, M.
anisopliae and M. anisopliae var. acridum conidia
exposed to artificial radiation (Moore et al. 1993;
Inglis et al. 1995a; Alves et al. 1998), but they did
not enhance survival in field settings (Inglis et al.
1995a). In contrast, a solar blocker (clay) and
a UV-absorbing optical brightener (Tinopal)
did increase the field persistence of B. bassiana
conidia on grass leaves exposed to sunlight (Inglis
et al. 1995a).
The microhabitat in which fungal BCAs are
deployed will also inftuence the persistence and
efficacy of fungal inoculum. Some habitats, such as
121
the underside of leaves and subterranean habitats,
offer better protection against harmful radiation.
Many major pests feed or exist for periods of time
in these habitats, however, effective delivery of
inoculum to these sites is often difficult or requires
specialised equipment (Sopp et al. 1990; Wraight
and Carruthers 1999).
2. Temperature
Temperature has a considerable infiuence on the
efficacy of entomogenous fungi (Inglis et al. 2001).
In laboratory based bioassays, entomogenous
fungi are infectious over a wide range of temper-
atures (1O-30°C), the optimum temperature is
between 20 and 25 oe. Above 30°C, vegetative
growth of most genera is inhibited and growth
usually ceases at 37 oe. The inability of an ento-
mogenous fungus to grow at mammalian bodyl
beehive temperature is an important considera-
tion in risk assessment and registration of fungal
BCAs. However, considerable variability exists
among genotypes in their thermal characteristics
(Fargues et al. 1997b) with little or no correlation
between the geographie origin of strains and their
thermal properties (McCammon and Rath 1994;
Fargues et al. 1997a; Ouedraogo et al. 1997; Vidal
et al. 1997). Furthermore, there is little informa-
tion on the physiological adaptations of strains to
extreme temperatures such as the production of
heat shock or cold-induced proteins (De Croos
and Bidochka 2001).
Temperatures which favour spore germina-
tion may not be optimal for growth. For example,
James et al. (1998) observed that temperature had
a significant effect on both germination rate and
vegetative growth of B. bassiana, with the fastest
germination occurring at 25-32°C and the fastest
growth occurring at 30 oe. Laboratory studies
have shown that fiuctuating temperatures (min.
10°C, max. 23°C), similar to those encountered in
the field, will interfere with M. anisopliae infec-
tions of mustard and ftea beetles (Butt, unpub-
lished observations). Low night temperatures may
reduce the percentage germination suggesting
that the timing of application of inoculum may be
crucial to the successful deployment of fungal
BCAs.
3. Relative Humidity
Water is critical for entomogenous fungi at all
stages of their life cyde (e.g. spore germination
and conidiogenesis at the surface of mycosed
122 T.M. Butt
cadavers), consequently, the ambient relative
humidity can have a profound influence on the
efficacy of entomogenous fungi including recy-
cling and transmission within pest populations
(Milner and Lutton 1986; Helyer et al. 1992;
Fargues and Luz, 2000). For most fungi, humidities
>96% are essential for spore germination and
mycelial growth (e.g. Ibrahim et al. 1999). Fargues
and Luz (2000) observed that a relative humidity
of at least 97% was required for production of B.
bassiana conidia on mummified cadavers of Rhod-
nius prolixus. Sporulation of M. anisopliae var.
acridum in mycosed cadavers of Schistocerca gre-
garia is a dynamic process dependent on the water
content of the locust (Arthurs and Thomas 2001).
Some strains of B. bassiana and M. jlavoviride will
form conidia within the haemocoel of cadavers
under conditions of low humidity (Fernandes et al.
1989; Prior and Greathead 1989).
Some studies show that dry conditions during
or immediately following application of fungal
propagules are less detrimental than previously
thought. For example, B. bassiana and M. aniso-
pliae var. acridum are able to infect their respec-
tive hosts under conditions of low ambient
humidity, presumably due to sufficient moisture
within the microhabitats (e.g. Ramoska 1984;
Marcandier and Khachatourians 1987; Fargues
et al. 1997b; Ferron 1997). Humidity may be high
in microhabitats such as the boundary layer sur-
rounding leaves and the intersegmental folds of
the insect integument.
Oil-based formulations appear to be superior
than water-based formulations for the control of
arthropod pests (Burges 1998; Prior et al. 1988).
Oils presumably reduce the dependency on satu-
rated conditions normallY required for successful
infections (Bateman et al. 1993). Since they
readily flow over insect surfaces they probably
carry spores to sites that are conducive for germi-
nation and infection such as the intersegmental
membranes (Ibrahim et al. 1999). Oils may have
many other useful properties such as releasing
stimulatory compounds in the host cuticle that
accelerate germination (Ibrahim et al. 1999).
4. Rainfall
Besides increasing humidity, rainfall can dislodge
and disperse conidia from plant and insect
surfaces within a relatively short period of time
(Inglis et al. 2000; Inyang et al. 2000). Significant
amounts of conidia of B. bassiana and M. aniso-
pliae can be removed from plant surfaces (Inglis
et al. 1995b, 2000; Inyang et al. 2000). New for-
mulation and application strategies ensure better
retention of conidia on leaf surfaces especially
the underside of leaves where most foliar pests
concentrate. Indeed, oil carriers appear to offer
greater rainfastness than aqueous carriers (Inglis
et al. 2000; Wraight and Ramos 2002). Inglis et al.
(2000) noted that conidia in direct contact with the
cuticle of leaves and beetle larvae were less prone
to be removed by rain than conidia in aggregates
that made little contact with the cuticle.
5. Edaphic Factors
Entomogenous, hyphomycete fungi are readily
recovered from soils from disparate habitats
including agricultural and forest systems (Barker
and Barker 1998; Bidochka et al. 1998). Usually,
far more fungi are recovered from habitats with
the greatest insect biodiversity such as woodlands
and hedgerows than disturbed, cultivated arable
fields (Chandler et al. 1997; Vanninen 1996).
However, the soil is an extremely complex envi-
ronment with several interacting factors influenc-
ing the persistence and/or efficacy of fungal BCAs
(e.g. Studdert and Kaya 1990a,b; Studdert et al.
1990). These factors include soil type (i.e. texture,
cation-exchange capacity, organic matter content,
pH, water-holding capacity etc.) and soil biota (i.e.
organisms that inhibit or feed on conidia). In addi-
tion, various formulation (e.g. conidia powder,
blastospores, lyophilised mycelium, inoculated
kerneis) and application (e.g. drilling, spraying,
injection) strategies can influence the success of
entomogenous fungi in the soil. On the whole,
conidia persist weH in soils in temperate climates
whereas the thin-walled blastospores are compar-
atively short-lived (Gaugier et al. 1989; Storey et
al. 1989; Inglis et al. 1997; Keller et al. 2000).
Soil texture appears to influence the distribu-
tion of the major entomopathogenic species
including M. anisopZiae, B. bassiana, P. farinosus,
P. fumoso roseus, V. Zecanii, and ToZypocladium
spp. (Vanninen 1996; Chandler et al. 1997;
Bidochka et al. 1998). It also appears to influence
the efficacy of these BCAs. For example, Lezama-
Gutierrez et al. (2000) observed that when M.
anisopliae conidia were applied to the soil surface,
it reduced the emergence of adult Mexican fruit
flies (Anastrepha Zudens) by 22 and 43% in sandy
loam and loam soils, respectively, compared with
the controls.
 Use of Entomogenous Fungi for the Control of Insect Pests
Soil moisture can influence the persistence
and efficacy of entomogenous fungi. Most soils are
kept at field capacity to prevent crops from
wilting. These conditions can either provide suffi-
cient moisture for host infection and disease
development or stimulate limited growth and
sporulation (Fargues and Robert 1985; Li and
Roldom 1993). Very little is known ab out the
effects of extreme soil wetting and drying on the
survival of fungal inoculum.
Rainfall, soil texture and organic matter
appear to be the most important factors deter-
mining vertical movement of fungal propagules
in water with soils with a high clay and organic
matter content retaining the most spores (Ignoffo
et al. 1977; Storey and Gardner 1988). Exactly why
these soils retain more spores is unclear but may
be related to their high cation-exchange capacity
and reduced pore sizes.
Soil pR appears to have little or no influence
on the distribution or efficacy of entomogenous
fungi (e.g. Rath et al. 1995). According to
Bidochka et al. (1998), the occurrence of M.
anisopliae and B. bassiana is not related to soil
type or pR. Butt and Shah (unpublished observa-
tions) found that M. anisopliae was highly effica-
cious for the control of vine weevil larvae in
composts and soils ranging in pR between 3.8 and
7. In contrast, Groden and Lockwood (1991)
reported that fungistasis against B. bassiana was
correlated with pR but not with other soil charac-
teristics such as texture and organic matter
content. Rowever, soil pR and nitrogen fertilisers
do impact on the germination of B. bassiana
conidia, but not on infection of the Colorado
potato beetle (Groden and Dunn 1996).
Both micro- and macroorganisms influence
the survival and distribution of entomogenous
fungi. Soil microbes will secrete antibiotics that
may inhibit the germination and development of
fungal BCAs. According to Lingg and Donaldson
(1981), fungistasis in non-sterile soils may have
been due to the prevalence of the soil fungus,
Penicillum urticae, which produces a water-soluble
inhibitor of B. bassiana in vitro. Elimination or
reduction of antagonistic soil biota by soil sterili-
sation or amendment with commercial peat and
peat-free composts considerably increases the
efficacy of fungal BCAs in the control of subter-
ranean pests (Butt and Shah, unpublished
observations). Other organisms that could have
a profound effect on the survival of fungal
BCAs include soil amoebae and collembola. For
123
example, the collembolan, Folsomia candida, will
consume and inactivate the insect pathogens B.
bassiana, M. anisopliae, Hirsutella spp., and V.
lecanii without suffering mortality, reproductive
disturbance or any other harmful effects (Broza
et al. 2001).
6. Rost Plant
Many plant traits will influence directly and indi-
rectly the survival of entomogenous fungi and
their efficacy in pest control programmes (Elliot
et al. 2000). Of particular importance are plant
growth, morphology and chemistry. Plants
produce a wide range of chemicals which, depend-
ing on their nature and concentration, can influ-
ence conidial vi ability and the susceptibility of
the arthropod pest to fungal BCAs (Rare and
Andreadis 1983; Ramoska and Todd 1985; Costa
and GaugIer 1989; Vega et al. 1997; Inyang et al.
1998, 1999a,b; Meekes et al. 2000). For example,
third instar nymphs of Trialeurodes vaporariorum
are highly susceptible to B. bassiana and P.
fumosoroseus on cucumber, but were less suscep-
tible to infection when reared on tomato plants
possibly due to the antimicrobial properties of
tomatine present in tomato leaves (Poprawski et
al. 2000). Similarly, Poprawski and lones (2001)
observed that Bemisia argentifolii reared on
cotton were less susceptible to infection by either
B. bassiana or P. fumosoroseus than when reared
on melon. They hypothesized that the cotton
plant produced a fungal inhibitor, gossypol,
that may have protected the whitefly. Germi-
nation of conidia of both fungi was strongly
inhibited «12% germination) on the cuticle of
nymphs reared on cotton but was >95 % on
the cuticle of nymphs reared on melon suggesting
that the inhibitor was sequestered by the insect
cuticle.
Cruciferous plants produce fungistatic isoth-
iocyanates such as phenylethyl, 2-chlorophenyl, 3-
butenyl and allyl isothiocyanates which can inhibit
germination of M. anisopliae conidia in vitro and
reduce its ability to infect insects (Inyang et al.
1999b). Tallamy et al. (1998) have shown that eggs
and larvae of the spotted cucumber beetle, Dia-
brotica undecimpunctata howardi, could sequester
cucurbitacin glycosides and, in turn, were less sus-
ceptible to infection by M. anisopliae. Rowever,
more re cent studies show that inhibition of fungal
growth attributed to cucurbitacin E glycoside may
actually be due to bacteria associated with CUCUI-
124
bits (Martin and Schroder 2001). This shows that
interactions with phylloplane microorganisms
cannot be ignored.
Plant morphology (e.g. leaf size, waxiness,
number of stomata, the veins, density of hairs on
the leaves and leaf shape) may directly influence
the pest species but more importantly it may affect
the micraclimate at the leaf surface which influ-
ences spore persistence (Inglis et al. 1993). For
example, conidia of A. aleyrodis were found to
retain infectivity for whitefly much better on
cucumber than poinsettias (Meekes et al. 2000).
Inyang et al. (1999a) observed that germination of
M. anisopliae conidia was more rapid and greater
on the surfaces of dewaxed crucifer plants than
intact leaves suggesting that epicuticular waxes
contain fungistatic compounds and/or act as a
barrier to the leaching of nutrients. Surface
leachates and soluble extracts of leaves increased
percentage germination of conidia and the viru-
lence of M. anisopliae to the mustard beetle,
Phaedon cochleariae. Germination increased on
insect cuticle that was pretreated with leaf
extracts/leachates, suggesting that the insect
cuticle will adsorb or sequester nutrients.
IV. Increasing the Efficacy
of Mycoinsecticides
Impraved production, formulation and applica-
tion strategies will increase the efficacy of fungal
BCAs. Introduction of quality control measures
will ensure that only viable, virulent inoculum is
applied in the field. For example, molecular
markers have been developed to detect attenu-
ated mutants and exclude these from the praduc-
ATTRACT ("P LL IN")
PE T INTO 01 C RD
TRlPrrRAP ROP MAI CROP
I G:
arietie favoured by I)csts Variety resistant to/less favoured
by in ecl pest
Pheromones
...
Gustatory limulanls
Treat witb aDtifeedaotJrepellents
Plant volatiles
INlJ ' nATE STRIP WIT .. FlJNGAL BCA.
T.M. Butt
tion system (Wang and Butt, unpublished data).
Effective targeting of fungal BCAs is needed for
efficacious control of pests because insect mortal-
ity is dose-related. Application strategies need to
be devised to ensure that sufficient amounts of the
pathogen contact the target. This section focuses
on some innovative strategies to increase the effi-
cacy of fungi for pest control. For example, recent
studies by Butt et al. (1998) have shown that
honeybees are more efficient than conventional
sprayers in delivering fungal inoculum to pest-
infested flowers. Trials with the insect pathogen,
M. anisopliae, increased pollen beetle (Meligethes
spp.) contral in oilseed rape and showed con-
siderable promise for the contral of other floral
pests such as the seed weevil (Ceutorhynchus
assimilis) and thrips (Butt, unpublished observa-
tions). Honeybees escape infection because of the
elevated temperatures in the beehive (Butt et al.
1994).
The "push-pull" strategy is another novel pest
contral method (Pickett et al. 1995, 1991). This
strategy entails insect pests being driven out of the
main crap through the use of feeding and ovipo-
sition deterrents and at the same time the pests
being drawn into an adjacent sacrificial or "trap"
crops where they can be controlled by inundation
with fungal BCAs (Fig. 7.3). To encourage pests
into the trap crop, lures such as favoured plant
varieties (i.e. those more attractive than the main
crap), and semiochemicals may be used. The latter
may include plant-derived attractants (e.g.
isothiocyanates attract certain crucifer pests) or
insect-derived attractants (e.g. aggregation
pheromones). The high density of pests in the trap
crop would attract predators and parasitoids
whose activities would augment those of the
fungal BCAs.
DI COURAGE
OR "PUSH"
PESTSO TOF
MAll ROP
Fig.7.3. The "push-pull" pest
contral strategy
 Use of Entomogenous Fungi for the Contral of Insect Pests
Semiochemicals and Conventional Pesticides.
Chemicals that alter insect behaviour can improve
the secondary "pick-up" of fungal inoculum from
treated leaves and subsequently improve pest
control. For example, when the entomogenous
fungus V. lecanii was used in conjunction with the
aphid alarm pheromone, (E)-ß-farnesene, ac-
quisition of conidia was enhanced as a result of
increased aphid movement, leading to high er
aphid mortality (Rockland et al. 1986). Other
chemicals (repellents and antifeedants) can also
increase insect movement and subsequently
increase the insect's acquisition of fungal inocu-
lum (Roditakis et al. 2000). Since mortality is
dose-related, the more spores acquired the
quicker the insects will be killed. Some insects
naturally feed on the undersides of leaves (e.g.
whitefiies), but it may be necessary to stimulate
other pest insects to move to the underside of
leaves with the use of repellents or antifeedants
(Amiri et al. 1999).
The enhanced efficacy of B. bassiana and M.
anisopliae applied in combination with the neuro-
toxin imidac10prid against the sugar-cane root-
stock borer weevil (Diaprepes abbreviatus) was
attributed to reduced mechanical removal of
conidia from the surfaces of larval cuticles
(Quintela and McCoy, 1998). Synergistic interac-
tions between conventional pesticides and fungal
BCAs have also been reported in the control of
several pests inc1uding termites (Boucias et al.
1996; Ramakrishnan et al. 1999), Colorado potato
beetle (Furlong and Groden 2001) and cock-
roaches (Zurek et al. 2002).
Host Plant Resistance and Trap Crops. As
discussed earlier, the host plant can infiuence the
susceptibility of insects to fungal infection.
Differences in insect susceptibility have been
observed depending on the type of host plant on
which insects have been reared (Rare and
Andreadis 1983; Ramoska and Todd 1985). Plant
breeding programmes have tried to confer traits
for insect resistance into many agricultural crops.
Insecticidal toxins have been genetically engi-
neered into several commercial crops (Jenkins
1999). These factors may stress insects making
them more susceptible to fungal infection.
Trap crops are usually grown in a narrow strip
to attract insect pests and thereby protect the main
crop from pest attack. The trap crop is also
referred to as a "discard" strip because, once the
125
insects are attracted to the trap crop, the strip is
inundated with a BCA or treated with a chemical
pesticide and ultimately destroyed. This decreases
or eliminates the need to treat the entire field.
Some of the desired attributes of trap crops are:
(1) they fiower before the main crop so concen-
trating fiowerlseed pests for treatment, (2) they
contain volatiles which draw pests away from the
main crop, and (3) when ingested they make the
pests more susceptible to fungal BCAs. It mayaiso
be possible to modify environment al conditions
within the trap crop to encourage epizootics in
pest populations. For example, forcing adults and
larvae of P. cochleariae to the underside of M.
anisopliae treated Chinese cabbage leaves with a
feeding deterrent increased the level of control
(Amiri et al. 1999). This may be due to higher
humidity levels at the underside of leaves which
favour fungal infection. Conidia on the underside
of leaves are less likely to be harmed by UV radi-
ation or dislodged by rain, thus improving the
chances of infecting susceptible insects (Inglis et
al. 1993; Inyang et al. 2000).
v. Potential Hazards
and Safety Concerns
One of the major hurdles in the registration, and
sub se quent commercialisation, of fungal BCAs is
risk assessment. Risk is defined as the magnitude
and likelihood of an adverse effect. This requires
estimation of the hazard (i.e. wh at harm will the
BCA cause) and the exposure (what population
will be exposed to the BCA, at what concentra-
tion, and fm how long). For fungal BCAs intended
for field release, human health and environmental
concerns require primary consideration. Risk
assessment studies are usually costly and contro-
versial and may have deterred many entrepre-
neurs from investing in the development of fungal
BCAs. Safety of fungal BCAs must be considered
at many levels. The primary concern is that the
agents and their residues (surface antigens and
metabolites) must not harm humans and other
non-target organisms. Potential safety issues
inc1ude: (1) competitive displacement of non-
target organisms, (2) allergenicity, (3) toxigenicity
to non-target organisms, (4) pathogenicity to non-
target organisms and (5) depletion of the target
host itself (Cook et al. 1996; Goettel et al. 2001).
126 T.M. Butt
A. Allergenicity
The prineipal fungal allergenie mOletles in the
environment are eonidia, these oeeur widely and
often in far greater eoneentrations than pollen
grains. Immunoglobulin E-speeifie antigens (aller-
gens) on airborne fungal spores induee type I
hypersensitivity (allergie) respiratory reaetions in
sensitized atopie subjeets, eausing rhinitis and/or
asthma (Horner et al. 1995). Little or no eonidia
of fungal BCAs are deteeted in volumetrie aero-
biological surveys (e.g. Beaumont et al. 1985).
Fungal BCAs are not among the species that are
responsible for produetion of eommon allergens
(Latge and Paris 1991). There are very few reports
of allergie reactions to entomogenous fungi
(Austwiek 1980; Ward et al. 1998). There is,
however, aneedotal evidenee of individuals devel-
oping mild allergies when exposed to high doses
of eonidia during mass produetion over long
periods of time. Beauveria bassiana is reported to
trigger moderate to severe allergie reaetions (Saik
et al. 1990). Metarhizium anisopliae has also been
shown to eause allergie immune and inflammatory
responses in mice (Ward et al. 1998, 2000).
Conidia and myeelia of the same fungi ean differ
signifieantly in allergen produetion and, eonse-
quently, allergenie responses. Whereas different
fungi do produee antigens with unique epitopes,
many fungal antigens from different species have
shared or eross-reaetive epitopes (Horner et al.
1995). Sinee all fungi are potentially a1lergenic,
it is neeessary to avoid exposing unproteeted
humans during produetion and applieation
(Goettel et al. 2001).
B. Toxicity
Fungi seerete a wide array of eompounds with bio-
logical aetivity against other organisms, mostly
produets of seeondary metabolism (Table 7.4).
Fungal metabolites serve different funetions,
depending on the eeologieal niehe of the fungus
(see Seet. ILD). Some metabolites may be antibi-
oties to protect the BCA against antagonistie
microorganisms, or may prevent growth of sapro-
phytie mierobes on the host after it is killed,
and thus improve the survival of the BCA.
Some bioaetive metabolites are also important
pathogenicity determinants and others have
antifeedant/repellent properties that presumably
deter myeophagous organisms. But the quantities
normally deteeted in infeeted inseets are usually
too small to aeeount for the above properties.
Vey et al. (2001) and Goettel et al. (2001) have
reviewed the properties and risks of major
metabolites öf fungal BCAs.
C. Pathogenicity
Entomogenous fungi are usually restrieted to
arthropod hosts, however, on extremely rare oeea-
sions they may infeet mammals and other
vertebrates. Representatives of the genera
Paecilomyces, Verticillium, Metarhizium and
Beauveria have been isolated from humans (Kisla
et al. 2000; Amici et al. 1994; Blaekwell et al. 2000;
Jani et al. 2001) and reptil es sueh as alligators and
tortoises (Fromtling et al. 1979; Gonzalez Cabo
et al. 1995). The pathogens are usually oppor-
tunists infeeting weak or immunosuppressed
patients (Blaekwell et al. 2000; Burgner et al. 1998;
Revankar et al. 1999). Certain strains of B.
bassiana, under laboratory eonditions, are patho-
genie to embryos of the inland silverside fish,
Menida beryllina, and the grass shrimp Palaemon-
etes pugio (Genthner and Middaugh 1992;
Genthner et al. 1997). Conidia of M. anisopliae
ean also adversely affeet embryos and the newly
hatehed larvae of the inland silverside fish
(Genthner and Middaugh 1995).
D. Depletion of Hosts
Although the goal of any biological eontrol pro-
gramme is to lower the pest population this re-
duetion may in turn detrimentally affeet other
non-target organisms that are dependent on this
pest. The damage to the non-target population will
depend on the extent and speed of the depletion
of the host (Goettel et al. 2001). Very little is
known about the repereussions of depleted hosts
on interaeting organisms in the food ehain.
E. Competitive Displaeement
Fungal BCAs have the potential to eompetitively
oceupy a niehe, thereby adversely affeeting one or
more native organisms within that niehe. In some
instanees this eompetition eould detrimentally
affeet a eomponent of the eeosystem or under-
mine IPM programmes. For example, fungal
 Use of Entomogenous Fungi for the Control of Insect Pests
pathogens and inseet parasitoids may eompete for
the same inseet host (Goettel et al. 1990, 2001).
Parasitoids may weaken the host immune system
making it more suseeptible to fungal infeetion
(Marris et al. 1999). Fungal BCAs usually work in
eoneert with arthropod predators and parasitoids
in suppressing pest populations rather than eom-
petitively displaeing them. But often these inter-
actions are extremely eomplex with predators and
parasitoids often avoiding fungal-infeeted insects
(Mesquita and Lacey 2001; Pell and Vandenberg
2002). Some parasitoids appear to produee anti-
fungal compounds that prevent colonisation of
a host insect by invading fungi (Jones and
Poprawski 1996). Studies by Pell and Vandenberg
(2002) show that the coceinellid, Hippodamia con-
vergens, prefers healthy Russian wheat aphids
(Diuraphis noxia) than those infected with P.
fumosoroseus. Furthermore, it will not consume
aphid eadavers from which the fungus was erupt-
ing or sporulating. However, predators eonta-
minated with fungal conidia (from spraying or
exposure to sporulating insect ca da vers ) will
readily transfer inoculum to healthy insects
contacting them (Pell and Vandenberg 2002).
Too little work has been done to evaluate the
impact of introduced exotic strains on natural
populations of entomogenous fungi. The develop-
ment of molecular markers has been extremely
useful to monitor parasexual recombination and
mutation in fungi (Leal-Bertioli et al. 2000). How-
ever, further studies are needed to establish if
exotie strains displaee indigenous strains, and if
the recombinant strains have altered in virulence
and host specifieity.
VI. Conclusions
Fungal BCAs will not be a eure-all for pest prob-
lems in horticultural, agricultural and forest
systems but they are likely to form a key eompo-
nent of IPM programmes in the future. This is
partly because researehers have identified some of
the major constraints (e.g. agronomie, technical,
industrial) limiting the development and use of
fungal BCAs (Butt and Copping 2000), and novo
strategies have been devised to inerease the effi-
eaey of these agents (Butt and Brownbridge 2001).
The development of a more effieacious pro-
duct will not ensure greater use unless combined
with a concerted effort to educate the end user.
127
Growers must be provided with effective, "easy-
to-use" strategies to ensure that fungi are applied
at the right time, in the right pi ace using the most
appropriate applieation techniques. Furthermore,
the fungal BCAs must be eompatible with other
crop protection strategies (e.g. entomogenous
fungi must be eompatible with bio or ehemical
pesticides used for plant disease eontrol). Part-
nerships between growers and extension person-
nel will be vital to demonstrate the viability of
fungal-based control strategies and increase their
uptake (Butt and Brownbridge 2001).
Acknowledgements. TMB thanks the EU for
partial fun ding of his research (grants FAIR6
CT98-4105 and QLRT-2000-01391) and Dr.
Michael Brownbridge, University ofVermont, and
Dr. Mark Goettel, Lethbridge Research Centre,
for kindly refereeing the manuscript.
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8 Biological Control of Weeds
HARRY C. EVANS
CONTENTS
I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . .
II. Classical or Inoculative Strategy . . . . . . . . . . .
A. The Principles .........................
1. Background. . . . . . . . . . . . . . . . . . . . . . . . .
2. Concept . . . . . . . . . . . . . . . . . . . . . . . . . . . .
B. The Mycota Involved ...................
C. Case Studies ..........................
1. Past Examples . . . . . . . . . . . . . . . . . . . . . . .
2. Current Examples . . . . . . . . . . . . . . . . . . . .
3. Future Targets. . . . . . . . . . . . . . . . . . . . . . .
III. Mycoherbicide or Inundative Strategy ......
A. From Conception to Inception ............
B. Current Status. . . . . . . . . . . . . . . . . . . . . . . . .
C. Potential Application . . . . . . . . . . . . . . . . . . . .
IV. General Discussion and Conclusions .......
References . . . . . . . . . . . . . . . . . . . . . . . . . . . .
I. Introduction
As technology advances and agricultural practices
are ever changing, weeds are having an increasing
rather than a decreasing impact on man's affairs.
It has been estimated that crop losses due to
weeds vary from 10-20%, which, in the USA
alone, would amount to over US $15 billion per
annum (Bridges 1994). However, a more re cent
and probably more accurate calculation puts the
annual cost of non-indigenous weeds to US agri-
culture at US $27billion (Pimentel et al. 2000). In
such agricultural ecosystems, there has been a sig-
nificant increase in the use of chemical herbicides
since the 1950s, when these comprised less than
20% of aH pesticides sold. By the 1980s, herbicides
accounted for over 50% of the pesticide market
(Jutsum 1988), and this is still rising due mainly to
radical changes in cultivation practices. This is weH
illustrated in rice-cropping systems where labour
shortages and escalating costs have resulted in a
CABI Bioscience, Silwood Park, Ascot, Berks. SL5 7TA,
UK
shift from the traditional transplanting culture to
135 direct see ding, with the consequent need to apply
136 both pre- and post-emergence herbicides. In addi-
136 tion, the development of hardy upland rice vari-
136
136 eties has led to an expansion of cultivation into
137 drier areas, previously considered as marginal for
137 rice growing, where the weed biota is markedly
137 different, and which has necessitated not only
140
141 alte ring the spectrum of herbicides used, but also
143 the application strategy.
143 The incidence of herbicide resistance is also
144
145
increasing due, in part, to the routine and exces-
147 sive use of the same herbicide, or herbicides
148 having similar modes of action (Moss and Rubin
1993; Jasienuik et al. 1996), particularly in cereal-
cropping systems such as wheat and maize. By way
of example, resistance to atrazine is now one of the
most economically significant problems facing
world agriculture (LeBaron 1991; Shanor 1995).
The other over-riding reason follows directly on
from basic changes in or modification of existing
agricultural practices. The continuous growing of
winter wheat in the UK, for example, using non-
plough tillage has favoured monotypic stands
of weeds such as black grass (Alopecurus
myosuroides) with the inevitable development of
herbicide-resistant populations (Orson 1999).
Similarly, advances in and expansion of inter-
national air travel and trade has led to the
increased intercontinental movement of actual
and potential weed species. The arrival of alien or
immigrant plant species (neophytes) in new
ecosystems, either accidentally or intentionally,
can have profound effects on both agricultural
as well as natural ecosystems. Although the econ-
omic impact on the latter habitats cannot be
quantified, exotic weeds represent a significant
threat to the earth's biodiversity, especially so in
small island systems (Cronk and Fuller 1995;
Binggeli 1996). In addition, Mack et al. (2000)
considered that these plant invaders not only have
a significant negative impact on the populations
of native species, but also drastically alter fire
The Mycota XI
Agricultural Applications
Kempken (Ed.)
© Springer-Verlag Berlin Heidelberg 2002
136 RC. Evans
regimes, nutrient cycling, hydrology and energy
budgets. Conventional control of these invasive
weeds, particularly in natural ecosystems, is often
extremely difficult due to financial, operational or
environment al constraints, or a combination of
these.
In order to address the existing as well as
future weed problems, alternative or new strate-
gies have to be sourced, evaluated and, hopefully,
eventually incorporated into a weed management
plan, either to supplement or even to replace
chemical herbicides. Biological control through
manipulation of a plant's natural enemies is one
such approach that is beginning to gain accept-
ance by weed scientists but one which is still
poorly resourced. Weed pathology, or the use of
pathogens (predominantly fungal) for weed
control, is still a relatively new discipline, with
little more than 30years of practical investigation
and experience (Wilson 1969; Charudattan and
Walker 1982; CuHen and Hasan 1988; TeBeest
1991,1993; Mortensen 1997; Evans 2000). The past
and present history, as well as the future prospects,
of the two seemingly distinct control strategies
which can be adopted - classical or inoculative
and mycoherbicide or inundative - are now criti-
caHy reviewed.
11. Classical or Inoculative Strategy
A. The Principles
1. Background
Potentially, classical or inoculative biological
control, which exploits coevolved natural enemies
from the centre of origin of the target pest species,
offers a practical, economic and sustainable solu-
tion for the management of alien, invasive weeds.
In the best-case scenario, the introduction of a
single natural enemy or biocontrol agent is suf-
ficient to destabilise or reduce the target weed
populations to a level where they no longer
present an environment al or an economic threat -
the so-called "silver bullet". There are many exam-
pIes, particularly with insect agents, ranging from
virtual eradication of Opuntia spp. (Cactaceae)
from over 20million ha of grazing land in
Queensland (Australia), some 60years ago (Dodd
1936), following the release of a South American
insect enemy, Cactoblastis cactorum (Lepidoptera:
Pyralidae), to the present-day spectacular control
of the neotropical aquatic weed, Eichhornia cras-
sipes or (water hyacinth) (Pontederiaceae), in
Lake Victoria after the introduction of coevolved
weevils, Neochetina spp. (Coleoptera: Curculion-
idae), from South America (Anon 2000).
The exploitation of fungi or, more specificaHy,
of coevolved fungal pathogens as c1assical biocon-
trol agents is of much more re cent origin and the
underlying concept employed and the protocols
followed (FAO 1996) owe much to entomologists
who developed and refined them over the last
century (McFadyen 1998). Over this period, more
than 600 insect agents have been transferred
between countries and continents for control of
immigrant plants (alien weeds) with a high success
rate and an equally impressive safety record
(Marohasy 1996; Julien and Griffiths 1998). How
and why this strategy works, and the ethics which
underpin it, are now expanded upon.
2. Concept
The concept central to classical biological control
is both simple and logical, and is based on the
premise that non-indigenous organisms when
freed of their natural enemy complexes become
ecologically fitter, compared to their indigenous
competitors. In the absence of such pressures, and
given the right environmental parameters, this can
lead to uncontrolled population explosions such
that the immigrant organism achieves pest status.
Classical or inoculative biological control aims to
redress the balance through the use of coevolved
natural enemies which have been left behind in
the evolutionary centre of origin of the target pest.
Both laymen and scientists alike frequently
find the concept difficult to grasp, questioning its
efficacy as weH as its safety. However, "conven-
tional" plant pathology affords many examples
of just how efficient and devastating a fungal
pathogen can be on its host in exotic situations,
dating back to the scientific beginnings of phy-
topathology in the mid-nineteenth century, when
the neotropical Phytophthora infestans (Mont.) de
Bary eventually caught up with its coevolved host
in the Old World (Large 1940). This threat has
long been recognised, particularly in the USA
(Klinkowski 1970; Kingsolver et al. 1983), but "the
classical problem of an introduced pathogen"
(Anagnostakis 1987) still continues in that country
as a new anthracnose disease, Discula destructiva
Redlin, imported on nursery stock from Asia, has
 Biological Contral of Weeds
quickly spread to native dogwoods, Cornus spp.
(Cornaceae), and threatens their existence as
weH as the species composition and community
dynamics of native forest ecosystems (Daughtrey
and Hibben 1994; Hiers and Evans 1997).
The dogwood example has been highlighted
he re since it illustrates an opposing concept to
classical biological control, the central tenet of
which opinions that the most efficacious natural
enemies are to be found in the centre of origin or
diversity of the target pest species, its genus or
tribe (Wapshere 1974a). The alternative view is
that evolutionarily new associations may, in fact,
be even more effective and increase the chances
of successful control (Hokkanen and Pimentel
1984; Hokkanen 1985). Whilst there is evidence
for this in crop pathology - for example, neo-
tropical cocoa (Theobroma cacao L.) has been
subject to several "new association" diseases in the
Palaeotropics (Thorold 1975) - the approach has
not been universaHy accepted in weed pathology,
probably because for most classical biocontrol
agents rigid host specificity is demanded. Indeed,
it is probable that such new natural enemy associ-
ations or encounters occur whenever an immi-
grant arrives in any exotic ecosystem and that
these constitute an additional cryptic pressure
which helps to prevent the establishment of many
alien plants, or at least to limit their populations.
B. The Mycota Involved
The mycota exploited thus far for classical biolog-
ical control of alien or invasive weeds are detailed
in Table 8.1. The majority of pathogens belong to
the rust fungi because they possess all the traits
demanded of a classical agent: high specificity;
virulence; and efficient, long-distance dispersal.
The narrow host range of most rust species, which
is an essential prerequisite of a classical agent,
reftects a close and long association with their
hosts to the extent that they are nutritionaHy
dependent on, and hence restricted to, a particular
plant biotype, or species, or genus. This obligate or
biotrophic condition is shared to some extent by
the U stilaginales, and both the "true" and the white
smuts are currently being assessed for their
biocontrol potential, although, in addition, these
fungi have the ability to grow saprophytically in a
yeast-like phase. This hemibiotrophic condition is
also characteristic of some of the Coelomycete
fungi that have recently been included in classical
137
biological control programmes against a diverse
range of alien weeds. Specific weed-pathogen asso-
ciations are now examined in more depth by
analysing case studies of past and present exam-
pIes, as wen as future targets.
C. Case Studies
1. Past Examples
a) Prickly Pear Revisited
The control of prickly pear (Opuntia spp.) in
Australia has been described as one of the most
spectacular achievements in the his tory of eco-
nomic entomology (Mann 1970). A complex of
neotropical cactus species, within 80years, had
invaded and occupied an area of grazing and
farming land in New South Wales and Queensland
the size of Great Britain: "that this plant invader
should be brought under control in the space of a
few years mainly by one introduced insect [a
pyralid moth from Argentina], is a twentieth
century miracle" (Mann 1970). However, prior to
this success, additional surveys in the USA identi-
fied a range of pathogens associated with Opuntia,
and at least 10 species were imported during the
1920s into Australia for host range screening.
There are no details of when or even if these
pathogens were ever released, which is why they
are not included in Table 8.1 or in the world cata-
logue of classical biocontrol agents (Julien and
Griffiths 1998). However, surveys in Australia in
the 1930s revealed that several of the American
diseases were already widely established, includ-
ing: shot-hole, caused by Gloeosporium lunatum
Ellis & Everh.; brown spot, due to Phyllosticta
concava Seaver; and a bacterial soft rot (Bacillus
cacticidis Johnston & Hitchcock). These were
often associated with Cactoblastis larvae in the
rapid decay of succulent segments and could also
be isolated from rotting basal portions, along with
various ubiquitous, opportunistic fungi. It was con-
cluded that prickly pear control resulted from the
activities of a complex of microorganisms follow-
ing Cactoblastis attack; with the soft rot bacteria
playing the principal role in the destruction of the
succulent joints, and the fungi appearing to be the
major cause of the lower stern and butt rot (Dodd
1936; Mann 1970). Thus, this could be interpreted
to be the first example of the classical introduction
of exotic fungi, either accidental or deliberate, as
biocontrol agents of an alien invasive weed.
Table 8.1. The mycota involved in classical biological control of alien invasive weeds ......
w
00

Mycota' Weed target Country of origin Country (and date of introduction)

Basidiomycota
Puccinia chondrillina Bubak Chondrilla juncea L. (Asteraceae) Italy Australia (1971, 1980, 1982, 1996)b
(Uredinales: Pucciniaceae) Turkey USA (1976)<
Argentina (1982)
Phragmidium violaceum (Schultz) Rubus constrictus Lefevre & P.I Mueller Germany Chile (1973)
G. Winter (Uredinales: R. ulmifolius Schott Europe d Australia (1984)
Phragmidiaceae) R. fruticosus L. agg. (Rosaceae) France Australia (1991, 1992)"
Puccinia xanthii Schwein. Xanthium strumarium L. (Asteraceae) USA Australia (1974Y
(U redinales: Pucciniaceae)
Uromyces galegae (Opiz) Sacc. Galega officinalis L. (Fabaceae) France Chile (1973)
(U redinales: Pucciniaceae)
Uromycladium tepperianum (Sacc.) Acacia saligna (Lab.) Australia South Africa (1987)
McAlpine (Uredinales: Wendland (Mimosaceae)
Pileolariaceae)
Puccinia carduorum Jacky Carduus nutans L. (Asteraceae) Turkey USA (1987)
(U redinales: Pucciniaceae)
Uromyces heliotropii Sred. Heliotropium europaeum Turkey Australia (1991)
(Uredinales: Pucciniaceae) L. (Boraginaceae) ::r:
Puccinia abrupta Dietel & Holw. var. Parthenium hysterophorus L. Mexico Australia (1991) (1
partheniicola (H.S. Jacks.) Parmelee rn
Puccinia melampodii Dietel & Holw. P. hysterophorus (Asteraceae) Mexico Australia (1999) <:
I"
::;
(U redinales: Pucciniaceae) '"
Puccinia cardui - pycnocephali Syd. Carduus pycnocephalus Italy Australia (1993)
(U redinales: Pucciniaceae) L. (Asteraceae)
Maravalia cryptostegiae (Cummins) Ono Cryptostegia grandiflora (Roxb.) Madagascarg Australia (1994)
(Uredinales: Chaconiaceae) R. Brown (Asclepiadaceae)
Diabole cubensis (Arthur & J.R. Johnst.) Mimosa pigra L. (Mimosaceae) Mexico Australia (1996)
Arthur (Uredinales: Raveneliaceae)
Puccinia evadens Harkn. (Uredinales: Baccharis halmifolia L. (Asteraceae) USA Australia (1998)
Pucciniaceae)
Puccinia myrsiphylli (Thüm.) G. Winter Asparagus asparagoides (L.) South Africa Australia (2000)
(Uredinales: Pucciniaceae) w.F. Wight (Asparagaceae)
Prospodium tuberculatum (Speg.) Lantana camara L. (Verbenaceae) Brazil Australia (200l)h
Arthur (Uredinales: Uropyxidaceae)
Entyloma ageratinae RW. Barreto Ageratina riparia (Regel) RKing Jamaica; Hawaii, USA (1975) South Africa (1989)
& H.C Evans (Ustilaginalcs; & H. Robinson (Asteraceae) New Zealand (1998) Australia (2001)h
Tilletiaceae)
Sporisorium ophiuri (Henn.) Vanky Rottboellia cochinchinensis (Lour.) Madagascar Costa Rica h
(Ustilaginales: U stilaginaceae) W. Clayton (Gramineae; Andropogoneae)
Ascomycota
Sphaerulina mimosae-pigrae H.C Evans Mimosa pigra Mexico Australia (1994)
& G. Carri6n (Dothideales:
Mycosphaerellaceae) Anamorph:
Phloeospora mimosae-pigrae
H.C Evans & G. Carri6n (Coelomycetes)
Deuteromycota
Colletotrichum gloeosporioides (Penz.) Clidemia hirta (L.) D.Don Panama Hawaii, USA (1986)
Penz. & Sacc. f.sp. clidemiae (Melastomataceae)
C. gloeosporioides f.sp. miconiae Miconia calvescens Blume Brazil Hawaii, USA (1997)
(Coelomycetes) (Melastomataceae)
Cercospora rodmanii Conway Eichhornia crassipes (Mart.) USA South Africa (1991) t:o
(Hyphomycetes) Solms-Laub. (Pontederiaceae)
o'
0-
(]Q
Phoma clematidina (Thüm.) Clematis vitalba L. (Ranunculaceae) USA New Zealand (1996) ('i'
Boerema (Coelomycetes) e:..
Septoria passiflorae Louw. Passiflora tripartita (Jussieu) Colombia Hawaii, USA (1996) ()
0
(Coelom ycetes ) Poir (Passifloraceae) g.
....
Septoria myricae Ellis & Everh. Myrica faya Aitoni (Myricaceae) USA (Mainland) Hawaii, USA (1997) 2-
(Coelomycetes) 0
Phaeoramularia eupatorii - odorati Ageratina adenophora (Spreng.) Mexico South Africa (1987) '""
(J.M. Yen) X.I Liu & Y.L. Guo k R King & H. Robinson (Asteraceae) ~
~
p..
'"
'Authors of scientific names quoted and abbreviated as recommended by Kirk and Ansell (1992).
bAdditional strains (pathotypes) introduced from Italy and Turkey against two remaining weed biotypes.
CReported in Canada in 1992 following natural spread.
dIllegal introduction from unknown European source (Marks et al. 1984), rust subsequently detected in New Zealand.
'Introduced after prolonged host specificity-virulence screening of a complex of European strains (Bruzzese and Hasan 1986a,b).
fNot deliberately introduced; illegal releases were made to circumvent the regulatory channels, viewed by some farmers as being too slow and cumbersome, and this
"accidental" introduction proved to be highly successful (Julien et al. 1979; Chippendale 1995).
gTwo strains introduced: initially, from northem Madagascar which proved to be "field incompatible"; secondly, from southwest Madagascar.
hprojected release date, subject to final approval.
;Both weed and pathogen naturalised in Jamaica: smut later discovered in Mexico (Veracruz State), the true evolutionary centre (Barreto and Evans 1988).
iThe original host was the North American species Myrica cerifera L.; the target weed (M. faya) originates from Macaronesia (Azores, Madeiras).
kDespite a redescription of this species (Morgan-Jones 1997), its identification remains in doubt due to confusion over host records; accidentally introduced into Australia
in 1954 on an insect biocontrol agent, later appeared in New Zealand in 1962, and has also been documented from China (Liu and Guo 1982). The mycologicalliterature
has confused the records of cercosporoid fungi recorded on the related genera Ageratina and Chromolaena (formerly assigned to Eupatorium) and it seems likely that P.
eupatorii-odorati is restricted to Chromolaena odorata (L.). RKing & H. Robinson, whilst the recently described Phaeoramularia nepalensis S.K. Singh & RK. Chaudhary
(Singh et al. 1995) is the species which occurs on A. adenophora.
>-'
w
'Ci
140 H.C. Evans
b) Skeleton Weed and Mistflower:
the Early Successes
Classieal biologie al control of alien weeds through
the exploitation of their coevolved mycota had
an auspicious beginning in the early 1970s with
the first two ventures proving to be spectacular
successes. N evertheless, there was more than an
element of risk involved since the pathogens were
imported and released before a formal code of
conduct or a strategy for evaluating the safety of
classieal biocontrol agents had been established.
The centrifngal, phylogenetic testing seqnence,
which now underpins all introductions of exotic
agents (FAO 1996), was only proposed and
adopted several years later (Wapshere 1974b,
1975), and modified since for microbials
(Weidemann 1991). The results of these host range
tests provide the basic data for the pest risk assess-
ment that is now central to any proposal to import
an exotie plant pathogen (Evans 2000). However,
the specificity screening for the two weeds dis-
cussed here was focused more on the threat posed
to crop plants in the release areas rather than on
genetic relatedness. Hence, these test lists were rel-
atively short (40-60 plant spp.), compared with the
phylogenetie lists compiled since for other alien
weeds, whieh average 80-120 spp. (Evans 2000).
The skeleton weed project in south-east
Australia, using the European rust Puccinia chon-
drillina, was also a pioneering example in that it
disproved the widely held view that the inocula-
tive approach was not applicable to tradition al
agro-ecosystems, especially annual crops. Never-
theless, weed populations in wheat, within a rela-
tively short period and over more than 1 million
ha, were reduced by more than 99% to den-
sities approximating those in the plant's native
Mediterranean range (Cullen and Hasan 1988).
The accrued savings, calculated on increasing crop
yields and reduced herbicides inputs, have been
estimated at Australian $260 million, based on
1975 priees and with a 10% discount rate up to
the year 2000 (Marsden et al. 1980), although
sub se quent estimates have varied considerably
(Wapshere et al. 1989). Whatever the exact figures
involved, the cost-benefit ratio is enormous
(112:1), particularly when compared to typieal
ratios for chemical herbicides of 4 or 5:1 (Tisdell
1990); moreover, control is sustainable. Subse-
quently, it was discovered that two, previously
unrecorded weed biotypes were resistant to the
rust strain (ex Italy) originally released. By
employing isoenzyme techniques to match
biotype to pathotype (Chaboudez et al. 1992),
additional strains were selected and imported
(Table 8.1). The rust has also been deployed suc-
cessfully against skeleton weed in the USA,
notably in the western states (Adams and Line
1984; Supkoff et al. 1988).
The other early success, which is still being
exploited today (Table 8.1), involves mistflower,
Ageratina riparia, and a white smut fungus,
Entyloma ageratinae. FoHowing the importation of
the smut (as "Cercosporella" sp.) from Jamaiea
into Hawaii, weed infestations were severely
depleted, with up to 80% population decline,
leading to the rehabilitation of upland pastures
and, perhaps more significantly, to the conserva-
tion of invaded or threatened indigenous forest
(Trujillo 1985). This introduction was made with
an incomplete knowledge of the biology and
taxonomy of the pathogen, now essential prereq-
uisites before any exotic agent can be considered
for importation, or even for host-range screening.
Subsequent investigations revealed that both host
and pathogen are naturalised in Jamaica and that
their true centre of origin, and hence coevolntion,
lies in a mountainous region of Veracruz state in
Mexico, where mistflower is a rare endemie
(Barreto and Evans 1988). The smut has since been
introduced into South Africa (Morris 1991) and
New Zealand (Frohlich et al. 1999), but only after
further host-range screening which more than
doubled the original test list, as this was considered
to be too short as weH as too disparate. Despite
earlier predictions that smut epiphytotics would
occur only in the New Zealand summer (Morin et
al. 1997), the agent has exceeded all expectations
and is active and damaging throughout the year.
2. Current Examples
a) Rust GaU in South Africa
Acacia saligna or Port Jackson willow is one of
many Australian Acacia spp. whieh have become
weeds in South Africa (Henderson 1998).
However, A. saligna was considered to be most
problematie because of the threat it posed to
the unique fynbos ecosystem of the Cape region.
Following studies in Australia on the biology and
host range of the gall-forming rust fungus Uromy-
cladium tepperianum (Morris 1987), the pathogen
was released in South Africa in 1987 (Table 8.1).
The early results, however, were disappointing
(Morris 1991). Although, somewhat prophetically,
the latter author added that: "As the pathogen
 Biological Contral of Weeds
completes only one life cycle per year, initial
spread is expected to be slow, and it may be 5-10
years before the fungus reaches an exponential
rate of spread and any influence on populations of
the weed can be observed". Within this 10-year
time frame, weed populations have been reduced
to 5-10% of the original densities, with trees being
weakened and even killed by gall infestations that
may reach up to 1500 galls per tree (Morris 1997).
Natural vegetation is now returning and it has
recently been concluded that A. saligna is under
control (Morris 1999).
b) Rubber- Vine Weed in Australia
The Madagascan climbing plant, Cryptostegia
grandiflora, has been described as the single great-
est threat to biodiversity in tropical Australia
(MeFadyen and Harvey 1990). Rubber-vine weed
is capable of smothering native forest, up to 30m
tall, and can become the dominant vegeta-
tive component of the landscape. In addition, C.
grandiflora also presents an agricultural threat,
particularly to the cattle industry of Queensland,
which sufters annuallosses in excess of Australian
$8million (Tomley and Evans 1995). Following
extensive studies on the novel life cycle, with two
teliospore stages (Evans 1993), and the host range
(Evans and Tomley 1994) of a Madagascan rust,
Maravalia cryptostegiae, several strains were
released in northern Queensland in 1994 (Evans
2000). Importation was approved despite the fact
that the rust had been found to sporulate on a
rare, endemie asclepiad (Evans and Tomley 1994);
although it was predicted, and later proven, that
infection was an artificial greenhouse response
and that this native species would not be a natural
field host (Evans and Tomley 1996). The results
from field evaluation trials have shown that the
rust has caused severe defoliation of rubber-vine
weed at all monitored sites and has reduced seed
production to almost nil. In addition, up to 25 %
of the plants have been killed by repeated defoli-
ation and almost no new seedlings have estab-
lished, despite suitable conditions (Anon 1999,
2000). Based on this provision al information, it is
confidently expected that rubber-vine weed will be
brought under substantial if not complete control
within the next decade.
3. Future Targets
This seetion summarises the projects still in
progress or in development, in order to demon-
141
strate the range of actual or potential weed targets
at which this management strategy can be aimed,
despite the increasing rather than decreasing
skepticism ("pathophobia") - especially in view of
the exemplary safety re cord - and the decreasing
rather than increasing investment, even though
the success rate has been high and the benefits,
both economic and environmental, have been
enormous. The examples are drawn from different
geographie regions and personal experiences, to
emphasise the specific regional, as well as logisti-
cal problems.
a) In the Palaeotropics
Most of the important invasive weeds in the
Palaeotropics are immigrants from Latin America
where they rarely constitute a problem; typically,
being regarded as minor, ruder al weeds only. In
particular, India has a number of neotropical plant
species which have become major invaders of
both natural and agricultural ecosystems (Evans
1998). For example, Parthenium hysterophorus or
"Congress grass", a relatively re cent arrival, is now
considered to be the principal terrestrial weed
(Evans 1997), whilst water hyacinth is the main
weed of aquatic ecosystems not only in India, but
also throughout the Palaeotropics (Gopal 1987).
Two rust fungi from Mexico have recently been
introduced into Australia against P. hysterophorus
(Table 8.1), as part of an integrated programme to
control the weed which currently poses a health
threat to urban populations in Queensland, as well
as being an on-going economic disaster for gra-
ziers (Evans 1997). However, although the project
has been extended to India, there are many prob-
lems to resolve befme either rust can be con-
sidered for release. In particular, India has no
established protocol or, indeed, no past history of
exploiting plant pathogens as weed biocontrol
agents, and there are relatively few examples in
which classical biological control using insect
agents has been employed as a management strat-
egy. Unfortunately, controversy still surrounds
the release in India of a Mexican weevil as a bio-
control agent of P. hysterophorus, despite the well-
documented evidence to prove that sporadic
insect attacks on sunflower crops were triggered
artificially by Parthenium pollen densely coating
the sunflower leaves and, therefore, that this is not
a true host (Evans 1997). In greenhouse tests, one
of the Mexican rust species (P. melampodii) also
showed an artificially extended host range, in duc-
ing symptoms in several related composite species.
142 H.C. Evans
However, following a pest risk assessment, the rust
was still approved for release by the Australian
quarantine authorities, i.e. the environmental and
health risks posed by the weed were adjudged to
be far greater than the perceived risks from releas-
ing the rust (Evans 2000). Nevertheless, public
awareness and acceptance of classical biological
control in Australia and India are poles apart and
the Indian public, in general, and the scientific
community, in particular, need to be educated
ab out the potential benefits and risks of such a
management strategy before a proposal to import
either rust can be submitted.
A more appropriate and immediate weed
target for classical biological control in India is the
climbing plant Mikania micrantha (Asteraceae),
which forms part of a complex of neotropical
invasive weeds - including Chromolaena odorata
(Asteraceae) and Lantana camara (Verbenaceae)
- that has seriously eroded, and still continues to
threaten, the biodiversity of the unique Western
Ghats (Muniappan and Viraktamath 1993; Evans
1998). M. micrantha is arecent arrival but the
other weeds have been present and dominant
members of the flora for many years so that their
true origins and negative environment al impact
have be co me obscured to such an extent that
the showy flowers of Lantana, as weIl as water
hyacinth, can be found adorning travel guides and
posters! This is another ever-occurring problem
that faces biocontrol practitioners, the perception
of what constitutes an alien pest and a useful intro-
duction: the conflicts of interest dilemma. Thus, in
parts of Africa, Chromolaena odorata is regarded
as a beneficial plant, making classical biological
control an impossible or at least a contentious
management strategy, even though it is regarded
as the worst alien plant species in subtropical
South Africa and is still spreading through the
continent (McFadyen and Skarratt 1996;
Zachariades et al. 1999). It was just such an
impasse in Australia that led to the tabling of the
Australian Biological Control Act of 1984 and
which provided for the first time a legal basis for
the introduction of exotic biocontrol agents
(Cullen and Delfosse 1985). In developing coun-
tries, like India, there is no such enabling legisla-
tion, whilst in developed countries, as for example
the UK and USA, classical biocontrol agents are
stilliegally classified as beneficial "pests" for pur-
poses of importation (Harris 1993; Tatchell1996).
Since M. micrantha is still in an invasive phase
in India, the chances of successful biological
control are considered to be greatly enhanced
(Zimmermann and Neser 1999). Moreover, two
rust species from the Neotropics, Puccinia
spegazzinii de Toni and Dietelia portoricensis
(Whetzel & Olive) Buritica & IF. Hennen, have
been shown to be extremely damaging and also
highly specific to this weed host (Cock et al. 2000).
Other, potentially exploitable fungal pathogens
have also been identified on both Lantana camara
and Chromolaena ordorata in the Neotropics
(Evans 1998), and, more recently, on Eichhornia
crassipes in the Upper Amazon (Evans and
Reeder 2001), suggesting that the management
of these invasive weeds in India, and the
Palaeotropics in general, through the use of co-
evolved fungi is a viable long-term strategy.
b) In the Neotropics
This region has a disproportionately low number
of invasive plant species compared to other
regions of the world (Cronk and Fuller 1995), pos-
sibly because most trade routes and plant exports
have been from rather than to the Neotropics.
However, the widespread establishment of alien
African grasses in the Amazon Basin could have
far-reaching global, as well as regional, conse-
quences, since their invasiveness is causing
massive ecosystem alterations which could even-
tually lead to the permanent conversion of this
biodiverse carbon sink to depauperate savanna-
like areas (D'Antonio and Vitousek 1992). Nev-
ertheless, such grasses are difficult targets for
biological control because of their relatedness to
many crop species; their protected meristems;
their multiple mechanisms of propagation and
perennation; notwithstanding conflicts of interest
with graziers (Evans 1991). The exception is itch
grass or Rottboellia cochinchinensis (Andro-
pogoneae), which has become a major invasive
weed, especially in graminaceous crops, through-
out Latin America (Evans 1995). Since seeds are
its only me ans of propagation and the seed bank
is short-lived, it is expected that a systemic head
smut, Sporisorium ophiuri (Table 8.1), could have
a significant impact on the population dynamics
of the weed (Smith et al. 1997). Astrain of the
smut from Madagascar is currently in the final
stages of screening for introduction into Costa
Rica. Depending on its powers of dispersal, the
smut could, of course, spread naturally throughout
the region; highlighting, perhaps, certain deficien-
cies in inter-country quarantine collaboration as a
 Biological Control of Weeds
biocontrol agent can be released unilaterally
by one country without consultation with its
neighbours.
Ironically, plant movements within a country
could also result in alien, invasive weed problems,
as, for example, in the Galapagos Archipelago of
Ecuador, where a number of the most important
weeds, such as Cinchona succirubra or red quinine
(Rubiaceae) and Lantana camara, originate from
mainland Ecuador (Cronk and Fuller 1995). Thus,
the only sustainable and, indeed, economic strat-
egy for their control may be to search for and to
intro du ce coevolved natural enemies from north-
western SouthAmerica. A potential candidate, the
rust Puccinia lantanae Farl., has already been iden-
tified on L. camara in Peru (H.C. Evans, unpubl.).
c) Europe
This region is included because it highlights some
of the main problems confronting biocontrol prac-
titioners, previously alluded to for India, namely,
the lack of public awareness of, and governmental
funding for, biological control, specifically the
classical approach. Thus far, there have been few
introductions of natural enemies of alien pests
and, until very recently, no initiatives to exploit
fungi for the control of invasive weeds. How-
ever, following increasing concern about the
spread of Japanese knotweed, Fallopia japonica
(Polygonaceae), in the UK, and its impact in ripar-
ian habitats, where it displaces the native flora, as
well as in urban situations, where it causes struc-
tural damage, a scoping study to evaluate the
potential of biocontrol agents is currently und er-
way. Preliminary surveys in Japan have identified
a number of potential biocontrol agents, includ-
ing a complex of fungal pathogens (H.C. Evans,
unpubl.). As with Parthenium hysterophorus,
however, it is envisaged that an integrated man-
agement approach, including the use of a guild of
natural enemies, will be necessary to bring this
problematic weed und er complete control.
111. Mycoherbicide
or Inundative Strategy
A. From Conception to Inception
In the very first review on the use of plant
pathogens for the control of weeds, Wilson (1969)
143
suggested that the time was ripe for the develop-
ment of microbial herbicides and that this could
be achieved either through the selection of highly
virulent pathogenic isolates - and the use of
"super pathogens" through genetic modification
was even touched upon - or by the exploitation
of their secondary metabolites, employing the
industrial technology developed by microbiolo-
gists. Indeed, whilst the inoculative approach has
been led by plant pathologists, the inundative
strategy has been pioneered by scientists with a
predominantly microbiological or biochemical
background.
Although both Russian and Chinese scientists
have been credited independently with establish-
ing "factories" for mass production of fungal
inoculum for the control of the parasitic weed
dodder (Cuscuta spp.: Convolvulaceae) in the
early 1960s, using Alternaria cuscuticidae Rudak
in the USSR (Wilson 1969) and Colletotrichum
gloeosporioides f. sp. cuscutae in China (Gao and
Gan 1992), and thus appear to have been the first
to have conceived and actually implemented the
mycoherbicide strategy, data are scarce and con-
flicting on the details and success of this new tech-
nology. It is really in the USA, a decade or so later,
that the strategy was exploited both practically
and commercially. Although there were concerns
at that time about the use and abuse of chemical
pesticides, this initiative was also environmentally
pioneering because it was conceived before the
U.S. Environmental Protection Agency (EPA)
officially endorsed (in 1979) a "biorational"
approach to crop protection through the use of
alternative products. Currently, in both Europe
(Denmark, Sweden, the Netherlands) and North
America, governments are committed to reducing
pesticide use by up to 50% (Haas 1989); provincial
legislation in Ontario (Canada) has even proposed
that this should be achieved by 2002 (Swanton et
al. 1993). The door has been opened to biora-
tionals, such as mycoherbicides, and the invitation
has been accepted, but have they succeeded in
mixing on equal terms with agrochemicals?
The first registered products were De Vine,
based on a wet spore (chlamydospore) formula-
tion of Phytophthora palmivora (Butl.) Butl. for
control of stranglervine (Morrenia odorata:
Asclepiadaceae), and Collego, a wettable powder
formulation of conidia of C. gloeosporioides f. sp.
aeschynomene for use against northern jointvetch
(Aeschynomene virginica: Leguminosae), in 1981
and 1982, respectively. However, the inception
144 H.C. Evans

phase for both products lasted more than a decade share of ups-and-downs, with different owners or
from the initial realisation of the biocontrol poten- manufacturers, as well as re-registration problems
tial of these plant diseases (Daniel et al. 1973; (Mortensen 1997; Boyetchko 1999). Thus, even
Burnett et al. 1974) to the commercialisation of though Collego has been extremely successful
the fungal products (Bowers 1986; Kenney 1986). locally, the small target-area - less than 3000ha
As Templeton (1992a) noted, it took 13years of treated annually according to Templeton (1992b)
research and development with Collego to - has meant that the market size, and hence profits,
demonstrate that mycoherbicides were a safe and are not attractive to agrochemical companies. In
practical alternative to agrochemicals: nine years other words, the effort and investment (inputs) do
ofwhich were spent in collaboration with the EPA not justify the returns. The market for De Vine is
to establish the protocol and guidelines for even more specialised and, therefore, sm aller,
product registration as these were non-existent at which is further complicated by the logistics of
that time. producing a fresh product on demand for an
The development of De Vine involved other erratic customer base. Ironically, this is partly due
novel concepts. Firstly, it employed an indigenous to the fact that the product is "too successful": the
fungus to control an alien plant species - strangler fungus has been shown to persist in the soil for
vine is of South America origin - and, secondly, many years and applications made in 1978 were
the selected strain was not specific to the target still having an impact (with 95-100% weed kill)
weed, but attacked other hosts, including several 8years later (Kenney 1986; TeBeest 1993). For
cucurbit crops and ornamentals (Ridings 1986). At commercial needs in a "throw-away society", a
this time, the current thinking was that biocontrol product has to have a limited life span in order to
agents had to be highly specific. However, label generate regular sales.
restrictions on the product, which has to be refrig- According to the most re cent literature and
erated and has a shelf-life of only 6weeks, ensures reviews on mycoherbicides, a number of products
that it is used only in orchard crops (citrus, are either on the market, about to be launched, or
avocado) and that a safety zone, of 1 mile or in the final stages of development (Mortensen
1.6 km, be established between the release site and 1997; Boyetchko 1999). Earlier, Charudattan
susceptible crops. Indeed, the "forma specialis" of (1991) provided a useful and comprehensive list of
C. gloeosporioides on which Collego is based was mycoherbicide projects then in progress, compris-
later shown to have a wider host range than ini- ing nearly 70 weed targets and over 100 fungal
tially predicted, and that this extended to nine taxa. However, few of these projects are included
genera within the Leguminosae (TeBeest 1988; here because the fate of the majority of them
Weidemann et al. 1988). Nevertheless, because remains unknown.
only jointvetch is actually killed by the product, Historically, almost all mycoherbicide candi-
which is targeted exclusively at rice-soyabean dates have been necrotrophic fungi pertaining to
rotations, then Collego still has EPA approval for the Deuteromycota, especially to the genera
use as a pre-emergence herbicide in this agro- Alternaria, Colletotrichum, Fusarium and Pho-
ecosystem. The product is usually applied aerially mopsis. However, one unique exception is the
and only once per season, as the weed emerges obligate rust fungus, Puccinia canaliculata
above the crop canopy. In fact, both these myco- (Schwartz) Lagerh., which comprises the active
herbicides have proven to be successful in the ingredient of the product Dr. Biosedge, registered
field, being highly effective, as well as environ- in 1993 for use against yellow nutsedge (Cyperus
mentally benign, and with good farmer accep- esculentus: Cyperaceae) in the USA, but marketed
tance. However, this is not the same as being a only recently (Mortensen 1997). Commercialisa-
commercial success, as will be shown. tion resulted from work over a 10-year period
which showed that a urediniospore inoculum,
mass-produced in greenhouses and formulated
B. eurrent Status with inert carriers, when introduced into the field
situation early in the season, induced premature
Collego and De Vine are, purportedly, still in use, rust epiphytotics which had a long-term impact on
although current information on their status is dif- weed population dynamies by significantly re duc-
ficult to obtain and at times can be conflicting. ing tuber formation (Phatak et al. 1983; Beste et
Suffice to say that both products have had their al. 1992). There are reports that mass-production
 Biological Control of Weeds
problems have resulted in the withdrawal of this
product from sale, but these are unsubstantiated.
Templeton (1992b) emphasised the impor-
tance of the genus Colletotrichum as a source of
potential mycoherbicides since these fungi can
be highly virulent plant pathogens and are rela-
tively amenable to mass production and formula-
tion (Green et al. 1997; Greaves et al. 1998). As
shown for Collego, if such aggressive necrotrophs
are applied inundatively and early in the
season, their poor powers of dispersal and inter-
crop survival are overcome and 90-100% weed
kill can be guaranteed (Bowers 1986). In addi-
tion to C. gloeosporioides, other speeies of the
genus, including: C. coccodes (Wallr.) Hughes; C.
dematium (Pers. ex Fr.) Grove; C. graminicola
(Ces.) Wilson; C. orbiculare (Berk. & Mont.) Arx
and C. truncatum (Schw.) Andrus & Moore, have
successfully been field-tested against a range
of annual weeds, mainly in North America
(Charudattan 1991; Boyetchko 1999). Neverthe-
less, apart from several formae speciales of C.
gloeosporioides, none of these appear to have
reached the final stages of commercialisation. A
dry conidial formulation of C. gloeosporioides f.sp.
malvae was registered in Canada in 1992 as
BioMal, for control of round-Ieaved mallow,
Malva pusilla (Malvaceae). This weed had
recently become an increasing problem in small
fruit and vegetable crops, such as lentils and straw-
berries, and had proved difficult to control with
conventional herbicides (Mortensen 1988).
However, the venture capital company which had
funded development of the product, in wh at would
now seem to be a classic example of poor initial
market research, pulled out before the final stages
of commercialisation, due to low anticipated sales
and high production costs (Mortensen 1997;
Boyetchko 1999). Latest updates indicate that the
market is currently being extended to the USA, as
weil as being expanded to related malvaceous
weeds - such as velvet leaf (Abutilon theophrasti),
a major agricultural weed causing annual losses
estimated at over US $300million (Mabberley
1997) - by the new product owners (K.
Mortensen, pers. comm.).
Similar production problems have sur-
rounded attempts to commercialise mycoherbi-
eides based on the genus Alternaria. Ten weed
hosts were listed by Charudattan (1991) against
which Alternaria spp. have been evaluated as
inundative biocontrol agents, including: A. alter-
nantherae Holcomb & Antonop; A. cassiae Jurair
145
& Khan; A. crassa (Sacc.) Rands; A. eichhorniae
Nag Raj & Ponappa; A. mascrospora Zirnrn.; A.
tenuissima (Kunze ex Pers.) Wiltshire; and A.
zinniae M.B. Ellis. In greenhouse and small-scale
field trials, 85-100% mortality of the target weeds
has been reported, the fungal formulations often
giving better levels of control than chemical
herbicides (TeBeest 1993). However, only one
product appears to have reached the registration
stage (Boyetchko 1999). This is CASST, which has
been developed in the USA as a post-emergence
herbicide of sicklepod, Cassia (Senna) obtusifolia
(Leguminosae), in soyabeans, as well as of related
weed speeies in various crops (Walker and
Boyette 1985; Bannon 1988). Several biotechnol-
ogy companies (Mycogen Corporation, Abbott
Laboratories) have been involved in product
development but commercially non-viable inocu-
lum loads - translating to 55 kg of product per ha
- were required to achieve consistent control
(R. Charudattan, pers. comm.).
C. Potential Application
Under the title: "Mycoherbicides - a major growth
area?", the problems and practice of exploiting
fungi for weed control were discussed over a
decade ago from an agrochemical viewpoint
(Anon 1988). The question still stands today.
The fact that mycoherbicides have failed to make
any impact on weed management in general, and
on the agrochemical industry in particular, despite
the initial optimism (Templeton and TeBeest 1979;
TeBeest and Templeton 1985), is obviously cause
for concern. Why has the inundative strategy not
been a commercial success, especially in view of
the potential environmental and economic bene-
fits? The timing could not be better, since, over the
past decade, there has been a dramatically increas-
ing acceptance of, and demand for, organic
produce - following concern over pesticide
residues - as well as increasing public awareness
about the use of "biorationals" as environmentally
friendly alternatives to chemicals.
Over the past two to three decades, there has
been considerable research on improving the effi-
cacy of mycoherbicides, as well as developing cost-
effieient, mass-production methods, from pilot to
commereial scale (Churchill 1982; Stowell 1991;
Morin 1992). Formulation is a key component of
the mycoherbicide package, which is designed to:
overcome environment al constraints by reducing
146 H.C. Evans
dew requirements and protecting against desicca-
tion and UV radiation; increase stability and thus
extend shelf-life; and help improve delivery
systems - making mycoherbicide easier to apply,
as weIl as more effective in reaching, covering,
impacting and being retained on the target host
(Green et al. 1997; Bateman 1998; Greaves et al.
1998). Much of this has been highly innovative,
often adapting the technology developed by the
food industry. Thus, for liquid formulations, the
use of invert emulsions (water suspended in oil)
and oil suspension emulsions (vegetable oils with
emulsifying adjuvants) has been extensively in-
vestigated, and an impressive array of adjuvants
(compounds which modify or increase the activity
of the fungal propagule) has been tested, includ-
ing: surfactants, stickers, inert carriers, humectants,
sun-blocking agents, anti-evaporating agents, as
weIl as micronutrients (Green et al. 1997; Greaves
et al. 1998). For those fungal pathogens which
act at the soil rather than the foliar level, solid or
granular formulations hold the most promise and
much of the research has concentrated on enclos-
ing the agents, typically in the form of dry fungal
propagules, within alginate pellets or pre-
gelatinised starch granules. Nonetheless, the
overall failure of many proto-mycoherbicides to
provide consistent control under varying climatic
conditions has usually been blamed on inadequate
formulation (Auld 1992). Thus, this is frequently
considered to be one of the major technological
constraints to successful product development and
hence to commercialisation of mycoherbicides
(Auld and Morin 1995).
As Greaves (1996) concluded, potentially
exploitable fungal pathogens can be isolated re la-
tively easily from most weed targets. However,
there is a long and difficult road ahead before any
initial la bora tory and greenhouse successes can be
transferred to the field situation, and even more
pitfalls, both financial and legislative, before a
commercially viable product, which can compete
with traditional chemical herbicides both in cost
and efficiency, reaches the marketplace. Many
decades ago, Petch (1925) came to a similar con-
clusion regarding the proposed development of
mycoinsecticides, " ... proposals of this nature are
still periodically put forward with no better foun-
dation than the discovery of another fungus on
another insect". Nevertheless, this perceived pes-
simism was also balanced by a pragmatic approach
to, and an on-going interest in, investigating the
potential of fungi for control of weeds and insect
pests, respectively. Petch's strategy was to analyse
epidemics and to monitor the role of fungi in the
natural control of insects, and here it is necessary
to distinguish between natural control, which is
cryptic and impossible to quantify, and biological
control in which man plays an active part. Whilst
Greave (1996), and others before hirn (Templeton
1992a; Auld and Morin 1995), have suggested that
the potential of mycoherbicides can be best
exploited, at least in the short term, in niche
markets where chemical herbicides are either
inefficient, environmentally undesirable, or
uneconomlC.
Currently, there are several good examples
where this potential has recently been realised and
both involve the control of non-indigenous, woody
weeds following the development of products
based on indigenous pathogens. Firstly, the myco-
herbicide Biochon is now registered in the
N etherlands for use against invasive tree species,
notably American black cherry, Prunus serotina
(Rosaceae), in both natural and plantation forests
(de Jong 2000). The product consists of a dry
mycelial preparation of the silver leaf fungus,
Chondrostereum purpureum (Pers. ex Fr.) Pouzar
(Meruliaceae), which is applied to newly cut
stumps to prevent re-growth. Although this fungus
causes a well-documented disease of stone fruits,
epidemiological data and simulation models have
been employed to predict inoculum movement
and hence the risks posed to susceptible crops fol-
lowing mycoherbicide application (de Jong et al.
1990, 1991). The analyses showed that airborne
basidiospores generated from the treated stumps
were a threat only to crops within a 500-m radius
and, therefore, that the product is safe to use as
long as stone fruit crops are more than 0.5 km
away from the treated sites. Whilst Biochon is a
commercial product, manufactured and devel-
oped by a private biotechnology company
(Koppert Biological Systems), and marketed as
"the environmentally friendly solution to undesir-
able tree re-growth", a similarly acting fungus,
Cylindrobasidium laeve (Pers. ex Fr.) Chamuris
(Hyphodermataceae), has been developed as a
mycoherbicide by a public sector initiative at the
cottage-industry level in South Africa (Morris et
al. 1999). Basidiospores of the fungus are formu-
lated in oil (both mineral and vegetable), and the
product, Stumpout, is applied as a stump treat-
ment to control invasive Australian species of
wattle (Acacia mearnsii, A. pycnantha). Stumpout
is supplied directly to the farmer or forester in
 Biological Contral of Weeds
lO-ml plastic sachets contammg ca. 2 x 106
basidiospores/ml. This low technology production
and delivery system is now being evaluated in
North America for the management of various
woody weeds, including red alder, Ainus oregona
(Betulaceae), in Canadian forests (Wall 1997),
with local strains of Chondrostereum purpureum
being employed as the active ingredient (de long
et al. 1996).
IV. General Discussion and Conclusions
McFadyen (1998) highlighted the environmental
and economic problems caused by alien or immi-
grant weeds and predicted that these can only
increase as the plant species introduced over the
last century become naturalised and begin to
spread unchecked. It was further emphasised that
classical biological control offers the only safe and
economically viable method for long-term or sus-
tainable control of these weeds, especially in
natural ecosystems. Whereas countries such as
Australia, New Zealand and South Africa have
endorsed enthusiastically this management
approach and have repeatedly shown just how
successful it can be, most other countries generally
regard the inoculative strategy with apathy and
even open hostility, believing that the use of exotic
natural enemies is inherently dangerous, particu-
larly with fungal pathogens ("pathophobia"), or at
the best ineffective. Indeed, some influential inter-
national bodies, such as the World Wildlife Fund
for Nature, actively eschew dassical biological
control, maintaining a policy that expressly con-
demns the movement of exotic organisms into
new ecosystems. Clearly, such policy makers, as
well as the public in general, need to be educated
about the concept, principles, protocols, and,
above all, the impressively safe and successful
track re cord of this strategy. Control of alien, inva-
sive weeds by the dassical approach is for the
public good, in that there is no ownership of bio-
logical agents and, potentially, everybody benefits.
Thus, funding has to be sought primarily from the
public sector; however, this is no easy task, given
the levels of ignorance or suspicion about biolog-
ical control, and the apparently shrinking financial
inputs into cataloguing and monitoring biodiver-
sity - despite the Rio Convention. There is no
better example than the gradual decline in myco-
logical expertise in the UK and worldwide, and of
147
taxonomists in general. Taxonomy underpins all
classical biological control programmes and, of
course, biodiversity cannot be measured without
it.
Ironically, in spite of the immense benefits
that have accrued to both agriculture and the envi-
ronment in Australia (McFadyen 1998), funds still
do not flow free1y into classical biological control
projects in that country in order to tackle the con-
tinuing threat from invasive weeds. There is a
danger that farmers or landowners will take the
initiative and illegally import untested biocontrol
agents into Australia, as has happened previously
on at least one occasion (Table 8.1; lulien and
Griffiths 1998; Evans, 2000). Inevitably, the immi-
grant plant species that have already established
around the world will only increase in importance,
whilst posing a continual threat to the biodiversity
of other land masses, particularly of small island
ecosystems (Cronk and Fuller 1995). The condu-
si on reached he re is that the dassical strategy
is safe, economic and sustainable, and, therefore,
that it should at least be considered in any long-
term weed management plan. As has been shown,
in the best-case scenario, the release of a single
coevolved fungus can provide the ultimate solu-
tion for control of an invasive immigrant weed.
For other, more problematic weeds, such fungal
natural enemies may have a supportive role to
play in any integrated management plan.
The recent successes with developing myco-
herbicides based on indigenous, broad-range fungi
to tackle non-indigenous weed problems also give
cause for optimism. It is concluded that this
approach, using either small capital venture com-
panies, or cottage industries created from the
public sector or by farmer cooperatives, is perhaps
the route that mycoherbicides will take, particu-
lady for difficult-to-control woody shrubs and
trees in deve10ped countries. Mycoherbicides for
mainstream agricultural use in such countries,
however, does not appear to hold much promise,
considering the constraints outlined above and
detailed by Auld and Morin (1995). Nevertheless,
re cent initiatives in developing countries, espe-
cially in Africa and Asia, where agriculture is tra-
ditionally on a much sm aller scale and pesticide
inputs are generally low, demonstrate how fungal
pathogens can best be exploited in an inundative
strategy at the village level for the control of
weeds which affect subsistence crops.
Watson et al. (2000) have now overcome a
number of the pitfalls previously alluded to in
148 H.e. Evans

order to develop any mycoherbicide, and have bicide, which varies from US $20-80million
implemented an inundative strategy targeted at (Woodhead et al. 1990) - this is still considered to
one of the major weeds of subsistence agriculture, be too high for a niche-market product.
in sub-saharan Africa, Striga hermonthica or Cook (1993), in his vision of the role of bio-
witchweed (Scrophulariaceae). This parasitic logical control in the twenty-first century, stated
plant has been controlled effeetively in grain erops that: " ... In spite of being the oldest component
in Mali utilising a simple inoeulum production of pest management, biological control is still very
system based on a liquid-fermentation proeess much a developing technology, possibly near or
employing loeally available substrates. Dried still in the lag phase ... agriculture may well come
inoeulum of Fusarium oxysporum Sehltdl., eon- 'full cirele' by again depending on biological
sisting of ehlamydospores applied at 80 gm/ha and control as the primary, if not dominant, compo-
produeed in traditional eooking pots, is dusted nent of pest management". He further concIuded
onto the erop seed previously eoated with arabie that, in order to exploit biological control to the
gum, prior to planting. The produet protects the full, the use of literally thousands of site - and pest
developing crop from attack by inhibiting the - specific biocontrol agents might be necessary.
emergence of Striga seedlings in the vicinity of sus- The challenge is how to achieve and then manage
ceptible root systems. Quality control is assured this quantity of"products". Target weed specificity
as the starter inoculum is produced centrally and is certainly essential for cIassical agents and thus
supplied in the form of gelatin capsules as part of each weed project is a "one-off", with no direct
an aid package. This project would appear to point profits involved, and thus has to be fu'nded from
the way for the future of myeoherbieides in devel- the public sector. The difficulty lies in attraeting
oping countries, employing cheap production and the fun ding, as the resulting benefits (economic
delivery systems operating at the village or coop- and environmental) will only become evident in
erative level. As the demand for organic produce the long term. This is not an attraetive proposition
increases in developed countries - in the UK for policy makers or politicians whose survival
within the last 5 years this has risen from practi- depends on short-term success. Site-specific myco-
cally zero to 15-17% - these "rustic" mycoherbi- herbicides seem to hold promise but only perhaps
eides could well find a market as alternatives to at the cottage-industry level, for no other reason
chemical herbicides, particularly in small-scale, than, at present, there is no evidence to indicate
high-profit cropping systems into which such prod- that major agrochemie al companies are investing
ucts could be integrated more effectively. in mycoherbicides, nor is it envisaged that niche-
Increasing the target range of mycoherbicides market produets will generate the profits neces-
would be a major requirement and mixtures of sary to satisfy their shareholders.
pathogens, possibly in combinations with
extremely low-dose, synergising chemical her-
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9 Molecular Variability Studies of Magnaporthe grisea
and Their Application in Disease Control
NICHOLAS J. TALBOT
CONTENTS
I. Introduction. . . . . . . . . . . . . . . . . . . . . . . . .
11. Life Cyde of Magnaporthe grisea . . . . . . . . .
III. Pathotype Variability and Stability
in M. grisea . . . . . . . . . . . . . . . . . . . . . . . . . .
IV. ?ispersed Repetitive DNA Families
m M. gnsea . . . . . . . . . . . . . . . . . . . . . . . . . .
V. Pot3/MGR586 Fingerprint Analysis
of M. grisea Populations ................
VI. Causes of Genetic Variability
in M. grisea . . . . . . . . . . . . . . . . . . . . . . . . . .
VII. The Gene-for-Gene Relationship
Between Rice and M. grisea . . . . . . . . . . . . .
VIII. Novel Cropping Systems
for Rice Blast Control .................
IX. Condusions and Future Prospects ........
References ..........................
I. Introduction
The blast fungus Magnaporthe grisea (Hebert)
Barr is an eeonomieally important pathogen of
more than 50 grass species and eauses severe
disease in a number of important erop speeies,
including riee, millets and wheat (Ou 1985;
Rossman et al. 1990). The fungus is an ascomycete
(anamorph Pyricularia grisea) and is closely
related to other pathogenic fungi such as Gaeu-
mannomyces graminis and Phialophora gramini-
cola within the Magnaporthaeeae (Mugnier 1999).
M. grisea eauses a foliar, leaf spot disease which is
recognised by the presence of large ovaliesions on
the leaf surfaee that ean coalesce in heavy infec-
tions and cause widespread neerosis of leaves. In
older plants, the fungus can spread to the sterns
and grain and in rice this causes node, neck and
panicle blast - signifieant diseases that can lead to
eomplete loss of the harvest (Fig. 9.1). In neck
blast, the rice stern beeomes rotten and the panicle
School of Biological Sciences, University of Exeter,
Washington Singer Laboratories, Exeter EX4 4QG, UK
containing the rice grain ean become detached
153 and fall to the ground. Panicle blast prevents
154 water and nutrient flow to developing seeds and
so inhibits their formation. In addition to sub-
155
stantial yield reduetions, poorly developed grain
156 from blast-infected plants will break apart during
milling, redueing the quality of the harvest. Rice
157 blast causes tremendous losses to rice production,
160
estimated to be 10-30% of the rice harvest (Baker
et al. 1997). Rice is the staple erop for about half
162 of the population of the world and the total rice
harvest was 573 million tonnes in 1997. The vast
164
165 majority of riee production (91 % ) is carried out in
166 Asia, in partieular China and India, which eollec-
tively produce 55% of the world's rice. The popu-
lation inereases projeeted for the next 25 years will
place enormous pressure on rice production and
it is antieipated that 880 million tonnes of rice will
be needed to feed 50% of the 10 billion people
that will constitute the world's population by 2025.
Losses due to rice blast therefore represent a very
signifieant problem. This is well illustrated by a
re cent riee blast epidemie that oecurred in Bhutan
in 1995. Although tradition al farming practices
prevailed in Bhutan with many different eultivars
und er cultivation - a situation that should theo-
retically limit the effects of a disease outbreak - a
serious blast outbreak eaused widespread devas-
tation. More than 700hectares (ha) was affected
by blast and yield losses of 1090tonnes of rice
were recorded (Thinlay et al. 2000).
M. grisea also eauses blast disease on a
number of millets (Echinochloa spp., Panicum
spp., Eleusine spp. and Setaria spp.), including
finger millet (Eleusine coracana) which is an
important erop in the developing world, particu-
larly in southern and east African countries and
India. Finger millet is an essential food security
crop, providing important nutrition including not
only stareh, but also minerals such as calcium,
phosphorus and iron, to very poor rural commu-
nities in Africa and India. Finger millet blast can
eause huge losses, and head blast ean lead to com-
The Mycota XI
Agricultural Applications
Kempken (Ed.)
© Springer-Verlag Berlin Heidelberg 2002
154 N.I Talbot
Fig.9.1. Rice blast disease symptoms. Magnaporthe grisea
eauses a leaf spot disease with the appearanee of many
ellipsoid lesions. The leaf beeomes ehlorotie and dis-
eoloured and the eentres of disease lesions are neerotie.
Under humid eonditions the fungus produees many eonidia
from eaeh disease lesion that spread to new plants by wind
and splash dispers al. In older plants the fungus ean eause
node, neek and panicle blast all of which ean severely affeet
the riee harvest
pIe te loss of the harvest if it occurs prior to grain
formation (Ekwamu 1991). Wheat blast has also
emerged as an important disease and has caused
significant problems in Brazil (Valent and
Chumley 1991).
The economic and social importance of blast
disease, in particular rice blast, has been the
impetus for a considerable amount of research
examining the pathology, molecular genetics, cell
biology and population biology of M. grisea. A
number of reviews covering these research areas
have recently appeared (Howard 1994; Howard
and Valent 1996; Zeigler 1998; Hamer and Talbot
1998; Talbot 1999) and recent progress in rice blast
research has been reviewed extensively in arecent
volume (Tharreau et al. 2000).
In this chapter, I have attempted to bring
together a number of different aspects of rice blast
research that are relevant to agricultural practices
and the control of the disease. The chapter con-
tains references to a number of re cent studies that
have demonstrated the enormous potential of
directed breeding strategies, genetic manipula-
tion, and novel cropping systems for control of
blast disease. The chapter focuses mainly on rice
blast, where the majority of work has been carried
out, but also describes a number of studies of the
less well-known host-limited forms of the fungus.
11. Life eyde of Magnaporthe grisea
M. grisea is propagated by conidiospores which
are wind and splash dispersed. The spores land on
the plant surface and germinate in the presence of
water. The fungus requires water to germinate
and, for this reason, humid conditions with heavy
dew and an ambient temperature of 15-28°C are
very conducive for infection. Conidiospores are
three-celled, pyriform (teardrop shaped) struc-
tures that produce a primary germ tube from one
ofthe apical cells ofthe spore (Howard 1994). This
emerges within 1 hand grows for a short distance
in close contact with the plant cuticle (Uchiyama
and Okuyama 1990; Gilbert et al. 1996). After 4 h,
the end of the germ tube thickens and bends, as it
becomes firmly attached to the cuticle (Bourett
and Howard 1990). This process, called hooking,
marks the first stages of infection structure devel-
opment. The germ tube apex then differentiates
into an appressorium, a dome-shaped cell that has
a specialised cell wall containing a thick layer of
melanin (Howard and Ferrari 1989; Chumley and
Valent 1990; Vidal-Cros et al. 1994). The appres-
sorium gene rates enormous turgor pressure
(Howard et al. 1991; Money and Howard 1996) by
accumulating a compatible solute, glycerol, which
draws water into the cell by osmosis (de Jong et
al. 1997). The hydrostatic turgor genera ted allows
the fungus to generate enormous invasive forces
that are concentrated at the appressorial pore, a
central region in contact with the host leaf surface
(Howard 1994). A penetration peg is forced
through the cuticle and into the epidermal cell
beneath, a process involving cytoskeletal reorien-
tation and localisation of actin at the penetration
peg apex. The penetration peg is narrow and can
appear to be wall-less or have a very thin unstruc-
tured cell wall in electron micrographs (Howard
1994). The peg so on extends and develops into
bulbous branched secondary infectious hyphae,
however, and the fungus begins to fill the epider-
mal cell with biom ass. M. grisea traverses the
initial epidermal cells in this way, filling them with
 Molecular Variability Studies of Magnaporthe grisea and Their Application in Disease Contra I
Fig. 9.2. Infection of a rice leaf by M. grisea. A conidium
has germinated and produced an appressorium. This spe-
cialised infection structure brings about direct penetration
of the plant cuticle, allowing the fungus entry to the plant
tissue. Appressorium development requires free water and
rice blast infection is therefore most prevalent under humid
conditions and at the dew point (Ou 1985). Bar 20,um
hyphae, before forming long runner hyphae which
infect adjacent cells (Heath et al. 1990). It is not
clear whether the fungus is truly intracellular
during the initial stages of development in plant
tissue, or whether the plant plasma membrane is
folded around the invading hyphae. Clearly,
however, there is an intimate association during
the initial asymptomatic phase of development
(Howard 1994). M. grisea grows rapidly and will
form disease lesions within 72-96 h following
infection. Under appropriate conditions (Fig. 9.2),
the lesions can grow to be very large and will
produce several thousand spores from aerial
hyphae that can be dispersed to new plants (Ou
1985). The disease can thus be fast and immensely
destructive when humid warm conditions prevail
and can spread quickly through rice fields, killing
plants at the seedling stage and severely affecting
older plants. The molecular basis of pathogenic
development by M. grisea has been the focus of a
significant amount of re cent research and has been
reviewed elsewhere (Howard and Valent 1996;
Talbot 1999).
111. Pathotype Variability and Stability
in M. grisea
M. grisea has long been known to be variable in
its ability to canse disease on different hosts, or on
different cultivars of a host (Ou 1985). In general,
host-limited forms of the fungus are restricted in
155
their pathogenicity to the host from which they
were isolated. Thus, M. grisea isolates pathogenic
on weeping lovegrass (Eragrostis curvula), for
example, do not cause disease on rice (Leung et
al. 1988; Valent et al. 1991). However, there are
some exceptions to this rule, as many rice patho-
genic isolates of M. grisea, for example, will also
infect barley (Lau and Hamer 1996). Although the
genetic basis for host specificity has been studied
for weeping love grass and rice (Sweigard et al.
1995), as discussed later, a detailed study of the
mechanisms of host range determination by
M. grisea has yet to be carried out (Zeigler 1998).
M. grisea isolates capable of parasitising a
given host also show cnItivar specificity. Rice
pathogenic isolates, for example, can be divided
into physiological races or pathotypes based on
their ability to infect host rice cultivars carrying
particular resistance genes (Leung et al. 1988;
Ellingboe et al. 1990; Valent et al. 1991). It is now
clear that M. grisea interacts with rice (and prob-
ably its other hosts) in a gene-for-gene interaction
(for reviews, see Baker et al. 1997; Hammond-
Kosack and Iones 1997). In gene-for-gene
interactions, host plants carry dominant disease-
resistance genes, which encode proteins that are
able to perceive and respond to proteins produced
by pathogens. If the resistance gene product
identifies the presence of a pathogen, then defence
reactions are induced and the pathogen is unable
to cause disease. Pathogen proteins that are re-
cognised by plant resistance gene products are
encoded by avirulence genes. These are so-called
because they are genetically dominant; virulence
can only result by mutation of the dominant
avirulence gene (Hammond-Kosack and Iones
1997). Rice blast disease was shown to involve a
gene-for-gene inter action during the very suc-
cessful plant breeding programmes of the 1960s
and 1970s, and by carrying out reciprocal genetic
analysis of the pathogen (Leung et al. 1988;
Ellingboe et al. 1990; Valent et al. 1991). Domi-
nant resistance genes have since been cloned
along with avirulence genes with M. grisea and the
genetics of the interaction are described below in
Section VII.
Rice-breeding programmes have been suc-
cessful in incorporating blast resistance genes into
high-yield cultivars, although a common feature
has been the reoccurrence of the disease within
the space of only a few growing seasons after their
introduction (Ou 1985; Zeigler 1998). For this
reason, pathotypic variation of M. grisea has
always been assumed to be very great by growers
156 N.J. Talbot
and this was confirmed in a number of studies (Ou
1985). These studies tested the pathotype of M.
grisea isolates on a number of rice cultivars dif-
fering in major blast resistance genes. Eight cul-
tivars are used as an internationally agreed
differential set for rice blast pathotype assignment
and pathotypes determined based on the spectrum
of disease and resistance observed. Independent
assessment of pathotype diversity by different
research groups led to diametrically opposed
views of the level of pathotype variability in rice
blast populations. On the one hand, the fungus
was described as being so variable that a single
disease lesion could give rise to many new patho-
types when single spores were taken from it (Ou
1985). Conversely, other studies showed that
pathotypes were not excessively variable and
moreover were very stable over a number of years
(Latterell 1971; Latterell and Rossi 1986). Many
explanations for these different results have been
obtained, induding the robustness of using the dif-
ferential set for pathotype assignment. It is now
apparent, however, that the researchers were
studying quite different rice blast populations; the
workers who found stable pathotypes were
working predominantly with isolates from the
USA, while those who found extraordinary levels
of variability worked with isolates from Asia. The
difference in the length of time rice has been cul-
tivated in these regions is very large, because rice
was only introduced into the USA in the last few
hundred years, compared to many thousands of
years of cultivation in Asia, dose to the centre of
origin of Oryza sativa (Zeigler 1998). These dif-
ferences in cultivation and the consequent effects
of host-dependent selection on the pathogen
population may well account for the differences in
variability observed and recent molecular studies
have recently shed light on the population struc-
ture of rice blast with much high er resolution than
was previously possible.
IV. Dispersed Repetitive D NA Families
in M. grisea
The rice blast genome contains a number of
repeated DNA families that have proved useful in
defining distinct populations of the fungus. There
are at least seven dispersed repeated DNA
sequences present in the M. grisea genome and a
number of other repeated elements present at
lower copy number (Skinner et al. 1993; Nitta et
al. 1997; Dioh et al. 2000). Two transposable
elements related to the Fotl element of Fusarium
oxysporum are present in the M. grisea genome
(see also Chap.10, this Vol.). Pot2 is an inverted
repeat DNA transposon, present within both rice
and grass pathogenic forms of M. grisea (Kachroo
et al. 1995a). This is related to the Pot3 element
(Farman et al. 1996) that is only found in high copy
numbers in rice pathogenic forms of the fungus.
Pot3 was originally identified as a dispersed
repeated DNA sequence, MGR586 (Hamer et al
1989), which has been used extensively to charac-
terise rice blast populations throughout the world
(see below). Arecent study aimed at identifying
avirulence genes by chromosome walking showed
that further Fot1-like elements are probably main-
tained within the rice blast genome. A RAPD
marker, OPE-ElO, used during a chromosome
walk towards an avirulence gene locus, was found
to be the result of a new Pot2-related transposon
within the rice pathogenic isolate Guyll (Dioh et
al. 2000). MGR586 (Magnaporthe grisea repeat)
was identified in a search for middle repetitive
DNA sequences (Hamer et al. 1989) and a
number of other elements were discovered in the
same screen. These indude the MGLlMGR583
sequence that shows homology to LINE-1 ele-
ments and a number of lower copy number
repeated DNA sequences. At least two Gypsy-
dass retrotransposons have also been found in the
M. grisea genome (Dobinson et al. 1993; Shull and
Hamer 1996). The Grasshopper element is found
specifically in millet (Eleusine) pathogens and is
present at between 30 and 50 copies per genome
(Dobinson et al. 1993). The Fosbury/MAGGY
element is amplified specifically in rice pathogenic
isolates of M. grisea and follows a similar dis-
tribution to Pot3/MGR586 (Farman et al. 1996b).
These elements have the potential to cause genetic
variability in a number of ways induding induc-
tion of translocations and deletions (Tal bot et al.
1993). They also of course have the potential to act
as mobile elements and both MAGGY and MGL
have been shown to be active transposons. MGL
was found to cause a developmental mutant by
insertion into the ACRl gene (Nishimura et al.
2000). Mutation of ACRl gives rise to an acropetal
phenotype where conidiospores are formed in
chains (acropetally) as opposed to in whorls (sym-
podially) in the wild type. The mutation also
brings ab out areduction in appressorium forma-
tion and reduction in pathogenicity (Lau and
 Molecular Variability Studies of Magnaporthe grisea and Their Application in Disease Contral
Hamer 1998). This highlights the potential for
transposable elements to cause genetic variability,
albeit probably at a low frequency.
V. Pot3IMGR586 Fingerprint Analysis
of M. grisea Populations
The discovery of repeated DNA elements in the
genome of M. grisea led to their use in population
studies. The most widely used element Pot3/
MGR586 has so far been used to characterise in
excess of 2500 isolates of M. grisea from most
rice-growing regions of the world (for review,
see Zeigler 1998). It was originally believed that
the presence of Pot3/MGR586 in the M. grisea
genome pre-dated the divergence of rice path-
ogenic forms of the fungus and that selective
amplification of the element had subsequently
occurred (Shull and Hamer 1996). The MGR586
sub-clone pCB586, however, was shown to contain
single copy DNA sequences Banking the Pot3
element and use of a Pot3-derived PCR product
as a probe showed no hybridisation to grass
pathogenic forms of the fungus (Farman et al.
1996a). It is likely therefore that acquisition of
Pot3/MGR586 occurred concomitantly with diver-
gence of rice pathogenic forms of M. grisea or
early in their evolution. The vast majority of rice
pathogenic isolates of M. grisea have the element,
indicating that the founder population of most of
the riee pathogenic isolates worldwide contained
Pot3/MGR586.
The first use of Pot3/MGR586 in a population
study of M. grisea examined 42 isolates of M.
grisea from the southern United States, represent-
ing the eight major pathotypes present in the
country, based on an archive collection of the
fungus. Southern blot analysis defined eight MGR
fingerprint groups (sharing at least 80% common
MGR586-hybridising fragments). Six of the eight
MGR fingerprint groups corresponded to isolates
sharing a given pathotype. In the other cases one
MGR fingerprint group was composed of isolates
showing two pathotypes, while another pathotype
could be divided into isolates classified into two
fingerprint groups (Levy et al. 1991). The presence
of such easily defined genotypic groups strongly
supported a clonal population structure for M.
grisea (Hamer 1991) and each fingerprint group
was therefore referred to as a clonallineage (Levy
et al. 1991). The high degree of lineage/pathotype
157
correspondence indicated that the American M.
grisea rice blast population was composed of a
limited number of discrete and stable clonallin-
eages. The limited sampie set used, however, was
based on an archive collection and was thus prob-
ably composed of the most commonly found blast
isolates (the modal type). For this reason, it may
not have been representative of the full range of
genetic diversity (Zeigler 1998). Further studies of
commercial rice fields and disease nurseries in
Arkansas showed that four main clonal lineages
predominated and confirmed a high degree of
correspondence to pathotype. There were excep-
tions, however, and considerable variability was
found in single-field sampling, indieating that the
original view of the US population was correct
in prineiple, but may have under-estimated the
degree of diversity present (Xia et al. 1993).
Combining Pot3/MGR586 with mitoehondrial
haplotype analysis has recently confirmed that the
M. grisea population in Arkansas is genetically
less diverse than populations of the fungus in Asia
(see below), with a single mitochondrial haplotype
predominating, highlighting the importance of
host-dependent seleetion (Xia et al. 2000). Recent
outbreaks of rice blast in California in 1996 and
1997 can be attributed to isolates of M. grisea
sharing an MGR-defined lineage with isolates
found previously in Arkansas (Zeigler 1998).
A detailed study at a riee blast nursery in
Colombia (the Santa Rosa Experimental Station)
was carried out using 151 isolates from 15 cultivars
of riee (Levy et al. 1993). MGR fingerprinting
revealed six major lineages showing 92% or
greater similarity within lineages. In contrast, the
similarity between lineages ranged from 32-85%.
The correspondence of genetic lineage to patho-
type was not found to be a simple relationship as
observed in the USA. Analysis of the virulence of
isolates on 23 riee eultivars and eomparison to
MGR586lineage showed correspondence of some
pathotypie groups to partieular lineages, but the
overall picture was of higher pathotypic variabil-
ity within lineages. Further studies in Colombia
using more than 1000 isolates have shown the
presenee of 17 discrete lineages in the country
(Correa-Vietoria and Zeigler 1993; Zeigler 1998).
In general, it was found that eaeh clon al lineage
was compatible with only a small number of riee
eultivars and that only a few lineages were re-
eovered from a single eultivar. This meant that if
a cultivar was cultivated that was resistant to the
pathotypes represented in a single lineage, then
158 N.J. Talbot

sampling in fo11owing seasons showed areduction defined and conversely the lineages exeluded by a
in the frequency with which the lineage was given cultivar could also be identified (Zeigler
observed in the pathogen population. Thus, host- 1998).
dependent selection is important in determining In China, MGR fingerprint analysis identified
the lineage diversity in a region; the most preva- 54 lineages from a co11ection of 473 isolates col-
lent cultivar will be associated with compatible lected at 144 different sites over a 16-year period
lineages. This observation gave rise to the idea (Shen et a1. 1993; Peiliang et a1. 1995). A similar
formulated by Zeigler and coworkers of using high level of diversity was observed in India where
MGR586 fingerprint analysis as a guide to rice- 29 lineages were identified, some of which were
breeding strategies. By analysing the resistance only found in particular regions (Sivaraj et a1.
spectrum of rice cultivars in association with the 1996). In Korea, 16 lineages were identified from
fingerprint groups within the Colombian M. grisea 62 isolates co11ected from commercial rice fields
population, they were able to identify cultivars (Zeigler 1998). A very detailed study in Thailand
that could completely exelude a particular identified 68 lineages from 527 isolates
MGR586lineage. That is, the cultivar carried resis- (Mekwatanakarn et a1. 2000). The isolates were
tance genes capable of defending the plant from found to represent 175 distinct pathotypes and
a11 the pathotypes present within the lineage. thus the relationship of lineage to pathotype was
When viewed in this way, there were a number of complex. Twenty-one of the pathotypes comprised
cultivars identified with potentia11y durable rice 53 % of the sampled population and were wide-
blast resistance. spread. The remaining 160 pathotypes were a11
Pot3/MGR586 fingerprinting was carried out rare and 117 of them were represented by a single
on 41 isolates from 5 rice-growing countries in isolate of M. grisea. Interestingly, the common
Europe. Roumen and co11eagues (1997) identified pathotypes could be isolated from many cultivars
five lineages and found that the most common and thus had widespread compatibility with the
lineage was found in numerous regions and over resistance genes deployed, while rare pathotypes
a 14-year co11ection period. Rice blast in Europe were recovered mainly from the non-indigenous,
therefore appears to fo11ow a similar pattern of a exotic cultivars used. Thus, it appeared that
pathogen population that is composed of a sma11 resistance genes in the exotic cultivars may have
number of discrete elonal lineages with a high acted as filters preventing the isolation of very
degree of correspondence to pathotype and prevalent M. grisea isolates in Thailand, therefore
broadly consistent with the resistance spectrum a110wing the detection of rare isolates that have
of prevalent cultivars. Thus, abundant lineages either reduced fitness or are not compatible with
represent isolates with particularly strong com- the indigenous cultivars tested.
patibility with the rice cultivars grown on the However, in a thoughtful study, Mek-
continent. watanakarn and co11eagues analysed the resis-
Pot3/MGR586 fingerprint analysis subse- tance spectrum of both indigenous and exotic rice
quently moved on to the large rice-producing varieties and showed that three resistance genes,
areas of Asia where rice cultivation is long- Pi-I, Pi z-5 and Pi ta 2 , were broadly effective
standing. First, in the Philippines, a study of two across the whole pathogen population. No single
disease nurseries showed a population composed isolate showed compatibility to both Pi-I and Pi-
of relatively few lineages. In an analysis of over z-5, and we11-represented lineages were normally
1500 isolates obtained from 38 rice cultivars incompatible to cultivars carrying one or other
planted in trap nurseries in both an upland and a gene. However, no combination of resistance
lowland site, 10 MGR586 lineages were defined genes analysed conferred resistance to every
(Zeigler et a1. 1995). The correspondence to lineage as small numbers of isolates within a
pathotype was more complex than observed in number of the lineages showed compatibility. The
Colombia with many pathotypes (defined by authors coneluded that lineage exelusion strate-
compatibility/incompatibility on a differential gies were not obvious for Thailand although
set of cultivars) present in a given lineage. The deployment of Pi-I and Pi z-5 might offer poten-
lineages, however, could be used to define the 'vir- tial for durable resistance, while combining Pi z-5
ulence capacity' of a group of isolates. Thus, by and Pi ta 2 might be useful in northeast and central
only considering the compatible interactions, the Thailand. The study also raised an interesting
cultivars compatible with a given lineage could be question concerning the likelihood of a fitness
 Molecular Variability Studies of Magnaporthe grisea and Their Application in Disease Control
penalty being associated with compatibility to rice
cultivars carrying Pi-l and Pi z-5 resistance genes.
Because isolates compatible with cultivars carry-
ing both of the genes appear to be very rare, a sit-
uation also observed in the Philippines (Chen et
al. 1995; Zeigler et al. 1995), it is possible that loss
of the avirulence genes corresponding to these loci
is detrimental to the fungus.
The use of Pot3/MGR586 fingerprinting has
therefore been informative in a number of ways
and has given at least an outline view of the popu-
lation structure of rice pathogenic forms of M.
grisea. Clearly, the prevalence of isolates carrying
high copies of the Pot3 element indicates that rice
pathogens are, in general, an isolated genetic
population that arose ancestrally and acquired
the Pot3 element at an early stage in this process.
Isolates with intermediate numbers of Pot3 ele-
ments, for example, have been isolated mainly
from dose to the centre of origin of M. grisea. Pot3/
MGR586 fingerprinting has also shown that, while
lB49A lB49B lB54 lC17 lB49A lB49B lB54 lCI7
159
pathotype diversity is relatively low in areas of
re cent rice cultivation such as the USA and
Europe, in Asia, where rice has been cultivated for
far longer (in Thailand archaeological evidence
suggests rice cultivation as long aga as 1l,OOOB.c.),
the situation is quite different and a high degree of
diversity is present in blast fungus populations.
In addition, there has not been widespread deploy-
me nt of exotic cultivars carrying novel, broad-
spectrum resistance genes in Thailand, Bhutan and
the Yunnan region of China, the areas which show
the highest levels of pathotypic diversity. The
pathogen has therefore not been exposed to the
same host-dependent selection (or bottleneck)
that has been imposed elsewhere by modern agri-
cultural practices and widespread monoculture.
Pot3/MGR586 analysis has thus provided a view of
the M. grisea population structure in traditionally
farmed communities in the developing world, and
the distinct situation in areas of re cent intensive
cultivation such as Europe and the USA (Fig. 9.3).
Fig.9.3. Pot3/MGR586 fingerprint
analysis of M. grisea. The presence
of the dispersed repetitive Pot3
transposable element (Farman et al.
1996b) is detected by hybridisation
of genomic DNA with the sub-clone
pCB586 (Hamer et al. 1989). Here
genomic DNA of eight isolates of M.
grisea from the United States has
been digested with Hind III and Sal
I respectively (left and right panels),
fractionated by gel electrophoresis
and hybridised with the pCB586
probe. RFLPs around each dis-
persed element can be detected. The
M. grisea isolates shown are of three
pathotypes: IB49, IB54 and IC17.
MGR586 fingerprinting classifies
these isolates into three groups
based on similarity in banding
pattern (Ne i and Li 1979). The IB49
isolates belong to two MGR586
groups denoted IB49A and IB49B.
The IB54 isolates are in the same
MGR586 group as IB49B isolates.
The IC17 isolates belong to a distinct
MGR586 group. In this form of
analysis Pot3/MGR586 is used to
define genotypically distinct groups,
or lineages, and has provided
insights into the population struc-
ture of M. grisea throughout the
world
160 N.J. Talbot
The sources of variation that have been the
engine of pathotype diversity in M. grisea have
been controversially debated for some time, but
are now also being examined using molecular
tools ineluding Pot3/MGR586 fingerprinting. An
interesting picture is beginning to emerge sug-
gesting a number of different mechanisms may
exist to allow the generation and subsequent
selection of newly virulent forms of M. grisea.
VI. Causes of Genetic Variability
in M. grisea
M. grisea is a heterothallic ascomycete that exists
in two mating types called MATl-l and MATl-2.
The mating-type genes have now been eloned and
characterised and are idiomorphic rather than
allelic with little sequence homology existing
between MATl-l and MATl-2 loci (Kang et al.
1994). Mating proceeds by production of flask-
shaped perithecia that contain the asci, elongated
bags containing eight ascospores that constitute
the products of meiosis (octads are produced by
meiosis followed by a mitotic division). Although
genetic crosses can be carried out in the labora-
tory, it has long been noted that rice pathogenic
strains of M. grisea lack fertility. This is normally
due to loss of female fertility, which me ans astrain
is unable to act as the female, forming a receptive
trichogyne structure, which, after fusion with the
male spermatium (in the form of a conidium or
hypha), goes on to form the ascogonium and
ascogenous hyphae. Hermaphroditic strains form
perithecia on both sides of a cross line when
paired together on agar plates, indicating that both
parents have the capacity to form either trichog-
yne structures or spermatia. In practice, female
sterile isolates can only be crossed with strains
that are hermaphroditic (have male and female
fertility) and, for this reason, hermaphroditic rice
pathogenic strains of M. grisea have been sought
after for their use in genetic analysis. Strains such
as Guyll, which is a hermaphroditic MATl-2
strain isolated by Leung and co-workers in
French Guyana in 1988, have consequently been
immensely useful as research tools and for intro-
gression of fertility into laboratory strains of M.
grisea (Valent et al. 1991).
The absence of fertility in field isolates of
M. grisea has been taken as evidence that sexual
reproduction is probably not important as a
source of genetic variation in the wild. Fertility
appears to be a complex trait involving a number
of genes, and is independent of mating-type gene
function. The sexual stage of M. grisea has not
been reported in the field, although reports of
induction of perithecial production on living rice
plants or rice straw have been made (Kato and
Yamaguchi 1982). It is elear, meanwhile, that the
predominant form of reproduction by M. grisea is
asexual production of conidiospores from disease
lesions and, taken together with Pot3/MRG586
fingerprint analysis, which showed numerous dis-
tinct lineages of M. grisea in the field, there is
strong evidence for a predominantly donal popu-
lation structure. This means that M. grisea is evolv-
ing as a number of isolates that exist as elonally
descended lineage with little or no gene flow
present between them (Levy et al. 1991). Again,
however, the evidence for universal elonality in M.
grisea has been largely derived from studying pop-
ulations of the fungus in areas of quite recent rice
cultivation. In these areas, often one mating type
prevails within a given area and certainly within a
Pot3/MGR586-determined lineage (Notteghem
and Silue 1992).
The prevalence of a single mating is obviously
a good indicator of a elonal population in a het-
erothallic organism and, furthermore, the analysis
of karyotype variation in M. grisea has shown that
chromosome-length polymorphisms are common
in the fungus, particularly in rice pathogenic
isolates (Talbot et al. 1993; Orbach et al. 1996).
Chromosome-length polymorphisms can be inter-
preted as evidence of the infrequency of meiosis
in an organism (Zolan 1995), due to the resulting
problems in non-sister chromatid alignment at
metaphase. The prevalence of small, meiotically
unstable mini-chromosomes, which contain
Pot3/MGR586 elements, has also been interpreted
as evidence that genomic rearrangements have
taken pI ace in M. grisea perhaps due to the abun-
dance of repeated DNA (Talbot et al. 1993). The
maintenance of such DNA molecules is also con-
sistent with a population that undergoes meiosis
very infrequently (Talbot et al. 1993). Interest-
ingly, chromosome-Iength polymorphisms were
rarer in Eleusine-pathogenic isolates and other
grass-pathogenic forms of M. grisea that are often
sexually fertile (Orbach et al. 1996).
Recent studies elose to the centre of diversity
of rice have provided evidence that sexual repro-
duction may have taken place, or be taking pI ace,
within rice blast populations. The main evidence is
 Molecular Variability Studies of Magnaporthe grisea and Their Application in Disease Control
the prevalence of isolates of both mating types in
the Himalayas and the southern Yunnan province
of China. In the Matli region of the Himalayas, for
example, 38% of isolates identified were MATl-l
and 13% were MATl-2 and male fertility and
hermaphroditic isolates were commonly detected
(Kumar et al. 1999). This indicates the populations
of M. grisea in these regions have the capacity for
sexual reproduction. The presence of Pot3/
MGR586 fingerprint profiles that could be inter-
preted as being recombinant forms of other
lineages, and the presence of isolates having
an intermediate copy number >40 copies of Pot3/
MGR586, is also consistent with a population
having been influenced by recombination. Kumar
and colleagues carried out a careful analysis in
order to determine the likelihood that M. grisea
populations in the Himalayan region were in-
fluenced by sexual recombination. Using both
Pot3/MGR586 and a number of single copy mole-
cular markers, they were able to study genetic
diversity levels. A number of valleys sampled
maintained highly diverse populations of the
fungus with many MGR-defined lineages present.
Using pairwise analysis of a1lelic associations,
based on probing with single copy RFLP markers
in addition to multi-Iocus variance analysis, they
were unable to reject the hypothesis of gametic
phase equilibrium - which would be expected for
a population undergoing sexual reproduction
(Kumar et al. 1999). Gametic phase equilibrium
analysis has been used to determine whether
recombination has influenced populations of
organisms that appear at the outset to be clon al
(Maynard-Smith et al. 1993; Burt et al. 1996). It is
based on allelic associations and the prob ability of
random associations of alleles, present in fully
recombining populations, as opposed to linkage
disequilibrium that occurs in clonally propagating
organisms (Maynard-Smith et al. 1993). The study
by Kumar et al. (1999) indicates that, although
M. grisea is a predominantly donal organism
which is propagated asexually by conidial pro-
duction from diseased plants, there may be popu-
lations dose to the centre of origin of rice where
sexual recombination has taken place and influ-
enced the degree of genetic variability present.
Although there is no clear-cut evidence that
sexual recombination still occurs in rice patho-
genic M. grisea isolates in this region, they clearly
have the capacity to cross. The absence of very
rare, or 'private alleles', only found in a given
isolate also shows that the lineages present in the
161
Himalayan region are not completely genetically
isolated (i.e. gene flow occurs in the population).
Thus, any analysis of genetic variability in the
fungus, particularly being carried out with a view
to disease control, should not overlook the possi-
bility that newly virulent forms of the fungus
might arise via sexual reproduction.
There is also the possibility that parasexual
recombination may have influenced M. grisea
populations, as discussed by Zeigler and cowork-
ers (1997), and it is a potential mechanism
for generating genetic variability. In parasexual
reproduction, hyphal anastamosis occurs between
compatible isolates and leads to production of a
heterokaryon. Karyogamy can subsequently occur
with production of diploid strains of a fungus.
Genetic variability is then brought about in two
ways. Haploidisation occurs due to random chro-
mosome loss, which is brought about by non-
disjunction during anaphase. Haploidisation will
thus result in a haploid individual having entire
chromosomes from one or other parent. This can
be detected readily in genetically marked strains
of a fungus because any two genetic markers
on the same chromosome will always be linked
in resulting progeny. Multiply repeated DNA
sequences, such as Pot3/MGR586, will appear to
be inherited in blocks of copies in progeny result-
ing from haploidisation. Rarely during mitosis of
a diploid strain can crossing over occur between
non-sister chromatids during metaphase. The
resulting recombinant chromosomes are then
inherited via haploidisation. In M. grisea, parasex-
uality can be induced in the laboratory. Crawford
et al. (1986) were able to produce heterokaryons
from pairing auxotrophic mutants of M. grisea
and, after sub-culture, faster-growing sectors were
isolated which appeared to represent strains that
had been through a diploid stage and haploidised
spontaneously during production of conidia.
Conidia always appeared to be haploid and all
recombinant classes expected from a cross could
be recovered among isolated conidia emerging
from sectors (Crawford et al. 1986). Parasexuality
has long been proposed to be a potential source
for variability of M. grisea (Genovesi and Magill
1976) and lagging chromosomes - taken as evi-
dence of non-disjunction during mitosis - have
frequently been observed in cytological studies
(Kato 1978; Kato and Yamaguchi 1982). Limited
evidence of parasexuality has been found for M.
grisea populations in the Himalayan region based
on analysis of MGR586 fingerprints and identifi-
162 N.J. Talbot
cation of potential recombinant progeny, consis-
te nt with previous parasexual exchanges having
taken pI ace (Zeigler et al. 1995). Distinguishing
these events from variation generated by sexual
reproduction is, however, not straightforward
(Kumar et al. 1999). Repeated parasexual events
could cause disruption of linkage disequilibrium in
the same way as sexual reproduction, for example.
Only by sequence analysis of large chromosomal
regions, or gene fragments spanning a whole chro-
mosome, could the results of haploidisation be
unambiguously proven, as long as potential
parental isolates are also extant in the population.
Mitotic recombination events, however, will be
difficult to distinguish from sexual events without
extensive sequence analysis.
VII. The Gene-for-Gene Relationship
Between Rice and M. grisea
The interaction between rice and M. grisea is a
gene-for-gene interaction involving major genes
for blast resistance and corresponding avirulence
genes in the fungus. The most accepted explana-
tion for gene-for-gene interactions is a receptor-
ligand model that involves the product of an
avirulence gene being recognised by the receptor
encoded directly, or indirectly by a plant resistance
gene. Activation of the resistance receptor leads
to a large number of responses by the plant includ-
ing localised cell death (called the hypersensitive
re action or HR), an oxidative burst, production of
antimicrobial compounds, lignin production and
cell wall thickening at the site of infection, and
increased transcription of pathogenesis-related
genes. The latter encode antimicrobial enzymes
such as glucanases and chitinase and a number of
abundant pro teins of less well-characterised func-
tion (Kombrink and Somssich 1997). During the
last 5 years, a number of disease-resistance genes
have been cloned from plants and the majority
share some common features. Many contain
leucine-rich repeat regions (LRRs) in common
with proteins from animals and yeast that are
involved with protein-protein interactions or
ligand binding. Plant LRR-type resistance genes
have been classified into two groups based on the
position of the LRR regions. Resistance genes,
including the tomato genes Cf-2, Cf-4, Cf-5 and Cf-
9 that confer resistance to leaf mould disease
caused by Cladosporium fulvum, contain extra-
cytoplasmic LRRs. A second group, including the
N gene that confers resistance to tobacco mosaic
virus, has intracellular LRR domains and a
nucleotide-binding domain (Hammond-Kosack
and Jones 1997). A further resistance gene class,
the Pto gene from tomato that confers resistance
to bacterial speck disease, encodes a protein
kinase (Martin et al. 1993) while the Xa-21 gene
contains both LRRs and a protein kinase domain
(Hammond-Kosack and Jones 1997). The struc-
tures of resistance genes therefore point to a role
in perception of a pathogen-encoded ligand that is
recognised at the cell surface or within the plant
cell cytoplasm. As a result a signal transduction
cascade is activated which is far from being under-
stood, but probably involves a phospho-relay,
transcriptional activation and induction of de-
fence responses (Scofield et al. 1996; Tang et al.
1996; Romeis et al. 1999).
The perception of pathogen proteins both at
the cell periphery and within plant cells has made
the characterisation of avirulence genes an impor-
tant goal. Those isolated to date, however, are very
diverse and do not appear to share common fea-
tures. Some bacterial avirulence genes appear to
encode elicitors that can directly induce HR when
applied to the outside of plant cells, while other
bacterial avirulence gene products are secreted
directly into plant cells using the bacterial type III
secretion pathway (Van den Ackerveken et al.
1996; for review, see Galan and Collmer 1999). The
fungal avirulence genes isola ted to date encode
elicitor peptides that appear to be perceived in the
apoplast (Van den Ackerveken et al. 1991; Joosten
et al. 1994; Rohe et al. 1995). These secreted
products do not show similarity but are smalI,
cysteine-rich peptides. In M. grisea, four aviru-
lence genes have been isolated to date, PWL2
(Kang et al. 1995; Sweigard et al. 1995), AVR-Pita
(Jia et al. 2000; Orbach et al. 2000), AVR-C039
(Farm an and Leong 1998) and AVRI-Irat7 (Dioh
et al. 2000).
The ability to grow on weeping love grass was
found to segregate as a single gene trait. Positional
cloning of the corresponding locus led to isolation
of PWL2 which encodes a 16kDa secreted,
glycine-rich, hydrophilic protein. The PWL2
allele confers non-pathogenicity (avirulence) on
weeping lovegrass and was found to be an unsta-
ble locus where re arrangements often led to loss
of the PWL2 gene and a gain in the ability to cause
disease on weeping love grass (Sweigard et al.
1995). However, another pwl2 allele differed by
 Molecular Variability Studies of Magnaporthe grisea and Their Application in Disease Control
only a single base-pair from the PWL2 gene sug-
gesting that even simple base-pair substitutions
can lead to loss of function and virulence on
weeping love grass. Thus, a host-specificity gene
appears to act in the same way as a classical
avirulence gene. PWL2 was found to be highly
polymorphic in strains of M. grisea and, subse-
quently, a PWL gene family was identified by
homology, including PWLl, PWL3 and PWL4.
Interestingly, PWL3 and PWL4 were non-
functional although PWL4 could be made func-
tional if expressed under control of the PWL2
promoter. This indicates that the genes are
expressed quite distinctly and may have diverse
potential as avirulence factors (Kang et al. 1995).
Perhaps further analysis of M. grisea hosts will
define resistance genes capable of recognising
each member of the PWL family, as in C. fulvum
where the pathogenicity factors ECPl and ECP2
have been shown to act as virulence gene products
(Lauge et al. 1998). The endogenous function
of the PWL genes, however, remains obscure
(Kang et al. 1995).
The AVR-C039 gene was cloned following an
extensive chromosome walk that led to generation
of a high-quality genetic map for M. grisea and
description of the organisation of repeated DNA
families (Kachroo et al. 1995a,b; Nitta et al. 1997;
Farman and Leong 1998). The gene has been iden-
tified but awaits detailed characterisation. The cul-
tivar CO-39, which carries the Pi a gene, is
susceptible to many rice pathogenic M. grisea iso-
lates and therefore loss of AVR-C039 function
does not appear to cause any fitness penalty to M.
grisea unless the mutation leading to virulence
does not aftect the gene's endogenous function.
The AVRI-lrat7 gene was identified following a
position al cloning that utilised a novel RAPD
screening method for selection of clones from
genomic libraries (Dioh et al. 1997). Three AVR
genes, AVRI-Irat7, AVRI-MedNoi' and AVRl-
Ku86, were mapped to individual loci and chro-
mosome walks initiated (Dioh et al. 2000).
Recently, the AVRI-lrat7 gene has been identified
and the protein product is currently being charac-
terised (M.H. Lebrun, pers. comm.).
The best characterised of the M. grisea aviru-
lence genes is the AVR-Pita gene which encodes a
neutral zinc metalloprotease. The AVR-Pita locus
was found to be located in a sub-telomeric posi-
tion and could not be isolated from conventional
genomic libraries. A library of chromosome ends
was constructed instead and this led to identifica-
163
tion ofAVR-Pita. The gene was shown to act as an
avirulence gene by introduction into strains that
are normally virulent on rice cultivars carrying the
Pi-ta gene such as Yashiro-mochi and YT14. Intro-
duction of AVR-Pita produced transformants that
were avirulent on Yashiro-mochi. Recently, the Pi-
ta gene has been cloned (Bryan et al. 2000) and
shown to encode a cytoplasmic pro tein of 928
amino acids containing an unusual LRR domain
and a centrally locate nucleotide-binding domain.
The Pi-ta gene is similar to the Arabidopsis RPMl
gene within the LRR domain (Grant et al. 1995)
but does not fit the consensus for LRR resistance
proteins (Jones and Jones 1997). The inter action
between Pi-ta and AVR-Pita has been studied and
it has been shown that the protein products of
these genes directly interact, based on analysis
with the yeast two-hybrid system. Moreover, when
AVR-Pita was transiently expressed in plant cells
it led to a Pi-ta-dependent resistance response
indicating that a physical inter action occurs in vivo
during the initial stages of infection. The interac-
tion involved the leucine-rich domain of Pi-ta and
the mature form of the AVR-Pita metalloprotease.
Interestingly, the interaction only occurred when
the mature processed form of the AVR-Pita-
encoded protein was used in far-western analysis
and required the presence of zinc, suggesting that
the metalloprotease was perhaps in an active form
during the interaction.
Direct biochemical evidence for the activity of
the AVR-Pita metalloprotease has not yet been
found, but the observation of an interaction
between AVR-Pita and Pi-ta raises a number of
interesting questions. For example, it seems likely
that Pi-ta encodes an intracellular pro tein and that
the inter action thus occurs within a plant cell. Yet,
although AVR-Pita is a secreted protein, it has
never been established whether M. grisea tra-
verses the plant plasmalemma during infection.
The fungus appears to grow intracellularly, but
may invaginate the plasma membrane of the plant
cell that is invaded in the same way as biotrophic
fungi that form haustoria. Also, it is not clear
whether the inter action between Pi-ta and AVR-
pita is a result of proteolytic cleavage of the resis-
tance gene product. Perhaps Pi-ta is an inhibitor
of HR which when cleaved will trigger a defence
re action. Alternatively, it may be that binding
AVR-pita and Pi-ta induces a conformational
change in the protein and this triggers a signal
transduction pathway for plant defence. It is also
possible that the dominant form of Pi-ta is resis-
164 N.J. Talbot

tant to proteolytic cleavage and the susceptible Glutinous rice cultivar monoculture
allele simply encodes a protein that is broken 0 0 0 0 0 0 0 0 0 0
down by AVR-pita. 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0
The interaction does show that resistance to 0 0 0 0 0 0 0 0 0 0
M. grisea requires an extremely intimate associa- 0 0 0 0 0 0 0 0 0 0
tion between plant and fungus that must occur 0 0 0 0 0 0 0 0 0 0 2.4 m
0 0 0 0 0 0 0 0 0 0
very soon after primary infection of the plant 0 0 0 0 0 0 0 0 0 0
cuticle. A study by Jia et al. (2000) has provided 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0
the first evidence for one of the mechanisms by 0 0 0 0 0 0 0 0 0 0
which host resistance against rice blast is initiated 15cm 20cm

and will serve as the model with which subsequent Hybrid rice cultivar monoculture
AVR-resistance gene associations are compared. x x x x x x x x
x x x x x x x x
x x x x x x x x
x x x x x x x x
VIII. Novel Cropping Systems x x x x x x x x
x x x x x x x x 2.4 m
for Rice Blast Control x x X x x x x x
x x x x x x x x
x x x x x x x x
Arecent study has highlighted that increasing tbe
x x x x x x x x
x x x x x x x X
genetic diversity in a cropping system can cause 15cm 30cm

reduction in rice blast disease and consequent Mixture

yield benefits. Mixed cropping has long been pro- x x 0 x x x x 0 x x
posed as an effective me ans of controlling disease x x 0 x x x x 0 x x
x x 0 x x x x 0 x x
and is weIl established in some agricultural com- x x 0 x x x x 0 x x
munities. However, there have rarely been exam- x x 0 x x x x 0 x x 2.4 m
x x 0 x x x x 0 x x
pIes of the efficacy of this form of disease control x x 0 x x x x 0 x x
except in glasshouse or limited field plot experi- x x 0 x x x x 0 x x
x x 0 x x x x 0 x x
ments. Zhu and colleagues have shown dramatic x x 0 x x x x 0 x x
reductions in rice blast in the Yunnan province in x15 cmx 0
15 cm
x x 30cm
x x 0 x X
China by planting mixed stands of rice cultivars
with a different spectrum of resistance to M. Fig. 9.4. Planting arrangements in arecent study that
grisea. Their plot design (Fig. 9.4) was planted in showed effective control of rice blast using mixed stands
of rice. In the experiment, monoculture fields of two rice
two growing seasons on 812 and 3342 ha respec- cultivars were plan ted in the Yunnan province in China.
tively. Such a large-scale experiment required the The first monoculture cultivar was a high-value rice culti-
cooperation of many farmers and pathologists var producing glutinous rice for specialty dishes, but which
within the area. The results were dramatic; rice is very susceptible to rice blast. The second cultivar was a
hybrid rice variety with good resistance to rice blast. Mixed
blast incidence fell enormously in the first growing stands afforded excellent disease control in large field
season, and in the second season the farmers aban- experiments carried out over 2 years and spanning 3342ha.
doned fungicide treatment because of the low inci- The dimensions of each plot and distances between
dence of disease. The two cultivars plan ted, one a each row are given. (Adapted from a study by Zhu et al.
2000)
blast-susceptible line producing high-value gluti-
nous or 'sticky' rice used in a large number of con-
fectionary products and specialty dishes, and the
other a hybrid variety with less susceptibility to to be extremely effective. Of course, it remains to
blast, were much more productive when grown be seen how the crop will cope under epidemic
together. For example, the glutinous rice variety conditions as in both years rice blast was not
produced 89% more rice as a result of mixed stand excessively damaging in this region. Nevertheless,
protection from rice blast. This meant that mixed there are clear lessons that can be learned from
stands produced 18.2% of the yield of a monocul- the study. First, the ability to reduce the area
ture field, even though only 9.2% of the area was planted to a given cultivar is always likely to limit
planted (Zhu et al. 2000). The mixed cropping pro- the risk that it will be seriously affected by a com-
cedure is now being reproduced over a 40,000 ha patible isolate, and second the mixed cropping
site in the current growing season and has proved itself appears to disrupt the incidence of disease,
 Molecular Variability Studies of Magnaporthe grisea and Their Application in Disease Contral
probably by disrupting the progression of new
disease foci within a field.
IX. Conclusions and Future Prospects
Molecular variability studies of M. grisea have
been extraordinarily informative in defining the
pathogen population and gaining an insight into
the me ans of disease propagation. It is clear that
M. grisea is predominantly a clonal organism,
reproducing by conidial production from disease
lesions. These spores are dispersed quickly within
rice fields and the importance of short-range dis-
persal from plant-to-plant, probably by splashes of
dew, is highlighted by the success of mixed crop-
ping for disease control. M. grisea epidemics
involve production of many thousands of spores
from diseased plants and it is therefore not sur-
prising that highly compatible, and presumably
aggressive, isolates predominate in some coun-
tries. The infiuence of agricultural systems is also
very apparent. In Europe and the Americas, where
rice cultivation is relatively new and dominated by
modern plant breeding, the introduction of culti-
vars carrying exotic resistance genes from numer-
ous genetic backgrounds has clearly exerted a
selective pressure on the pathogen population
such that a few compatible clon al lineages of the
fungus predominate. This is obviously also infiu-
enced by the founder population of the pathogen
that may have also limited genetic variability. A
converse situation exists in Asia where the long
history of rice cultivation and the huge number of
tradition al cultivars grown have meant that the
pathogen population is more diverse, although
predominantly spread as successful clonally
propagating lineages. Rice pathogenic isolates are
characterised in both regions as being diverse
in terms of electrophoretic karyotype and the
genomes may therefore tolerate, or accumulate,
genomic re arrangements in the absence of
meiosis. Such changes have the potential to gen-
erate new virulence forms by disruption of aviru-
lence gene function, and may thus infiuence the
population by subsequent selection. Finally, at
the centre of origin of rice, and by inference the
pathogen, there is evidence of sexual recombina-
tion infiuencing the variability of M. grisea popu-
lations, perhaps as it did before the widespread
cultivation of rice and consequent propagation of
the disease over a very large region of the planet.
165
What are the consequences of these discover-
ies for control of M. grisea on rice and other hosts?
One immediate consequence is the use of lineage
exclusion breeding which has already been very
successful. The cultivar Oryzica Llanos 5, for
example, has shown durable resistance to rice
blast for over 10 years of cultivation in Colombia
(Zeigler 1998).
Combinations of resistance genes identified in
Thai populations of rice show similar promise and
the leadership of CIAT and the International Rice
Research Institute in promoting the incorporation
of these methods has been central to their pro-
motion and dissemination to national breeding
programmes.
Bringing together the information gained
from population studies and the novel cropping
procedure tested in the Yunnan province, China,
mayaiso prove to be extremely beneficial for rice
production in developing countries. For example,
if lineage assignment can be used to define the
virulence capacity of a related group of M. grisea
isolates, and thus the exclusion capacity of the
prevalent rice cultivars, then this information can
be assimilated into breeding programmes. Where
possible, combinations of resistance genes can be
introgressed into high-yielding cultivars with the
milling qualities and culinary characteristics
required. Where complete exclusion of the lin-
eages prevalent in a given region cannot be
achieved in this way, then mixed cropping of cul-
tivars with distinct resistance spectrums can be
used instead to exclude the majority of pathogen
genotypes in the area (Zhu et al. 2000). This type
of disease management, combined with seed
hygiene and continual disease monitoring (Teng
1994), may provide the best type of durable
protection from rice blast out breaks in these
countries. In this regard, the monitoring of nitro-
gen levels in a field and the application of silicon-
containing compounds can also be important
factors for disease management (Long et al. 2000;
Seebold et al. 2000).
The development of durably resistant rice
varieties by genetic manipulation of the defence
processes using cloned avirulence genes as induc-
ers shows enormous potential but remains
untested commercially. The degree of consumer
resistance in the main rice-growing regions of the
world is also unknown and the distribution of such
cultivars to the developing world may not be suf-
ficiently lucrative for private sector investment.
Developing an understanding of the molecular
166 N.J. Talbot
interaction between the pathogen and its host
during a resistance response is the first critical step
in developing biotechnological strategies for
disease control, and the re cent report by Jia et al.
(2000) is hugely significant in this context. The
prospect of utilising avirulence genes for induction
of plant resistance to a large variety of pathogens
is an extremely worthwhile goal, with some
prospect of success (Kamoun et al. 1999), and one
which offers perhaps the greatest long-term hope
for effective durable control of this destructive
pathogen.
Acknowledgements. I am grateful to Morris Levy,
Robert Zeigler, Jim Correll, Mark Farman, Jean-
Loup Notteghem and Marc-Henri Lebrun for dis-
cussions and writings that have greatly infiuenced
my thinking regarding the population dynamics of
rice blast. I am also grateful in particular to John
Taylor, James Brown and Mark Macnair for dis-
cussion of this in the wider context. My own
research regarding pathogenicity mechanisms of
M. grisea is funded by the Biotechnology and
Biological Sciences Research Council.
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10 Transposable Elements in Fungal Pathogens: New Diagnostic Tools

DIANA FERNANDEZ 1 and THIERRY LANGIN2

CONTENTS iors and life histories that have direct implications

I. Introduction .......................... .
11. Transposable Elements in Fungi .......... .
A. Description .......................... .
1. Class I Elements .................... .
2. Class 11 Elements ................... .
B. Isolation Strategies .................... .
111. Transposons as Repeated Sequences
for Fingerprinting ..................... .
A. Introduction .......................... .
B. Characterizing Clonal Lineages in Fungal
Species with Exc\usive Asexual
Reproduction ........................ .
1. Magnaporthe grisea .................. .
2. Fusarium oxysporum ................ .
C. Identifying Clones in Fungal Species
with Sexual and Asexual Reproduction .... .
1. Sclerotinia sclerotiorum ............... .
2. Mycosphaerella graminicola ........... .
D. Typing Pathogenic Isolates:
Afutl in Aspergillus fumigatus ........... .
IV. Transposon-Inserted Sequences
as PCR Targets ....................... .
A. TE-Based Diagnostic PCR Tests:
Presence/ Absence of an Amplified Fragment
1. Introduction ....................... .
2. The Fusarium oxysporum Paradigm ..... .
B. Rep-PCR in Magnaporthe grisea .......... .
V. Conc\usions .......................... .
References ........................... .
I. Introduction
Knowledge of the population genetics of fungi has
increased notably in the last decade thanks to the
use of molecular markers that have made possible
more precise identification of the fungal genetic
entities composing natural populations (Leung
et al. 1993; Brown 1996; McDonald 1997). Within
fungi there is a great diversity of biological behav-
1 Unite Resistance des Plantes aux Parasites (UR 075),
Institut de Recherche pour le Developpement (IRD), 911,
avenue Agropolis, BP5045, 34032 Montpellier, France
2 Laboratoire de Phytopathologie Moleculaire, Institut de
Biotechnologie des Plantes (IBP), Bat. 630, Universite
Paris-Sud, 91405 Orsay Cedex, France
171 for the genetic structure of fungal populations.
172 Many filamentous fungi are capable of reproduc-
172 ing both sexually and asexually and the relative
172
172 contribution of each of these modes of reproduc-
172 tion can have major implications for their genetic
population structure (Brygoo et al. 1998). In par-
174 ticular, many pathogenic fungi displaya predomi-
174
nantly asexual reproductive phase in their life
cycle; as a consequence, large fungal populations
175 may be composed of a relatively sm all number of
175 genetically distinct clones. New clones may arise
179
through infrequent sexual reproduction and indi-
182 viduals within clones may evolve through the
182 accumulation of mutations.
183
Assessment of the level of genetic variability
184 in pathogenic fungal populations is extremely
important for disease management and epidemi-
184 ology (Rogers 1995; Milgroom and Fry 1997).
184 Furthermore, identification of individuals may be
184 required to differentiate and track different clones
185 in a population and to detect long-distance dis-
187 persal of clones between subpopulations. Migra-
187
188 tion of pathogenic isolates may be an important
source of contamination and may seriously affect
the genetic structure of recipient fungal popula-
tions (McDermott and McDonald 1993; Rogers
1995).
Molecular markers that can discriminate
clones in a population with significant clon al
reproduction are a great help when examining the
genetic population structure of fungal species on
a fine spatial scale. In addition, in plant and animal
pathologies, diagnostic tests are required for the
identification of a pathogenic fungal species or a
pathogenic group within a species. Tests need to
be quick, accurate and reliable and also highly
sensitive in order to detect the lowest possible
number of fungal propagules.
Transposable elements (TEs) combine several
advantages as molecular markers. First, they are
considered to be neutral markers and, as such,
highly suitable for analyses of population genetics.

The Mycota XI
Agricultural Applications
Kempken (Ed.)
© Springer-Verlag Berlin Heidelberg 2002
172 D. Fernandez and T. Langin
Second, they gene rally have a moderate to high
eopy number dispersed in the nudear genome
thus providing a multilocus signature (fingerprint)
when used as DNA probes in Southern experi-
ments. Because of the evolutionary dynamics of
TEs, intraspecifie polymorphism levels displayed
by TE-fingerprinting are usually high, enabling
population studies at a fine spatial scale. Finally,
isolation of TE copies and their flanking-genomic
regions can provide useful sequenee data for the
development of monolocus analyses or poly-
merase chain re action (PCR)-based detection
tests.
Since their characterization in fungal species,
some TEs have been widely used as molecular
markers (Table 10.1) to (1) identify clones and
clonal lineages in populations (Levy et al. 1991;
McDonald and Martinez 1991; Kohli et al. 1992;
Milgroom et al. 1992; Tantaoui et al. 1996;
Debeaupuis et al. 1997), (2) develop diagnostic
tests to detect plant pathogenic isolates
(Fernandez et al. 1998; Chiocchetti et al. 1999), (3)
type strains of industrial interest (Kempken and
Kück 1996), (4) detect sub divisions within fungal
species (Hamer et al. 1989; Giraud et al. 1997,
1999), and finally (5) genetically map potentially
useful genes (Romao and Hamer 1992; Dioh et al.
2000).
Several reviews on TEs in fungi have been
published in the last decade (Daboussi and Langin
1994; Daboussi 1996, 1997; Kempken and Kück
1998; Kempken 1999). The aim of this chapter is
to give an overview of the significant advances
conferred by using TEs as diagnostic tools in
fungal species. We will focus he re on studies rele-
vant to analysis of population genetics, i.e., analy-
ses of the genetic diversity within species and its
distribution among populations, and assessment of
the evolutionary dynamics of fungal populations.
11. Transposable Elements in Fungi
A. Deseription
Since their discovery, transposable elements (TEs)
have been detected in all organisms in which they
have been studied. Moreover, they probably
represent more than 10% of the genome. While
some authors consider TEs to be selfish or para-
site DNA, many data suggest they can generate
genetie novelties that may be retained by natural
selection. As a major and probably ancient com-
ponent of all genomes, TEs influence their evolu-
tion, size, behavior and functioning. The impact of
TEs on the host genome, generating genetic vari-
ability, is directly due to their ability to transpose
or indirectly by homologous recombination events
involving ectopic copies of TEs.
1. Class I Elements
According to their mode of transposition, TEs can
be divided into two major dasses (Finnegan 1989;
Capy et al. 1997). Mobile genetic elements that
use RNA intermediates to transpose are collec-
tively called retroelements or dass I elements.
Retroelements fall into two major subclasses,
depending on the presence or the absence of long
terminal repeats (LTRs). The non-LTR retroele-
ments are supposed to represent the more primi-
tive type of elements. LTR retrotransposons are
structurally similar to integrated retrovirus. They
are divided into two different subgroups on the
basis of gene order and sequence similarities.
2. Class 11 Elements
Class 11 elements are nucleic acid sequences that
are able to move from one chromosomal site into
a new site via a DNA intermediate. These ele-
ments contain inverted repeats (ITRs) at their
termini and encode a transposase that catalyzes
transposition. These TEs are classified using dif-
ferent criteria such as their transposase sequences,
the size and the composition of their ITRs, and the
nature of their target site duplication.
In re cent years, a great deal of data has been
accumulated concerning the occurrence and the
distribution of TEs in fungal genome. These data,
mainly obtained through works developed in field
or industrial strains, provide evidence that TEs are
ancient and common components of all fungal
genomes. Now, TEs identified in different species
of fungi reflect the whole spectrum of known
eukaryotic transposable elements (Daboussi 1997;
Kempken 1999). Some of these TEs are still active
and they will provide a valuable tool to tag genes.
B. Isolation Strategies
Fungal TEs have been identified using different
strategies: cloning of repetitive sequences, trans-
poson trapping approach or heterologous
hybridizations (Daboussi 1997; Kempken 1999).
Table 10.1. List and some characteristics of transposable elements used as diagnostic tools in fungi

Transposable Species Class/family Size (kb) Copy no. Diagnostic for Reference( s)
element

MGR586 Magnaporthe grisea IIlFot1-Pogo 1.86 40-50 in rice pathogens M. grisea pathogenic to rice Hamer et al. (1989);
Farman et al. (1996a)
Pot2 Magnaporthe grisea IIlFotl-Pogo 1.86 -100 M. grisea pathogenic to rice Kachroo et al. (1994);
George et al. (1998)
MAGGY Magnaporthe grisea I1gypsy 5.64 1-100 M. grisea pathogenic to rice Farman et al. (1996b);
and Setaria Shull and Hamer (1996)
Grasshopper Magnaporthe grisea I1gypsy 8.0 >10 M. grisea pathogenic to Eleusine Dobinson et al. (1993); ;::;i
Viji et al. (2000)
~
Fotl Fusarium oxysporum II1Fotl-Pogo 1.92 0-100 F. oxysporum f. sp. albedinis; Daboussi et al. (1992); '"0
'"'"
F. oXY5porum f. sp. Fernandez et al. (1998); 0
dianthi races Chiocchetti et al. (1999) '"ü
'(D"
Impala Fusarium oxysporum II1Tc1-Mariner 1.28 <10 F. oxysporum f. sp. dianthi races Langin et al. (1995);
Chiocchetti et al. (1999) tTl
(D
Palm Fusarium oxysporum I Not known 0-30 F. oxysporum f. sp. elaeidis Mouyna et al. (1996) S
(1)
TfoI Fusarium oxysporum II1hAT 2.76 5-25 F. oxysporum pathogenic Namiki et al. (1994); ::;.
to cucurbits Okuda et al. (1998) '"Ei"
Crypt-I Cryphonectria II1hAT C. parasitica clones Milgroom et al. (1992);
'Tj
parasitica B.I. Hillman (pers. comm.) ~
~
pLK44.20 Sclerotinia sclerotiorum I1non-LTR -12 S. sclerotiorum clones Kohn et al. (1991); Baller (1992) IJQ
AfutI Aspergillus Jumigatus I1gypsy 6.91 -10 A. Jumigatus strains Girardin et al. (1993); eo.
'"0
pathogenic to humans Neuveglise et al. (1996)
Boty Botrytis cinerea I1gypsy 6.0 0-15 distinguish transposa, Boty and Diolez et al. (1995) '"SC0
IJQ
vacuma B. cinerea strains (1)
~
Flipper Botrytis cinerea 11 1.84 0-20 distinguish transposa and Levis et al. (1997) ~
vacuma B. cinerea strains Z
(1)
Restless Tolypocladium infiatum 11 4.1 15-20 T infiatum ATCC34921 Kempken and Kück (1996) :e
identification tJ
pSTL70 Mycosphaerella I Not known M. graminicola clones S;.
McDonald and Martinez «1991); IJQ
graminicola Goodwin and Cavaletto (1999) ~
0
;!;.
(S.

f-'
--..)
v.>
174 D. Fernandez and T. Langin
The two most important and fruitful strategies are
the analysis of repeated sequences and the use of
a trapping approach. Cloning of dispersed repeti-
tive sequences is based on the fact that TEs
represent probably the major component of repet-
itive DNA sequences in many eukaryotes. This
strategy does not provide information about the
activity of the TE-like sequences identified. The
trapping approach is based on another property of
TEs, i.e., their capacity to generate spontaneous
mutations through their insertion in known gene
sequences. For example, the use of the nitrate
reductase structural gene (nia), as a target gene,
has led to the identification of active transposons
in different fungal species.
Some fungal TEs have been identified
through extensive DNA sequencing. The major
sequencing initiatives now underway into the
sequence determination of the complete genome
of different fungal genomes may extend our
understanding of the diversity of fungal TEs and
their importance in the structural and functional
organization of fungal genomes.
111. Transposons as Repeated Sequences
for Fingerprinting
A. Introduction
Initially, the term DNA fingerprinting described
a technique for detecting genetic differences
between human individuals at a large number of
hypervariable DNA loci in a single test (Jeffreys
et al. 1985). Since then, the term has been
extended to any multilocus analysis that can dif-
ferentiate between individuals or groups of closely
related individuals. The similarity of the finger-
prints generated by analyzing the DNA of dif-
ferent individuals gives an estimate of their
relatedness. DNA fingerprints have proven to be
highly efficient tools for addressing important
questions in clinical, forensic and evolutionary
dynamic studies. In plant and animal pathologies,
fingerprinting methods have direct applications
for epidemiological surveys and for the detection
of sources of inoculum, the identification of clones
and clonallineages, and for the control of migra-
tion (estimation of gene fiow).
Repetitive sequences such as TEs have been
used as fingerprinting probes for assessing rela-
tionships among individuals because they provide
data on a large number of loci simultaneously.
Nevertheless, some basic concepts have to be
taken into consideration, especially in diploid
organisms, when using fingerprint data to estimate
individual relatedness (Lynch 1988). First, alter-
nate alleles should be identified at each locus (and
each locus must be precisely identified, which is
difficult to ascertain because of possible comigra-
tion of nonallelic markers); second, fingerprint loci
must be genetically unlinked in order to avoid
redundancy of information; and, third, Mendelian
inheritance of markers is required. In fungal
species, stability of fingerprints through asexual
reproduction is also aprerequisite.
Theoretically, TEs should fulfill these require-
ments. In a simple model of transposition, there
should only be two alternate alleles at each locus
based on the presence or absence of a TE copy.
This is generally true if truncated copies that
might provide additional length alleles are
excluded. If unidentified, the truncated copy might
be scored as a new locus with two alleles. Never-
theless, in F. oxysporum f. sp. albedinis, where a
truncated copy of Fot1 was isolated and used as a
target for specific peR detection of the parasite,
the full-Iength TE copy could not be detected but
only the two alleles corresponding to presence or
absence of the truncated copy that were present
in the whole sampie tested (Fernandez et al. 1998).
Traditionally, DNAs sampies to be finger-
printed should be digested with an endonuclease
which does not cut the TE, and restricted frag-
ments should then be hybridized with the TE
sequence after separation by electrophoresis and
Southern transfer onto a Nylon membrane. Each
hybridizing band should then correspond to the
"presence" allele of each locus, the migration dis-
tance on the gel characterizing the locus. This is
in contrast to other repetitive probes used for
fingerprinting, where allelic fragments cannot
be identified with certainty. Ideal TEs to use as
fingerprinting probes should hybridize with an
average number of 10-30 DNA fragments to give
easily scorable data (Table 10.2).
Because ofthe mobile nature ofTEs and their
dispers al on several chromosomes, no genetic
linkage among copies should be detected;
however, more data are required to determine if,
for instance, new copies ofTE could arise from the
same "active" copy or if preferential insertion or
deletion of transposons could occur at particular
loci ("hot-spots" of transposition). In Sclerotinia
sclerotiorum, a high level of homoplasy has been
 Transposable Elements in Fungal Pathogens: New Diagnostie
Table 10.2. Suitability of transposable elements as diag-
nostie tools depending on their copy number in the fungal
genome
Technique TE copy no. Reference( s)
<5 5-30 >30
Fingerprin ting * Levy et al. (1991);
McDonald and
Martinez (1991);
Kohli et al. (1992);
Milgroom et al.
(1992); Tantaoui
et al. (1996);
Debeaupuis et al.
(1997)
* fragments cosegregated in each progeny set. In
PCR-based * * Femandez et al.
detection (1998); Chiocchetti
et al. (1999)
Rep-PCR * * George et al. (1998)
*Indieates suitability of TE
detected in the fingerprint data set, suggesting that
parallel insertion or deletion of the TE has
occurred at the same loci in strains with distinct
patterns of descent (Carbone et al. 1999). In
strictly asexually reproducing fungi (or those
assumed to be), it is expected that all fingerprint
loei will be linked, except for those with a high rate
of transposition. Fingerprints should thus provide
a multilocus signature that would be useful to
identify individuals, clones or clonal lineages
within fungal populations. Clon al lineage refers
here to groups whose members share relatively
re cent common ancestry and are related by clonal
descent. Within-lineage diversity may reflect
only moderate accumulation of mutations and
transpositions.
Finally, concerning Mendelian inheritance of
fingerprint markers, this may be true among
closely related strains within a species, but hori-
zontal transmission between species has been
demonstrated for the P and mariner elements in
Drosophila (Daniels 1990; Maruyama and Hartl
1991). However, alternative explanations have
also been proposed that fit with Mendelian in-
heritance of TEs (Capy et al. 1994a,b). In fungi,
severallines of evidence suggest the occurrence of
interspeeific exchange of TEs (Daboussi 1997;
Langin et al. 2002).
These prerequisites are important in fungal
populations undergoing meiosis, and in diploid or
multinucleate species where heterozygous indi-
175
viduals must be differentiated from homozygous
ones. A few studies involving TEs have confirmed
these points. In Cryphonectria parasitica, a haploid
ascomycete that causes chestnut blight, Milgroom
et al. (1992) investigated the use of the Crypt-1
transposon, which belongs to the hAT family of
Activator (Ac)-like transposable elements (B.!.
Hillman, pers. comm.), in population studies of the
parasite. They tested the segregation pattern of
fingerprint fragments in a laboratory cross as weH
as from progeny from a single peritheeium col-
lected from a field population. A 1: 1 ratio was
obtained, and only two out of the 12 hybridizing
Magnaporthe grisea, the MGR586 sequences were
shown to segregate in a Mendelian manner and to
be dispersed on several chromosomes (Hamer et
al. 1989).
We will now review some of the studies that
have been conducted in fungal species using TEs
as fingerprinting probes. The majority of the
studies were carried out on plant pathogenic fungi.
B. Characterizing Clonal Lineages in Fungal
Species with Exclusive Asexual Reproduction
1. Magnaporthe grisea
a) Transposable Elements Isolated
M. grisea (Hebert) Barr (anamorph Pyricularia
grisea) is a heterothallic ascomycete that is patho-
genie to a wide variety of gramineous hosts, and
causes one ofthe most devastating diseases (blast)
of cultivated rice (Ou 1985). Isolates collected in
the field show a limited infection spectrum and
develop the most severe disease symptoms on
their host of origin (Dobinson et al. 1993). The
pathotype of a rice blast isolate is typicaHy deter-
mined by assaying its infection spectrum on a set
of differential rice cultivars. Examination of the
diversity in virulence of the rice blast pathogen
has produced conflicting results and the level of
diversity of the pathotype and its stability over
time was the subject of much debate over aperiod
lasting several years, which consequently handi-
capped the development of strategies to control
the disease (Hamer 1991).
The search for repetitive DNA sequences as
genetic markers able to characterize the riee blast
fungus led to the successive isolation of several
active TEs (Table 10.1). Two dass 11 elements
were isolated, MGR586 (also called Pot3) and
176 D. Fernandez and T. Langin

Pot2 (Hamer et al. 1989; Kachroo et al. 1994; i. Lineage Designation and Population Genetic
Farman et al. 1996a) and at least five class I ele- Diversity of M. grisea
ments were recorded: MAGGY (alias Fosbury)
The MGR586 element occurs in 40 to 60 copies
and grasshopper, two gypsy-like LTR retroele-
that are weIl distributed throughout the genome
ments (Dobinson et al. 1993; Farman et al. 1996b;
of M. grisea pathogenic to rice (Hamer et al. 1989;
Shull and Hamer 1996a), and three non-LTR TEs
Romao and Hamer 1992). The extensive poly-
displaying characteristics of LINE elements
morphism of MGR586-hybridizing frag~ents
(MGR583) or SINE elements (Mg-SINE and
provided diagnostic RFLP patterns (fingerpn~ts)
MGSRl) (Hamer et al. 1989; Sone et al. 1993;
(Fig.10.1) and isolates can be clustered accordmg
Kachroo et al. 1995, 1997). Only Pot2 and Mg-
to their similarities into statistically robust groups
SINE elements were conserved among most ofthe
(Fig.1O.2). Given that M. grisea is believed to
M. grisea isolates tested. The other elements like
reproduce asexually over most of its geographical
MAGGY generally showed a discontinuous con-
range, groups sharing more than 80% similarity
servation pattern (Tosa et al. 1995), some of them
were referred to lineages and have been inferred
being restricted to certain subgroups: MGR586 is
to reflect clonal derivation from a common ances-
only present in rice pathogens and grasshopper
tor (Levy et al. 1991). There was a good corre-
was found exclusively in finger millet isolates.
spondence between these MGR586 lineages and
These features have the potential ability to differ-
groupings obtained by using VCG (Correll et al.
entiate between pathogens (Dobinson et al. 1993;
2000) or other molecular tools such as RFLP with
Farman et al. 1996b; Shull and Hamer 1996a; Viji
et al. 2000), and the strict association of MGR586
with rice pathogenicity has led to speculation that
most rice blast isolates diverged from those infect- A
ing nonrice species (Hamer et al. 1993).
Although the sexual stage of M. grisea can be
induced in the laboratory, it has not been reported
23.1kb A A A B B B B B C C 0 0 0 ODE E F G H H H H
in the field. Until recently, asexual reproduction
was thus believed to be the predominant if not the
9.4
exclusive mode of reproduction of the fungus over
most of its geographical range (Zeigler 1998). 6.6

Studies on M. grisea offer a good example of the

significant advances conferred by usin~ TEs ~s 4.4

multilocus DNA probes not only for dIagnostIc

purposes, but also to examine a range of fun?a-
mental questions concerning fungal populatIon
biology and genetics. 2.3
2.0

b) MGR586 and the Rice Pathogens

By far the most widely used fingerprinting pro.be
to characterize population diversity of the nce
blast pathogen was the MGR586 transposon B
cloned in a rice-infecting isolate (Hamer et al. 14.4kb
3.7
1989' Farman et al. 1996a). This class II element 2.3
was 'first described as a dispersed repetitive 1.9

sequence belonging to the wider family of Mag-

naporthe grisea repeated (MGR) sequences. One
of the key features of the MGR586 element
was the characteristic differential hybridization
pattern displayed between rice blast isola~es
and other nonrice-infecting M. grisea speCles
(Hamer et al. 1989; Borromeo et al. 1993; Viji et
al. 2000).
0.7
AAABBBBBCCDDDDDEEFGHHHH
Fig. 10.1. Photographs showing A MGR586 fingerprin~ing
and B Pot2 rep-PCR of 23 reference Pyricularia grisea 1S0-
lates. (Reprinted with permission from Correll et al. 2000)
 Transposable Elements in Fungal Pathogens: New Diagnostic 177

0.. 2 0.4
I 0.. 6 1;0
BhRl-1
BhRl-2
BhRl-3
BhRl-4
BhRl-5
L---BhRl-6
L-----BhRl-7
BhRl-8
BhRl-9
BhRl-l0 Unl Paro
BhRl-11 Th'

~
BhRl-12 Impu
BhRl-13
BhRl-14
BhR1-15
BhRl-l6
BhRl-17
BhRl-18
r----IC ~~~g6
. - - - - BhRl-2l
L-_ _ BhRl-22
BhR2-l ]
BhR2-2
BhR2-3 Un2 Thlmpu
BhR2-4 Punakha

]
BhR2-5
L______ ~~~=~~~~ ~~~r.~ Un3 Thimpu, Wangdi
BhR4-1
BhR4-2
, - - - - - BhR4-3
BhR4-4
BhR4-5 Paro
BhR4-6
BhR4-7 Lin4 Thlmpu
BhR4-8 Wangdi
BhR4-9
BhR4-l0
BhR4-1l
L_____ ~~==~==== BhR4·12
BhR4-l3
BhR5-1 ::J UnS Punakha
r - - - - - - ; -____-C==BhR6-1 ]
,-----t-------1- BhR6-2
BhR7-1
BhR7-2 ]
Un6
.
Lm7
Wangdi, Trongsa
Punakha, Trongsa
' - - - - - - - - L - - - - - - - 1 C ~~~1~ ] Un8
Punakha, Wangdi
~~~iJl:J Lin9 Wangdi
L - - - - - - - - - - - t - - - - - r...._ _ _ BhRl().2] Linl0 Punakha
BhR1().3
[ :r============:;:=======BhRll-l:J Linl1 Tro~sa
~------ _____r-____ BhR12-l:J Lln12 Tronasa
~~BhR1~1 ,~

~~~fr.~J Lin13 Wangdl

Fig. 10.2. Phylogenetic tree for Pyricularia grisea isolates based on the hybridization of EcoRI-digested DNA with
collected in Bhutan in 1995. The phenogram was derived the transposablc element MGR586. (Reprinted from
from restrietion fragment length polymorphism band data, Thinlay et al. 2000b with permission from Elsevier Science)

the cloned avr gene PWL2 (Zeigler et al. 1995) or
anonymous single- and low-copy probes (Kumar
et al. 1999), RAPD (Hong et al. 1996), and rep-
PCR using Pot2 (Fig.10.1; George et al. 1998;
Correll et al. 2000).
The MGR586 element proved to be a useful
tool far determining the genetic population struc-
ture of M. grisea pathogenic to rice at both micro-
and macro-geographic scales. DNA fingerprinting
studies conducted on some M. grisea populations
from the Americas, Europe and Asia showed that
pathogen populations were generally composed of
a limited number of clonal lineages (Levy et al.
1991; Xia et al. 1993; ehen et al. 1995; Sivaraj et
al. 1996; Roumen et al. 1997; Kumar et al. 1999;
Correll et al. 2000). Most of these lineages are
restricted to specific geographical areas, and only
a few of them are found dispersed in more than
one country or growing region. Considerable
variation in the degree of lineage and fingerprint
diversity was observed between geographically
distinct populations. The highest level of genetic
diversity was recorded in the center of rice diver-
sity, in the Indian Himalayas, where 157 MGR586
fingerprints (representing 45 lineages) were de-
tected among 222 isolates (Kumar et al. 1999).
In contrast, only eight lineages were identified in
the whole United States and only four are COill-
178 D. Fernandez and T. Langin

monly found in contemporary isolates (Levy et al. contrast, observations of populations in the
1991; Xia et al. 1993,2000; Correll et al. 2000). Philippines indicate that they may have arisen
only through asexual propagation of a few
found~r lines that accumulated aHelic diversity by
ii. MGR586 Lineages and Recombination
mutatIOn and/or transposition (Kumar et al. 1999).
in M. grisea
The simple population structure observed in M. iii. MGR586 Lineages and Pathotypes
grisea pathogenic to rice was interpreted as the
Regarding virulence, c1assification of isolates
result of predominantly asexual reproduction.
withi~ MG R586 lineages was complex: lineages
In an exc1usively asexual reproductive mode,
were generaHy made up of multiple pathotypes
molecular evolution within a c10nal lineage is
but some pathotypes were also found in several
supposed to occur through accumulation of muta-
lineages (Levy et al. 1991, 1993; Xia et al.
tions and/or transpositions. Spontaneous minor
1993; Zeigler et al. 1995; Roumen et al. 1997;
changes in MGR586 fingerprints have been
Gnanamanickam et al. 2000). Diversity of patho-
detected under both field and laboratory condi-
types within lineages was dependent on the geo-
tions (Xia et al. 1993; Wu and Magill1995; Xia and
graphical populations and related to the history of
Correll1995). Shull and Hamer (1996b) examined
rice cultivation (Levy et al. 1991, 1993; Xia et al.
the meiotic and mitotic behavior of some
1993; Zeigler et al. 1995; Roumen et al. 1997;
MGR586 RFLPs in order to understand the mol-
Mekwatanakarn et al. 2000; Thinlay et al. 2000a).
~cula~ basis of this novel RFLP creation. They
. ?b~ervations that distribution of pathotypes
ldenhfied a hypervariable genetic locus that was
wlthm hneages was nonrandom indicated that the
subject to recurrent re arrangement during asexual
p.attern of virulence of the lineages might be spe-
propagation. Instabilities at this locus are caused
~lfic to hosts and allowed the definition of specific
by a number of molecular-genetic mechanisms
hneage-host combinations (Levy et al. 1993; Chen
such as re arrangement involving the MGR586
et al. 1995; Zeigler et al. 1995; Xia et al. 2000).
element and nearby single-copy sequences and
These data have been used to minimize testing for
insertion/deletion of another transpo~able
pathogenicity and to propose a plant-breeding
element (Fosbury) related to MAGGY (Farman et
approach termed "lineage exc1usion" in which
al. 1996b; SchuH and Hamer 1996a). Some authors
several resistances would be combined in one or
have pointed out that MGR586-fingerprint data
several rice genotypes to exc1ude available P.
should thus be interpreted with caution when
gris~a lineages from infection in a target region
making inferences regarding the c10nality versus
(Zelgler et al. 1994; Zeigler and Correa-Victoria,
sexuality of field populations. Unstable loci could
2000!. In the Kerala region of India, development
provide a false estimate in gametic equilibria tests
of thlS strategy by pyramiding the major resistance
with an apparent intermediate allelic frequency
~enes Pi-l and Pi-2 exc1uded the entire popula-
(Schull and Hamer 1996b).
tIon composed of 29 MGR586 fingerprints
Genetic recombination by heterokaryosis as
(Gnanamanickam et al. 2000). However as
well as by sexual reproduction mayaiso contribute
pointed out by Zeigler and Correa- Vic~oria
to generate new fingerprints. Evidence for recom-
(20~0), this breeding tool might not be appropri-
bination occurring in natural populations has been
ate m areas where populations of the fungus are
intensively explored in M. grisea (Zeigler et al.
complex. In Thailand, no combination of resis-
1997; Zeigler 1998; Kumar et al. 1999). Differences
tance genes would confer resistance across all
in MGR586-lineage composition and complexity
lineages (Mekwatanakarn et al. 2000). The
between populations may be explained by differ-
possibil~ty ?f virulence gene exchanges through
ent models of population dynamics and evolution
recombmatIOn between isolates is a serious threat
that depend on the geographical region. Tests for
to the durability of the resistance and cannot be
gametic equilibrium using single- or low-copy
exc1uded in these regions.
RFLP prob es indicate that M. grisea populations
from the Indian Himalayas may have undergone
iv. Conclusions
recombination (Kumar et al. 1999). These authors
c~nc1uded that sexual reproduction of M. grisea The MGR586 element has proved to be a useful
mlght occur in this region where fertile isolates too~ for a~alyzing M. grisea populations patho-
of the two mating types have been isolated. In gemc to nce and will likely be used for further
 Transposablc Elements in Fungal Pathogens: New Diagnostic
analysis such as tracking changes in local popula-
tions. To cite one example, the MGR586 probe was
recently used to investigate the possible causes of
the first rice blast epidemic in Bhutan (Thinlay et
al. 2000b). The high MGR586 haplotypic diversity
detected in M. grisea collections suggested that the
blast epidemic was not caused by the emergence
or introduction of a new highly virulent isolate of
M. grisea in this region. Examination of weather
data indicated that climatic conditions might be
the most likely parameter to explain the outbreak
of the disease (Thinlay et al. 2000b).
However, MGR586 fingerprinting is not well
adapted to infer relationships between world
populations pathogenic to rice. Although similar-
ity between lineages can be high in local popula-
tions, most MGR5861ineages share a few common
bands and do not provide suitable data for com-
parison of geographically distant populations.
Other tools such as vegetative compatibility
groups (VCGs) (Correll et al. 2000) or sequence
characterized amplified region (SCAR) markers
(Soubarere et al. 2000,2001) have been developed
that are more efficient for the detection of allelic
variation between isolates on the global scale.
These additional neutral markers might be useful
to infer relationships between strains in ancient
populations where MGR586 analysis have shown
a complex population structure (Zeigler and
Correa-Victoria 2000).
2. Fusarium oxysporum
a) Transposable Elements Isolated
Fusarium oxysporum is a common soil-borne
fungus that can cause destructive vascular wilt
disease in many agricultural crops (Messiaen and
Cassini 1981). Despite its highly conserved
morphology, extreme pathogenic divergence is
observed within the species and strains have been
classified into special forms based on their host
specificity (Armstrong and Armstrong 1981). The
formae speciales are distinguished by the ability
of their members to attack a limited taxonomic
range of host plants. F oxysporum also includes
nonpathogenic strains able to persist through
asymptomatic colonization of plant roots and
saprophytic growth on dead organic matter.
The sexual stage of the fungus has never been
observed, and F oxysporum is thus believed to
have an exclusively asexual mode of reproduction
(Gordon 1993; Gordon and Martyn 1997). As is
179
true in many soilborne fungal pathogens, the
apparent absence of a sexual stage may be com-
pensated by other processes that ensure genetic
exchange and diversity build-up. In F oxysporum,
genetic exchanges may occur via hyphal anasto-
mosis between strains but may be restricted
primarily to within VCGs. Hence the relative
importance of transposition as a mechanism that
generates variability was considered to explain the
diversification of the species observed (Daboussi
et al. 1992; Daboussi and Langin 1994).
The search for transposable elements was
successfully initiated by transposon trapping in
the nia gene co ding the nitrate reductase enzyme.
Selection of nia mutants is easily made through
resistance to chlorate, an analog of nitrate for this
enzyme, and four class 11 elements (Fotl, Fot2,
Impala and hop) were isolated (Daboussi et al.
1992; Daboussi and Langin 1994; Langin et al.
1995; Hua-Van et al. 1998). At the same time, the
Tfol transposon (hAT element) and the Foret
and Palm retroelements were obtained through
screening for repetitive sequences (Julien et al.
1992; Namiki et al. 1994; Mouyna et al. 1996;
Okuda et al. 1998). Using a third strategy (screen-
ing with a transposase sequence), Anaya and
Roncero (1995) isolated the re tropos on Skippy,
which belongs to the same gypsy family of TEs as
Foret (Julien et al. 1992). Recently, a family of
SINEs retroposons called Foxy was identified
through analysis of a gamma-irradiated mutant of
F oxysporum f. sp. lycopersici (Mes et al. 2000).
Two additional elements have been reported, but
have not yet been fully characterized, Folyt,
another hAT element (Gomez-Gomez et al.
1999), and Joyrider (Rosewich et al. 1999). Finally,
a detailed study of the genomic organization of a
family of Impala elements revealed that they were
located in regions containing some new transpos-
able elements: Han, which has several features of
retroposons, and several new class 11 elements
(Fotl-like, Ac-like and mimp) (Hua-Van et al.
2000). All these elements appeared to be tightly
juxtaposed or nested within others, forming clus-
ters of repetitive sequences that might be prone to
rapid reorganization (Hua-Van et al. 2000). The
large number ofTEs isolated in F oxysporum may
not be intrinsic to that species but may rat her
refiect the considerable research investment in
this pathogen.
Several of these elements, for example, Fotl,
Pa Im , and TfoI, have been successfully used in
studies of population genetics and in the diagno-
180 D. Fernandez and T. Langin

sis of F. oxysporum forms pathogenic to important and molecular marker variations were examined
agricultural crops (Mouyna et al. 1996; Ouinten between F. oxysporum f. sp. albedinis isolates
1996; Tantaoui et al. 1996; Okuda et al. 1998). We collected over the entire geographical range of
will highlight some examples below. In addition, distribution of the disease. Though high genetic
Fotl and Impala transposons were used as molec- similarity was demonstrated with several markers
ular targets to develop PCR tests to identify F. (VCG, RFLP, RAPD), hybridization of the Fotl
oxysporum f. sp. albedinis (Fernandez et al. 1998) probe on EcoRI-digested genomic DNAs of
and to differentiate some F. oxysporum f. sp isolates allowed the production of numerous
dianthi races prevalent in Italy (Chiocchetti et al. polymorphic fragments (Ouinten 1996; Tantaoui
1999), respectively (see Sect. IV). et al. 1996; Fernandez et al. 1997). More than
64 dosely related Fotl-hybridization patterns
were evidenced among 120 F. oxysporum f.
b) Fotl and Microevolution ofF. oxysporum
sp. albedinis isolates (Fig.10.3; Ouinten 1996;
J. sp. albedinis Tantaoui et al. 1996). Examination of the genetic
The DNA transposable element Fotl was first relatedness of strains based on common Fotl hap-
doned in F. oxysporum f. sp. melonis by transpo- lotypes grouped all isolates at 78% similarity. The
son trapping in the nia gene (Daboussi et al. 1992). authors thus conduded the occurrence of a single
This dass 11 element is a member of the new pogo donallineage within the African F. oxysporum f.
superfamily of TEs, distributed in insects, humans sp. albedinis populations (Ouinten 1996; Tantaoui
and fungi.
Several lines of evidence indicate that FotI
is an ancient component of the F. oxysporum
genome (Langin et al. 2002). Active copies have
been identified for several strains and chromo- 1 2 3 4 5 6 7 8 9
somal rearrangements due to transposition have
been demonstrated (Deschamps et al. 1999;
Migheli et al. 1999). Southern blot tests have
showed that FotI is widely distributed in F.
oxysporum but that it displays a variable number
of copies (0 to more than 100) depending on the
strains or special forms considered. The discon-
tinuous distribution of this element may reftect
stochastic losses and/or re cent introduction by
horizontal transfer in several strains (Langin et al.
2001). Study of Fotl distribution could thus
provide insights about the evolutionary pattern
of pathogenic F. oxysporum strains. To give an
example, in F. oxysporum f. sp. vasinfectum, the
causal agent of Fusarium wilt in cotton (Gossyp-
ium spp.), race A isolates were totally devoid of
Fotl copy, in contrast to race 3 and 4 isolates which
harbored 7 and 3 copies, respectively (Dubois
1997). This, together with other genetic and mole-
cular marker results (Assigbetse et al. 1994;
Fernandez et al. 1994), indicates that the three
F. oxysporum f. sp. vasinfectum races have distinct
phylogenetic origins and may have independently
acquired pathogenicity to cotton species (Dubois
1997; Langin et al. 2000).
Use of FotI as a potential fingerprinting
probe has been successfully developed for F.
oxysporum f. sp. albedinis, the causal agent of
Fig. 10.3. Fotl-hybridization patterns obtained with 9
the Bayoud, a devastating disease of date palm isolates of F. oxysporum f. sp. albedinis. (D. Fernandez,
(Phoenix dactylifera L.) in North Africa. Genetic pers. comm.)
 Transposable Elements in Fungal Pathogens: New Diagnostic 181

et al. 1996; Fernandez et al. 1997). These observa- A

tions were consistent with the scenario involving
evolution in Morocco of a virulent clone and M 1 2 3 4 5 6
its subsequent geographic spread to Aigeria
(Louvet and Toutain 1981). The occurrence in dif-
ferent oases of F. oxysporum f. sp. albedinis iso-
lates with similar Fotl haplotypes was partially but
significantly correlated to the historical records of
the spread of the Bayoud disease in different
Aigerian regions over aperiod of one century
(Ouinten 1996).
Depending on the F. oxysporum f. sp. albedi-
nis strain, 9-13 copies of the transposon Fotl were
identified (Ouinten 1996; Fernandez et al. 1998).
Hybridization of a Fotl probe on chromosome
bands separated by pulse-field gel electrophoresis
showed the TE was weIl distributed throughout
the genome (Fig.10.4; D. Fernandez, pers. comm.).
B
Mitotic stability of haplotypes in the field has not
been directly measured. However, severallines of
evidence regarding the distribution and frequency
of several common haplotypes provided an
indirect estimate (D. Fernandez, pers. commun.;
Ouinten 1996; Tantaoui et al. 1996). Recovery of
representatives of the same Fotl haplotype over
30years and across long distances is a strong argu-
ment for the suitability of the Fotl probe for F.
oxysporum f. sp. albedinis population studies.
In conclusion, use of Fotl has proved helpful
in characterizing the population genetic structure Fig. 10.4. Distribution of Fotl transposable element on
of this important pathogen. It allowed confirrna- F. oxysporum f. sp. albedinis chromosomes. A Pulsed-field
tion of the existence of a single clonal lineage of gel electrophoresis separation of chromosomal DNAs of
10 isolates. B Southern hybridization of Fotl sequence of
F. oxysporum f. sp. albedinis in N orth Africa but gel presented in A. (D. Fernandez, pers. comm.)
the presence of multiple Fot1 haplotypes is also an
indication of the possible development of patho-
genic populations at a local scale. Consequently,
future efforts should be directed towards con- oil palm pathogen was needed to replace labori-
tinuously surveying local populations for tracking ous pathogenicity tests. InitiaIly, the Palrn element
changes of the date palm pathogen over time. As was selected as a RFLP probe for the numerous
a result of these studies, a PCR-based diagnostic DNA polymorphisms generated between F. oxys-
test was created to facilitate identification of the porum f. sp. elaeidis isolates (Mouyna et al. 1996).
fungus (Fernandez et al. 1998; see Sect. IV). As in the case of several TEs, the real nature of
this element was revealed through further analy-
ses after fingerprinting of the isolates had been
c) Palm and F. oxysporum f sp. elaeidis
achieved; sequence analysis showed that Palm
The transposable element Palm was randomly has the characteristics of a LINE-like, non-LTR
isolated from a genomic DNA library of F. oxys- retroelement (Mouyna 1994).
porum f. sp. elaeidis (Mouyna et al. 1996). This Irrespective of the geographical origin of the
fungus causes the most serious wilt disease of oil isolates, patterns of hybridization of the Palm
palm in many West African countries and has element sueeessfuily differentiated isolates that
recently spread to Brazil and Ecuador (Renard are pathogenic to oil palm from other nonpatho-
and Ravise 1986) but not yet to Asian countries genie F. oxysporum isolates isolated in palm grove
where oil palm is actively grown, which remain soils. Complex patterns of 8-29 bands of the F.
free of the parasite. A method of identifying the oxysporum f. sp. elaeidis isolates contrasted with
182 D. Fernandez and T. Langin
the simple banding pattern displayed by nonpath-
ogenic isolates (Mouyna et al. 1996). Besides char-
acterizing the oil palm pathogen, Palm haplotypes
refined previous VCG analyses by revealing the
geographical structuralization of F. oxysporum
f. sp. elaeidis populations in Africa (Dossa et al.
1991; Mouyna et al. 1996). Groups of Palm haplo-
types sharing more than 80% similarity generally
corresponded exactly to the geographical origin of
the isolates. African F. oxysporum f. sp. elaeidis
isolates showed the highest diversification
whereas American (Brazil, Ecuador) isolates dis-
played the same Palm haplotype as some isolates
from Cote d'Ivoire (Mouyna et al. 1996). Given
that American isolates also shared vegetative
compatibility alleles with the F. oxysporum f. sp.
elaeidis from Cote d'Ivoire (Dossa et al. 1991;
Dossa 1993), Mouyna et al. (1996) concluded that
they were probably of African origin and thereby
demonstrated the usefulness of the Palm retro-
transposon for detecting possible migration of the
pathogen.
However, this TE shows a wide distributiou
among F. oxysporum; by examining a reference
collection, Mouyna (1994) showed that complex
Palm haplotypes were also displayed in several
other special forms. In addition, nonpathogenic F.
oxysporum isolated from soil or roots of agricul-
tural crops in France also exhibited repetitive
patterns of hybridization with the Palm element
(Edel et al. 1995). Palm may thus be of interest for
fingerprinting F. oxysporum populations but
caution should be exercised if it is used for the
identification of F. oxysporum f. sp. elaeidis. Sub-
sequent pathogenicity tests will still be required
unless thorough DNA analyses have been carried
out on an extended set of isolates.
d) Tfol Separates Cucurbit-Injecting
F. oxysporum
As for the Palm element, Tfol was further char-
acterized from a randomly cloned DNA probe
used to fingerprint pathogenic F. oxysporum
(Namiki et al. 1994; Okuda et al. 1998). The
repeated dispersed probe FORL3 was isolated in
F. oxysporum f. sp lagenariae and used to detect
variation in F. oxysporum forms that cause wilt in
cucurbits (Namiki et al. 1994). Later, Okuda et al.
(1998) showed the probe contained the TjoI
element that belongs to the hAT family of trans-
posons, for instance, the maize transposon Activa-
tor (Ac) (Pohlman et al. 1984).
F. oxysporum spp. causing wilt in cucurbits
have been subdivided into six formae speciales
that are basically host specific and can be distin-
guished by their host species (Armstrong and
Armstrong 1981). However, the infection spectra
of cucurbit-infecting F. oxysporum is complex thus
rendering identification of forma specialis labori-
ous. In addition, some cross-infectivity of these
formae speciales has been detected (Kim et al.
1993). Genetic (VCG) and molecular (mtDNA
RFLP) analyses detected variability both within
and between these special forms but no direct cor-
relation with host specificity could be evidenced
until TfoI was cloned (Jacobson and Gordon 1990,
1991; Larkin et al. 1990; Kim et al. 1992; 1993;
Namiki et al. 1994). The different copy number of
Tfol and DNA banding patterns between isolates
allowed the six cucurbit-infectingjormae speciales
to be distinguished when tested with the probe
FORL3 (Namiki et al. 1994). The wide distribu-
tion of this TE in strains infecting cucurbits led the
authors to speculate that Tfol may have invaded
the genome of F. oxysporum prior to pathogenic
specialization, and that clonal organization of
these special forms may have resulted in further
independent evolution (Okuda et al. 1998).
However, this analysis was limited to 50 strains
collected exclusively in Japan. Although it may
be of interest for controlling wilt diseases in that
particular region, it needs to be validated on an
extended set of isolates from different geographi-
cal areas. To illustrate this point, variability in Tfol
haplotypes was detected within the special forms
(Namiki et al. 1994); in particular, F. oxysporum f.
sp melonis was clearly subdivided into several
TfoI-haplotype groups that corresponded to path-
ogenic groups (Namiki et al. 1994, 1998). It is
likely that new Tfol-fingerprinting groups will be
detected within formae speciales such as melonis
or niveum where variation has been detected
in pathogenicity, vegetative compatibility and
mtDNA RFLPs (Larkin et al. 1990; Jacobson and
Gordon 1991; Kim et al. 1992, 1993).
C. Identifying Clones in Fungal Species
with Sexual and Asexual Reproduction
1. Sclerotinia sclerotiorum
Despite the fact that Sclerotinia sclerotiorum
(Lib.) deBary infects many crop and weed species,
little work has been devoted to developing
 Transposable Elements in Fungal Pathogens: New Diagnostic
molecular tools for diagnosis and early detection.
Evidence for genetic heterogeneity in Canadian
S. sclerotiorum populations, based on mycelial
incompatibility interactions between strains iso-
lated from a single field, led Kohn's team to
analyze the genetic structure of the pathogenic
populations at a local scale (Kohn et al. 1991;
Kohli et al. 1992, 1995; Kohn 1995; Cubeta et al.
1997). This ascomycetous fungus has a haploid
somatic phase and can reproduce both asexually
via sclerotia and sexually by self-fertilization.
The major goal of the study was to determine if
clonality was the major reproductive system con-
trolling the evolution of agricultural S. sclerotio-
rum populations, and, subsequently, if novel
genotypes arose by mutation alone or also by
occasional recombination.
The authors used the cloned probe pLK44.20
containing a repeated dispersed element of
nuclear DNA trom S. sclerotiorum that dis-
plays similarities to the 3' end of a non-LTR-
retrotransposon (Baller 1992), to "fingerprint"
impressive quantities of S. sclerotiorum isolates.
The pLK44.20-containing element has proved to
be the most effective probe for detecting genetic
variation in S. sclerotiorum (Kohn et al. 1991;
Carpenter et al. 1999). An average of 12 hybridiz-
ing bands was observed on BamHI-restricted
DNAs (Kohn et al. 1991). The repetitive patterns
generated with the probe pLK44.20 were used to
define clones in association with mycelial compat-
ibility tests. A high level of genetic variation was
detected in agricultural populations, showing that
each field was infested by several S. sclerotiorum
clones (Kohli et al. 1992,1995; Kohn 1995; Cubeta
et al. 1997). Clone frequencies for petals (inocu-
lum phase) and disease lesions were significantly
different in one out of two years of sampling, indi-
cating either selection for some clones or that
immigration waves may occur during the disease
cycle (Kohli et al. 1995). In addition, fingerprint-
ing of the isolates indicated that clonal propaga-
tion of the pathogen can occur over long distances
in Canada (2000km), but not in North Carolina
(USA) or in New Zealand where local movement
of isolates was only suggested (Cubeta et al. 1997;
Carpenter et al. 1999). Evidence for common
origins of these fingerprint genotypes and an
understanding of their evolutionary relatedness
was only obtained with multilocus DNA sequence
data (Carbone et al. 1999; Carbone 2000).
Linkage disequilibrium tests were performed
on pairs of fingerprint loci to test for random asso-
183
ciation among fragments, and therefore to assess
the possible occurrence of recombination among
loci (Kohli and Kohn 1998). The random associa-
tion of a high number of pairs of fingerprint loci
was not consistent with exclusively clonal dyn am-
ics in S. sclerotiorum populations. Although the
possibility was not excluded that some of the
clonal variability of the pathogenic populations
may be brought about by transposition of the
pLK44.20-containing element, the authors con-
cluded that occasional genetic exchange and
recombination may be a source of new genotypes
in S. sclerotiorum (Kohli and Kohn 1998).
However, phylogenetic analysis detected a high
level of homoplasy (independent acquisition of a
character in two species) in the fingerprint data,
suggesting parallel transposition or excision of the
pLK44.20-containing element in strains that were
not phylogenetically related. In addition, phyloge-
netic analysis performed on another data set
including two genes and two nuclear sequences
detected little homoplasy and indicated a pre-
dominantly clonal mode of evolution in S. sclero-
tiorum with no evidence for contemporary
recombination between individual genotypes
(Carbone et al. 1999).
This result shows the limits in using TEs as
genetic markers for inferring phylogenetic rela-
tionships among strains and for deducing any
evolutionary factor shaping fungal populations.
The pLK44.20-containing element has proved
useful as a fingerprinting probe in identifying very
closely related strains (clones) and has helped in
discerning re cent episodes of divergence that had
not previously been detected in gene genealogies
(Carbone et al. 1999). In the analysis of divergent
populations, TEs will not be able to tell us more,
due to their intrinsic transposition properties and
the resulting drawbacks, such as the high level of
homoplasy in the generated data set.
2. Mycosphaerella graminicola
In the same way, McDonald and Martinez (1991)
examined the usefulness of fingerprinting Myco-
sphaerella graminicola isolates to discriminate
among different clones in a field population.
M. graminicola is an ascomycete causing leaf
blotch disease of wheat in many parts of the world.
Few data were available on the relative impor-
tance of asexual and sexual reproduction in the
genetic structure of M. graminicola populations
and the contribution of immigration to modifying
184 D. Fernandez and T. Langin
fungal diversity in local populations. The authors
used the repetitive dispersed DNA probe pSTL70
(McDonald and Martinez 1990, 1991), which was
shown to contain a partial TE sequence (Table
10.1; Goodwin and Cavaletto 1999). The probe
pSTL70 identified 21 of the 22 multilocus haplo-
types genera ted with eight anonymous single-
locus RFLP probes in a sampie of 93 M.
graminicola isolates. lsolates having the same mul-
tilocus haplotype and pSTL70 fingerprint were
assumed to be individual members of the same
clone (McDonald and Martinez 1991). However,
pairwise comparisons among clones based on
shared fingerprint bands did not provide the same
estimate of genetic similarity between isolates as
pairwise comparisons based on shared alleles at
individual RFLP loci (McDonald and Martinez
1991).
Hence, the probe was only used to quickly and
easily identify clones resnlting from asexual prop-
agation of the pathogen during the course of an
epidemic (Chen and McDonald 1996; Zhan et al.
1998). Data obtained showed that a very limited
portion of the population was clonal, which indi-
cates that the pathogen might display a high level
of sexual reproduction under field conditions and
may have the potential to rapidly recombine
new combinations of virulence genes (Chen and
McDonald 1996; Zhan et al. 1998).
As for the S. Sclerotiorum pLK44.20 probe,
pSTL70 has proved useful to estimate the number
of different clones in a natural population and to
c1arify the genetic population structure of an
important wheat pathogen, but the limits to its
usefulness as a neutral genetic marker have also
been demonstrated.
D. Typing Pathogenic Isolates:
AJutl in Aspergillus Jumigatus
Among human-infecting fungi, Aspergillus fumi-
gatus is an opportunistic fungus causing invasive
aspergillosis (lA) in immunocompromised pa-
tients in hospitals. Due to its presence in the
atmosphere, control of IA is becoming one of the
major tasks in bone marrow transplant units, but
the source of contamination is still unclear. The
AJutl retroelement was isola ted when searching
for repetitive sequences to fingerprint strains for
epidemiological purposes (Girardin et al. 1993).
An average of ten hybridizing fragments was
found using Afut1 as a probe on EcoRI-digested
DNAs of A. fumigatus isolates. The cloned
element displays features of gypsy retroelements
of Drosophila and is defective because of the accu-
mulation of stop co dons in all six reading frames
(Neuveglise et al. 1996). As a result, repetitive
hybridization patterns were highly conserved in
strains repeatedly subcultured for 20years or
passed several times through experimentally
infected mice (Neuveglise et al. 1997).
An extensive epidemiological study of A.
fumigatus and aspergillosis was conducted using
AJutl as a molecular probe and more than 1500
isolates from several sources and different hospi-
tals were typed (Debeaupuis et al. 1997; Chazalet
et al. 1998). Extremely high genetic variability was
observed among strains collected from all sources.
Strains were considered different when their
Afut1-hybridization patterns differed by at least
one fragment. Afutl fingerprinting of the isolates
yielded significant outcomes. First, the results
unexpectedly showed that IA outbreaks were not
caused by particular genotypes of A. fumigatus
and that practically any A. fumigatus strain was
potentially pathogenic; second, there was no
spatial structuring of isolates in the hospital,
showing that nosocomial infection of distinct
patients with the same Afut1 genotype could be
the result of independent inhalation of the same
strain at one distinct location rather than direct
contamination from one patient to another
(Debeaupuis et al. 1997; Chazalet et al. 1998).
These studies could have important implications
for controlling IA outbreaks in hospitals in that
they indicate that preventive measures should be
applied to any environmental A. fumigatus
conidia (Debeaupuis et al. 1997).
IV. Transposon-Inserted Sequences
as peR Targets
A. TE-Based Diagnostic PCR Tests:
Presence/Absence of an Amplified Fragment
1. lntroduction
Some TEs may be ideal targets for developing a
PCR-based diagnostic tool at the subspecies level
(special form, race, etc., we hereafter refer to path-
ogenic versus nonpathogenic strains). The strategy
developed to design specific primers is based on
the hypothesis that (1) one copy of the element
 Transposable Elements in Fungal Pathogens: New Diagnostic
would be inserted at a genomic location (locus)
which is common to all the pathogenic strains to
be identified, and that (2) no copy of the TE would
be present at this locus in other strains. Design of
primers overlapping the 3' or 5' end of the TE and
the corresponding genomic region of insertion
would lead to specific amplification of a chimeric
sequence (Fig.lO.5). Neither the TE sequence nor
the genomic sequence is specific to the pathogen
but amplification of this chimeric region would
only be achieved in strains bearing the TE at this
particular locus (Fig.10.5).
To enhance the reliability of a diagnostic
system based on the specific insertion of a TE copy
that might be still active, it is advisable to develop
at least two primer pairs targeted at two different
A pathogenic nonpathogenic
... ...
B F~
C t PCR
1----1
Amplilication of a o amplification
chimeric fragment
Fig. 10.5. Strategy for designing a PCR-based diagnostic
test using Fotl in F. oxysporum f. sp. albedinis. A Schema-
tized Fotl-hybridization patterns obtained with four path-
ogenic isolates and four nonpathogenic F. oxysporum
isolates. Arrow indicates common band in F. oxysporum f.
sp. albedinis isolates. B Example of genomie loeus harbor-
ing a Fotl eopy in pathogenie isolates and not in nonpath-
ogenie F. oxysporum. Arrows indieate primers designed
from the Fotl copy and the genomie loeus to amplify a
ehimerie region. C Sehematized eleetrophoresis showing
results of PCR amplifieation on DNAs of pathogenic and
nonpathogenie isolates using primers designed in B
185
copies and insertion sites. The pathogen can then
be detected by multiplex peR, using the different
primer pairs in combination in a single re action
tube.
Design of a TE-based PCR test thus requires
prior knowledge of the genomic distribution of the
element in pathogenic strains as well as in a large
sampie of nonpathogenic strains. This can be
assessed by Southern tests using the TE as the
probe on restrietion enzyme-digested DNAs.
Identification of hybridizing bands that are con-
served between pathogenic strains provides
preliminary evidence of potential for the develop-
ment of a TE-based diagnostic too1.
The main step for designing specific PCR
primers thus relies on obtaining the sequence of
the TE-inserted genomic regions. Two strategies
are available that have been successfully tested in
F oxysporum (Fernandez et a1. 1998; Chiocchetti
et a1. 1999): either (i) cloning of the TE copies
present in the genome of the pathogen together
with their fianking genomic sequences by con-
structing a genomic library, or (ii) cloning of DNA
fragments fianking TE copies by inverse PCR. The
second strategy is far more attractive because it
avoids constructing a genomic library and could
be used in pathogens where only a small number
of TE copies have been detected. After restrietion
of genomic DNA with an endonuclease that has
no site within the transposable element, restricted
fragments are self-circularized, and used as tem-
plates in PCR with two outward facing oligonu-
cleotides specific for the TE. The chimeric
amplified fragments contain the 3' and 5' fianking
regions delimited by the endonuclease recognition
site (Fig.10.6).
Further sequencing leads to subsequent
development of specific oligonucleotides to use in
combination with primers targeted at the 3' or 5'
end of the TE. PCR assays for identifying the
pathogen should be as exhaustive as possible.
2. The Fusarium oxysporum Paradigm
a) Identifying Fusarium oxysporum
J. sp. albedinis
Bayoud, the Fusarium wilt of date palm tree
(Phoenix dactylifera L.), has caused the death of
millions of trees. The disease has occurred since
1870 in Morocco and since the beginning of the
twentieth century in the western and central parts
of the Aigerian Sahara. It has never been reported
186 D. Fernandez and T. Langin

lates. In addition, the pathogen can easily be dis-

seminated through the exchange of contaminated
material, although, at present, strict phytosanitary
regulations are applied at the borders of date-
GENOMIC DNA palm growing countries that are free of Bayoud.
~ RESTRICTION
Advantage has been taken of the high genetic
relatedness among isolates pathogenic to date
P4 P1 P2 P3 palm (Quinten 1996; Tantaoui et al. 1996;
-i;,.mU+1·W'
~~ ~~

Fernandez et al. 1997; cf. Sect. IILC.1) to develop

a sensitive peR assay (Fernandez et al. 1998).
~ SELF-LlGATION
Genomic analyses had shown that, depending on
the F. oxysporum f. sp. albedinis strain, 15-26
EcoRI fragments hybridized with the Fotl
sequence (Tantaoui et al. 1996). Several of the
Fotl-hybridizing bands were conserved among the
isolates and might be good candidates as peR
targets. In addition, molecular analyses clearly dif-
ferentiated the date palm isolates from nonpatho-
I IPCR
genic F. oxysporum commonly isolated from
t (PlIP2) palm-grove soils (Tantaoui and Fernandez 1993;
P1 P4 P3 P2 Fernandez and Tantaoui 1994; Tantaoui 1994).
Fotl-hybridization patterns of F. oxysporum f. sp.
albedinis and other nonpathogenic F. oxysporum
~ NESTED
isolates were very different, with the non-
PCR
(P31P4)
pathogens exhibiting only 0-8 EcoRI-hybridizing
P4 P3 bands (Tantaoui 1994).
"""'" ..0=0
DNA clones containing a copy of the trans-
posable element Fotl were isolated from a
~ genomic library of the date palm pathogen
(Fernandez et al. 1998). Sequence analysis showed
SEQUENCING
and that one of the Fotl copies was truncated, lacking
PRIMER DESIGN
182-bp at its 3' terminus. Because this copy might

-
~ primer pair amplifying 204-bp overlapping the
P4
.:. ....
R ACE-SPECIFIC
PCR
Fig. 10.6. Strategy for isolating DNA fragments fianking
Fotl or Impala homologous sequences in Fusarium oxys-
porum by inverse polymerase chain reaction (IPCR). PI to
P4 correspond to primers in Fotl or Impala sequences.
(Reprinted with permission from Chiocchetti et al. 1999)
in any other area of date palm cultivation through-
out the world (Louvet and Toutain 1981).
Detection and identification of Fusarium
oxysporum f. sp. albedinis remains difficult, mainly
because inoculation tests are still required to
assess the pathogenicity of the F. oxysporum iso-
be unable to transpose, the authors selected a
Fotl truncated-copy and its 3' region of insertion
in the albedinis genome. More than 300 F. oxys-
porum f. sp. albedinis and 98 isolates encompass-
ing 17 other special forms and nonpathogenic F.
oxysporum were tested. The primer pair enabled
identification of 95% of the albedinis isolates. An
additional peR primer pair was selected from
another Fotl-containing clone to specifically
amplify the DNA of 100% ofthe albedinis isolates.
Less than 100pg of purified albedinis DNA was
detected. The combination of the two primer pairs
used in peR assays thus provided a useful diag-
nostic tool for F. oxysporum f. sp. albedinis
(Fernandez et al. 1998).
b) Detecting F. oxysporum j. sp. dianthi
F. oxysporum f. sp. dianthi is the most important
carnation pathogen and a sensitive detection tech-
 Transposable Elements in Fungal Pathogens: New Diagnostic
niqne was needed to produce certified pathogen-
free cuttings. Several genetic subgroups of the
pathogen had previously been characterized using
genetic and molecular markers, and ten races had
been reported worldwide (Manicom et al. 1990;
Manulis et al. 1994; Migheli et al. 1995, 1998;
Baayen et al. 1997). Foll and Impala genomic
insertions in F. oxysporum f. sp. dianthi were used
as targets to detect the pathogen at the level of the
race in Italy (Chiocchetti et al. 1999). The authors
started from the preliminary evidence that distri-
bution of the two elements Fotl and Impala was
associated with race in the 72 isolates tested: the
first hybridizing group included isolates of races 1
and 8, the second group included isolates of races
2, 5 and 6, and the third grouped ra ce 4 isolates.
All the isolates belonging to the same race shared
identical Fotl or Impala profiles. In contrast to the
situation prevalent in F. oxysporum f. sp. albedinis,
Fotl- and Impala-hybridizing signals varied
between one and six and three to eight, respec-
tively, in F. oxysporum f. sp. dianthi DNAs. An
inverse-PCR strategy was thus developed to
obtain TE-Banking sequences, and three indepen-
dent sets of primers showing specific race amplifi-
cation were designed. Two primer pairs were
derived from Fotl insertions and were diagnostic
for race 2 or races 1 and 8, respectively. The third
primer pair was obtained from an Impala insertion
and was diagnostic for race 4 isolates. In addition,
these primers were successfully used in multiplex
PCR to efficiently identify the pathogen at the
level of the race, and to allow direct detection of
the pathogen in diseased tissues (Chiocchetti et al.
1999).
B. Rep-PCR in Magnaporthe grisea
The repetitive-element-based PCR (rep-PCR)
allows random amplification of DNA sequences
lying between repeated dispersed sequences in
a genome by using specific primers directed
outward from each repetitive sequence. This tech-
nique thus detects length-polymorphisms between
interspersed seqnences at amplifiable distance and
can reveal differential insertions of TEs.
George et al. (1998) successfully applied rep-
PCR to the detection of sequences separating the
Pot2 copies in the M. grisea genome. Pot2 is a 1.8-
kb DNA transposon shared by rice and nonrice M.
grisea isolates and it is present at a copy number
of approximately 100 in each genome (Kachroo et
187
al. 1994). By using long PCR conditions and opti-
mized electrophoresis protocols, the high number
of Pot2 copies resulted in the amplification of a
number (ranging from 2 to 32, depending on the
isolates) of variable-length DNA bands that could
be easily scored, thus generating a multilocus
haplotype for each isolate (George et al. 1998).
Polymorphie patterns of amplification were dis-
played that differentiated riee from nonrice iso-
lates. In rice-infecting M. grisea, the patterns
obtained group the strains according to their pre-
viously determined MGR586lineages in the USA
and in the Philippines (Fig.10.1; George et al.
1998; Correll et al. 2000), but not in India (Gnana-
manickam et al. 2000). Analysis of inheritance of
PCR products showed that sequences Banking
Pot2 elements segregated as single-locus markers,
but tests for random association between locus
pairs suggested that 6 out of the 14 markers ana-
lyzed could be linked (George et al. 1998).
If carefully designed, the Pot2 rep-PCR thus
provides rapid diagnostic fingerprints that can
facilitate large-scale population studies of the rice
blast pathogen. Such an application of the PCR
technology is useful for TEs whose number of
copies is high, because it can be expected to
provide a greater number of repeated sequences
at amplifiable distance and thus to reveal poly-
morphisms between isolates.
The rep-PCR could then be successfully
applied to TEs that show complex hybridization
profiles on digested DNAs, such as Fot2, Fot!,
Afttt1, etc.
v. Conclusions
The numerous studies presented here clearly show
the potential benefit of using TEs as diagnostie
tools in fungal pathogenic species. TEs have been
found in many ascomycetous species and probably
exist in all fungi. Arecent report on TEs in a sym-
biotic basidiomycete (Murata and Yamada 2000)
indieates it is likely that such tools will also be
developed to study populations of ectomycorrhizal
fungi. In oomycetes, fungal-like organisms, several
retrotransposons have been recently reported (F.
Panabieres pers. comm.; Tooley and Garfinkel
1996; Panabieres and Le Berre 1999). Extensive
studies have been conducted on the potato late
blight pathogen, Phytophthora infestans, using the
dispersed repetitive sequence RG57 (Goodwin et
188 D. Fernandez and T. Langin
a1. 1992, 1994; Drenth et a1. 1994; Forbes et a1. 1998)
but its sequence did not reveal any obvious char-
acteristics ofTEs (S. Goodwin, pers. comm.). Such
prevalence of TEs in the fungal genome might
contribute to the genetic variability of the fungal
species. Further investigations are needed to
elucidate the extent to which TEs contribute to
the genetic ftexibility responsible for population
adaptation (Hua-Van et a1. 2000).
When used as fingerprinting prob es, TEs have
considerable potential to identify clones or clonal
lineages within populations. However, we have
seen that inferences about the evolutionary
biology of fungal populations are difficult to ascer-
tain using TE-multilocus data. Another approach
would be to use a TE at each individual genetic
loci for monolocus analyses in PCR-based experi-
ments by designing locus-specific primers from the
ftanking regions. This would give a plus/minus
polymorphism at each locus: plus when the trans-
posable element is present and minus when it is
absent. Allelic variation at each locus would
provide easily scorable data for studies of popula-
tion genetics.
In addition to their utility for fungal popula-
tion analysis and the development of diagnostic
tests, transposons are powerful research tools for
genetic analysis because of their ability to insert
into the genomic DNA of their host. For years now,
transposons have been engineered to carry and
insert a DNA sequence of interest into the
genomes of microorganisms, for applications such
as creating gene "knockouts" (insertion al mut-
agenesis), and genome mapping. Recently, a
highly efficient in vitro Tn5 transposition system
was developed (Goryshin and Reznikoff 1998)
which paved the way for further applications for
TEs such as sequencing large DNA fragments
without subcloning (http://www.epicentre.com/
transposomics.htm). Genetic manipulation of
pathogenic fungi is often difficult because of the
lack of a sexual stage; it would thus be desirable to
establish a tagging mutagenesis system to facilitate
the isolation of mutated genes. The fungal trans-
poson Impala is being used as a genetic tool to tag
genes involved in plant-fungal interactions in F.
oxysporum (Migheli et a1. 2000) and also in M.
grisea where the transposon is autonomous (Vil-
lalba et a1. 2001).11 is likely that other tools will be
developed from fungal TEs in the near future.
Acknowledgements. We are very grateful to IC.
Correll and L. Kohn for critically reviewing earlier
versions of this manuscript. IC. Correll, S.
Goodwin, B. Hillman, M. Milgroom, F. Panabieres
and D. Tharreau generously shared unpublished
data and information.
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11 Disease Management of Phoma Infections

KERSTN VOIGT 1 and JOHANNES W. WÖSTEMEYER2

CONTENTS I. Introduction
I. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . 193
11. Agricultural Importance of Brassica . . . . . . . 194
III. Epidemiology of Blackleg Disease . . . . . . . . 194 Agriculture has developed from isolated small-
IV. Genetic Variability of Phorna Ungarn scale units scattered around fertile soil areas to a
Supports a Multispecies Concept ......... 195 global, intensified industry. Reductions in yield
A. Variability in Pathogenicity Patterns ...... 195
B. Variability in Geographie Distribution . . . . . 196
and quality result in substantial financial losses
C. Variability in Mating Behavior . . . . . . . . . . . 196 and may affect whole continents. Therefore,
D. Variability at the Level of DNA schemes for effective disease management play
Fingerprints. . . . . . . . . . . . . . . . . . . . . . . . . . 196 a crucial role in modern agriculture. Disease
E. Variability at the Karyotype Level . . . . . . . . 197
F. Variability at the Chemotype Level ....... 198 management comprises all approaches to control
G. Nonaggressive Strains Are Related diseases in agro-ecosystems: disease prevention,
to Phorna wasabiae . . . . . . . . . . . . . . . . . . . . 198 pathogen exdusion, pathogen eradication, im-
V. Development of Novel Pathotypes: proved diagnosis and forecasting systems, breed-
Phylogeny of Phorna Ungarn. . . . . . . . . . . . . 199
VI. The Blackleg Complex: ing for resistance, and others. Appropriate methods
Infection, Colonization and Symptoms. . . . . 199 can be different in nature between cultural,
VII. Targets for Plant Proteetion ............. 201 chemical or biological measures. They aim at
A. Gene-for-Gene Interactions ............. 201 reduction of primary inoculum and at increased
B. Chemical and Structural Barriers
of the Host .......................... 202 plant resistance.
C. Chemical Response of Leptosphaeria Species of the genus Phoma are among the
rnaculans . . . . . . . . . . . . . . . . . . . . . . . . . . . . 202 most common pathogens of agricultural crops
D. The Use of Toxin Resistance worldwide. They cause many seed- and soil-borne
in Plant-Breeding Programs ............. 203
E. Canola Quality and Plant Proteetion ...... 203 diseases in the field. Minimizing the damage from
F. Phytoalexin Detoxification . . . . . . . . . . . . . . 204 these fungal agents is important to maintain pro-
G. Evaluation of Blackleg-Resistant ducer profitability, the viability of the food and
Cultivars ............................ 204
VIII. Diagnosis of Blackleg Disease ........... 205
feed industries, and the integrity of exports.
A. Conventional Disease Assessment ........ 205 Phoma comprises more than 2000 species.
B. Molecular Probes and Assays . . . . . . . . . . . . 205 Many Phoma species cause diseases on a wide
IX. Management of Blackleg Disease . . . . . . . . . 206 variety of crops, such as potato (P. exigua), beet
A. Cultural Control ...................... 206
B. Chemical Control ..................... 207 (P. betae), legurnes (P. medicaginis), ornamentals
C. Biological Control . . . . . . . . . . . . . . . . . . . . . 207 (P. chrysanthemicola, P. aquilina) and citrus
D. Breeding for Resistance ................ 208 plants (P. tracheiphila). The related species P.
E. Forecasting Blackleg Epidemics . . . . . . . . . . 210 lingam, P. siliquastrum, P. oleracea, P. brassicae,
X. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . 210
References .......................... 211 P. napobrassicae, P. wasabiae, P. nigrificans and
P. eupyrena attack crucifers and often appear in
mixed infections. P. lingam is economically the
most relevant species as an important pathogen
of crucifers worldwide. It causes Phoma leaf spot
1 Pilz-Referenz-Zentrum, Institut für Mikrobiologie, and stern canker associated with blackleg disease.
Friedrich-Schiller-Universität, Neugasse 24, 07743 Jena, P. lingam and its perfect form, Leptosphaeria
Germany maculans, have a pronounced pathotype structure,
2 Lehrstuhl für Allgemeine Mikrobiologie und
Mikrobengenetik, Institut für Mikrobiologie, Friedrich- which renders phytopathological assessment diffi-
Schiller-Universität, Neugasse 24,07743 Jena, Germany cult. Therefore, dia gnosis and analysis of genetic

The Mycota XI
Agricultural Applications
Kempken (Ed.)
© Springer-Verlag Berlin Heidelberg 2002
194 K. Voigt and IW. Wöstemeyer
variability in L. rnaculans and neighboring species
is highly important for understanding the spread
and management of the disease.
Although Phorna lingarn is the major
causative agent of blackleg disease, evidence is
increasing that a differentiated species complex
has to be taken into account. The wealth of geno-
types in the blackleg complex makes disease
management complicated. Defining appropriate
measures and targets for plant protection requires
understanding of infection biology and plant/
pathogen interactions at the molecular level.
Few reviews also encompass aspects in the
management of blackleg diseases caused by P.
lingarn (Gabrielson 1983; Gugel and Petrie 1992;
Kharbanda 1992; Kharbanda and Tewari 1996;
Gladders et al. 1998). For reviews dealing with the
disease management of plant pathogens other
than Phorna spp., see the appropriate chapters of
this volume.
11. Agricultural Importance of Brassica
Brassicas are economically among the most
important crops worldwide. Enormous amounts
of vegetable brassicas, such as broccoli (Brassica
oleracea var. botrytis), cauliflower (B. oleracea var.
italica) , kaIe (B. oleracea var. acephala) and a wide
variety of cabbages (B. oleracea) are consumed
annually. Oilseed brassicas constitute the third
largest source of edible vegetable oils, and Indian
mustard seeds (E. juncea) are a widely used
condiment. Brassica oiIs are also produced for
industrial use as the chemical basis for lubricants,
for soap production and for use as fuel (bio-diesel)
after methyl-esterification of the fatty acids.
Rapeseed (B. napus and B. rapa) ranks third in
the production of seed meal, which is used as a
protein source in animal nutrition (Pedras 1998a).
111. Epidemiology of Blackleg Disease
The causative agent of blackleg disease in Brassica
crops is Phorna lingarn (Tode ex Fr.) Desm. (syn.
Plenodornus lingarn), whose sexual form is
Leptosphaeria rnaculans (Desrn.) Ces. et de Not.
This loculoascomycete is a minor pathogen of
many crucifers but, in its stern canker or blackleg
phase, it is a devastating pathogen in oilseed rape
cultivation (Brassica napus L. var. oleifera). Black-
leg disease was first reported at the turn of the
nineteenth century (Hughes 1933), when the pro-
duction of cabbage, turnips or swedes domina ted
human nutrition, and rapeseed played a minor
role as pro tein and oil source for animal feeding.
Today, the disease occurs endemically in all
regions of the world where rapeseed cultivation
has a history. Major epidemies have occurred in
Germany (Krüger and Wittern 1985), Poland
(Jedryczka et al. 1999), France (Brun and Jacques
1990), the United Kingdom (Humpherson-Jones
1986; Gladders 1995; West et al. 1999; Zhou
et al. 1999), Canada (Hall et al. 1993; Rimmer
et al. 1995; Juska et al. 1997) and Australia
(McGee 1977; McGee and Emmett 1977; Plummer
et al. 1994; Salisbury et al. 1995).
Severe epidemics have been described on
winter oiIseed rape in Europe since 1950 (Gugel
and Petrie 1992). Blackleg became a problem in
autumn-sown oilseed rape in England in 1977
when the susceptible cultivars Primor and Rapora
were widely grown. Since then the disease has
become endemic in many rapeseed-growing areas.
The disease affected up to 90% of the winter
oilseed plants (Gladders 1995), threatened the
rapeseed production, and yield losses of up to 60%
were reported (Parry 1990). A lower incidence of
blackleg in 1979 coincided with the introduction
of the canker-resistant cultivar Jet Neuf, followed
by a long period of moderate disease incidence
until 1992. The problem became severe again
during 1993-1995. A detailed account of blackleg
occurrence in England in the years 1977-1995 is
given by Gladders (1995).
In Australia, rapeseed farming began in the
mid-1960s with varieties introduced from Canada
and increased rapidly until the early 1970s, when
farmers recognized the potential of this alternative
cash crop (Salisbury et al. 1995). The Canadian
cultivars, however, proved to be highly susceptible
to blackleg, and, by 1972, two years after the intro-
duction of Canadian spring rapeseed cultivars,
severe blackleg disease epidemics virtually elimi-
nated the rapeseed industry in Western Australia.
An area of 49,000ha shrank to 2000ha in 1974
(Gugel and Petrie 1992; Salisbury et al. 1995). Due
to the release of the first blackleg-resistant culti-
vars in 1978-1980, farmers regained interest in
rapeseed, and production increased considerably.
In 1995, an estimated 430,000ha were sown with
oilseed rape, with a production exceeding 600,000
tons (Wratten et al. 1995).
 Disease Management of Phoma Infections 195

In Canada, prevalence and severity of stern

canker were greater in winter than in spring
oilseed rape: Over a 4-year period between 1986
and 1989, maximum estimated yield los ses in
a total of 256 fields examined were 29.2% in
winter rapeseed and 8.8 % in spring rapeseed (Hall
et al. 1993). Since 1975, blackleg disease has
spread throughout the Canadian provinces of
Saskatchewan and Manitoba and into large areas
of Alberta (Rimmer et al. 1995). By 1978, the
pathogen had been reported worldwide, in 49
countries on all continents (Gabrielson 1983).

IV. Genetic Variability of Phoma

Ungam Supports a Multispecies
Concept

The taxonomy of P. lingarn and related species is

still a matter of debate. The taxon appears to com-
prise several species that are morphologically
similar (Purwantara et al. 2000), but differ in path-
ogenicity and in cultural, molecular and isozyme
characteristics (Rouxel et al. 1994). A general
view has been expressed and substantiated at dif-
ferent experimental levels that aggressive (A) and
nonaggressive (NA) isolates of Phorna lingarn
may belong to different species (Koch et al. 1991;
Taylor et al. 1991; Williams 1992; Morales et al.
1993a,b; Rouxel et al. 1994,1995; Voigt et al. 2001).
Genetic variability at different levels supports the
multi-species concept of P. Ungarn.

A. Variability in Pathogenicity Patterns

Isolates of P. lingarn possess a considerable vari-
ability in pathogenicity and aggressivity. Based on
aggressivity on oilseed rape, the isolates of P.
lingarn were divided into two major pathogenicity
groups, aggressive (A) and non-aggressive (NA;
Hammond and Lewis 1987; Badawy and Hoppe
1989; Koch et al. 1989; Williams and Fitt 1999).
Although the NA-isolates comprise a broader
host range on crucifers and their importance
may have been underestimated in field studies
(Johnson and Lewis 1994), the A-pathogenicity
group is economically more relevant (Hall 1992).
Exclusively, isolates of the A-group are capable of
systemic spreading in plants, where they cause leaf
spots, corticallesions and stern canker formation
(Fig. 11.1; Gabrielson 1983). The A-group leads
to severe losses in seed yield and seed weight
Fig. 11.1. Symptoms of blackleg disease on Brassica napus
(Hall et al. 1993) exceeding, in Canada alone, $30
million annually (Pedras 1995). In the United
Kingdom, losses have been estimated as in excess
of BO million in winter oilseed rape (Brassica
napus var. oleifera) for each season between 1993
and 1996 (Fitt et al. 1997). The NA-pathogenicity
group of P. lingarn is recognized by superficialleaf
and stern lesions, normally without major effect on
crop yield.
In pathogenicity tests with Brassica cotyle-
dons, NA-strains cause no differential reactions,
whereas the A-pathotype group can be divided
into five subgroups according to lesion phenotype
and pathogenic reaction on Brassica cultivars with
196 K. Voigt and IW. Wästemeyer
different susceptibility (Badawy et al. 1991,1992).
This matches with statistieal analysis of DNA
fingerprinting patterns, which also allowed the
grouping of A-isolates into different clusters
(Mahuku et al. 1997). Other research groups
(Williams 1992; Rouxel et al. 1995; Taylor et al.
1995; Purwantara et al. 1998; Sexton et al. 1999)
have also demonstrated the differential patho-
genicity within the A-group.
Similar to the differential aggressivity of
A-isolates on different Brassica genotypes
(Badawy et al. 1991), NA-strains express differen-
tial host-specificity and pathogenicity patterns
(Rouxel et al. 1995). The "Puget Sound" type
(Pound 1947) is found exclusively on senescent
plants or stubble of B. napus and B. rapa
(synonymous to B. campestris). The"'Sisymbrium"
type occurs on mature plants or stubble of
Sisymbrium spp. and the "Thlaspi" type originates
from stinkweed (Thlaspi arvense). Isolates of
these groups have also been recovered from
Descurainia sophia and Brassica crops (Rouxel
et al. 1995). Unlike any other A- or NA-strains,
whieh are non-aggressive on T. arvense (Johnson
and Lewis 1994), isolates of the 'Thlaspi' type
affect stinkweed plants severely by the formation
of stern cankers, although they do not produce
stern cankers on rapeseed (De March et al. 1986).
Data inferred from genomie DNA polymorphisms
indieate that "Thlaspi" isolates are intermediate
between A- and NA-strains (Morales et al. 1993b),
but are more closely related to NA-isolates
(Morales et al. 1993a). The "Erysimum" and the
"Lepidium" types were isolated from species of
their respective host genus (Rouxel et al. 1995).
Although morphologically not clearly distinguish-
able (Fig. 11.2), all subgroups of P. lingam can be
differentiated by several molecular approaches
(see Sects.IY.D, E).
B. Variability in Geographie Distribution
The relative frequencies of the two major patho-
genicity groups vary geographically. Aggressive
lines predominate in Australia, whereas non-
aggressive lines are more frequent in Canada
(Humpherson-Jones 1986), where A-isolates have
been monitored since the mid-1970s in only a
few areas (Petrie 1978). Kutcher et al. (1993)
compared isolates of P. lingam from Canada
(Saskatchewan and Manitoba) and Western
Australia. Despite variations in pathogenicity
within a fungal population collected from a single
field, the Australian isolates were more pathogenic
than those from Canada. Within the A-group,
European and Australian populations are
separate adjacent clusters, while the North
American population partially overlaps with both
(Purwantara et al. 2000). The similarity between
North Dakota isolates collected in 1995-1996 and
Western Canadian isolates collected in the 1980s
suggests that P. lingam was introduced into North
Dakota from Western Canada, and that the popu-
lations have remained essentially unchanged over
the past 10 years (Pongam et al. 1999).
C. Variability in Mating Behavior
Attempts to mate A-and NA-isolates have failed
regardless of geographie origin. Attempts to cross
within the NA-pathotype group have also been
unsuccessful (Salisbury et al. 1995). The sexual
ineompatibility within the NA-group may not only
provide an additional argument for different
biological species comprised in the NA-group,
but mayaiso reftect the physiological or genetic
reasons for the imperfect character of these
Phoma isolates. Genetic analysis of mating-type
genes and perhaps of additional incompatibility
factors at the molecular level is required to clarify
the reproduction biology of NA-isolates. There is
no reason, however, to divide the inter-breeding
A-isolates into different species.
D. Variability at the Level of DNA Fingerprints
DNA fingerprinting by the random amplified
polymorphie DNA technique (RAPD; Schäfer
and Wöstemeyer 1992,1994) and by mierosatellite
fingerprinting (Meyer et al. 1992) has been used
to distinguish between A- and NA-isolates. A high
degree of genetic similarity among A-isolates was
observed by these techniques and by analysis of
restriction fragment length polymorphism (RFLP;
Koch et al. 1991). At the molecular level the
A-isolates form aseparate group, equivalent to a
species distinct from all NA-isolates. NA-strains
differ to a much higher degree and can be divided
into three distinct subgroups (Koch et al. 1991).
The high heterogeneity within the NA-group
supports the assumption of different species.
Based on differences in host range on cruciferous
weeds, several subtypes were determined (Fig.
11.2), which all are non-aggressive on oilseed rape
(De March et al. 1986; Rouxel et al. 1995).
 Disease Management of Phoma Infeetions
Fig.ll.2. Cultures of Phoma lingam isolated from Brassica napus and erueifer weeds after 14-day ineubation on pea
extraet agar
RFLPs of he at shock responsive genes
(Patterson and Kapoor 1995) and of the ribosomal
RNA repeat (Plummer et al. 1994) also distinguish
between the pathogenicity groups. Differences in
the internal transcribed spacer (ITS) regions cor-
relate with pathogenicity grouping (Xue et al.
1992; Morales et al. 1993a; Balesdent et al. 1998).
The ITS 1 regions of A- and NA-isolates share
only 66.5% identity, whereas the 18S and 5.8S
rDNA regions of both pathogenicity groups are
97.7% identical (Xue et al. 1992).
Amplified fragment length polymorphism
(AFLP) assays readily discriminate between path-
ogenicity groups and subgroups and also provide
individual fingerprints for isolates (Purwantara et
al. 2000). In comparison with pathogenicity data,
AFLPs assess variation and relatedness of popu-
lations distributed over different rapeseed pro-
duction areas worldwide (Pongam et al. 1999).
E. Variability at the Karyotype Level
The electrophoretic separation of the chromo-
somes of P lingarn reveals significant differences in
197
the karyotypes of A- and NA-isolates (Plummer et
al. 1994). While only small variations in chromo-
some size and apparent number were detected
among isolates of the same pathogenicity group,
dramatic differences in both chromosome number
and size were found when A- and NA-isolates
were compared (Taylor et al. 1991). Although a
high degree of chromosomal size variation was
observed among individual genomes, predominat-
ing karyotypes of both A- and NA-strains were
described (Morales et al. 1993b). NA-strains have
a tendency towards sm aller chromosomes, while
A-isolates expose a more evenly distributed
pattern between 0.7 and 3.7Mb (Morales et al.
1993b). The differences between A- and NA-
isolates were used as an argument to separate both
pathogenicity groups into different species (Taylor
et al. 1991; Morales et al. 1993b). However, kary-
otyping is not generally suited for defining the
boundaries of species at the molecular level and
should be handled with caution in the context of
species definition (Plummer and Howlett 1995;
Zolan 1995; Wöstemeyer 1997). Major chromoso-
mal length polymorphisms were found in field
isolates of P lingarn after meiosis (Plummer and
198 K. Voigt and IW. Wöstemeyer
Howlett 1993). Size differences of 35% between
homologous chromosomes could be attributed
to repeat variations of rDNA units. Mutations
accumulate faster than homogenization occurs
(Howlett et al. 1997). Dispensable B chromosomes
contribute to karyotype alterations by escaping
Mendelian segregation and also due to losses
during meiosis. B chromosomes in P. Ungarn vary
in size between 650 and 950kb (Howlett 1996;
Leclair et al. 1996). Extrachromosomal elements
of different sizes correlate with pathogenicity
groups and may contribute to genetic variability
(Hass an et al. 1991; Lim and Howlett 1994).
F. Variability at the Chemotype Level
Aggressive isolates of the blackleg fungus are
characterized by the synthesis of epipolythiodiox-
opiperazines (Koch et al. 1989; Pedras 1998a),
also known as sirodesmins. Sirodesmins were
first detected in Sirodesrniurn diversurn, a wood-
degrading saprophytic fungus, which farms
sirodesmin G, a toxin with similar chemical struc-
ture (Curtis et al. 1977). Sirodesmin PL is the
maj or compound (50--70% ) of the toxins produced
by the A-group of P. Ungarn (Badawy and Hoppe
1989; Pedras 1998a). Similar to sirodesmin G from
S. diversurn, sirodesmin PL from P. Ungarn is a
disulfide-bridged epidithiopiperazine (Boudart
1978; Pedras 1998a). Sirodesmin PL, its corre-
sponding deacetyl derivative (deacetylsirodesmin
PL), and other toxie compounds (sirodesmin H, J,
K and phomalirazine; Pedras and Seguin-Swartz
1992) are naturally occurring mono- or polysulfur-
bridged dioxopiperazines.
Furthermore, the A-group synthesizes the
toxin phomalide, a cyclic depsipeptide and thus
structurally unrelated to the sirodesmins (Pedras
et al. 1993). Toxicity and selectivity of phomaIide
were determined utilizing cell suspension cultures
of B. napus and B. juncea (Pedras and Biesenthai
2000). While phomalide decreased the cell viabil-
ity of B. napus to 32 % after 4 days and to 14 %
after 8 days of incubation, it is significantly less
toxic to B. juncea (Indian mustard), a species resis-
tant to P. Ungarn. The biosyntheses of sirodesmins
and phomalide interact by an inhibitory effect of
sirodesmin PL on the biosynthesis of phomalide
(Pedras and Biesenthai 1998). This inhibitory
effect explains why phomalide is produced for
an unusually short period (24--60h) until siro-
desmin production predominates in older cultures
(Pedras et al. 1993). A third compound, the cyclic
dipeptide phomamide, is also formed exclusively
by the A-group (Pedras 1998a). Phomamide has
no toxic activity and is known to be a sirodesmin
precursor (Pedras and BiesenthaI1998).
The culture filtrates of A-isolates are unpig-
mented. NA-strains, however, produce a variety
of polyketides (Taylor et al. 1995) and a water-
soluble yellow pigment in liquid cultures, which
appears reddish-brown in higher concentrations
(Gabrielson 1983; Koch et al. 1989). This pigment
was recognized as a complex containing colorless
(phomaligadiones, phomaligols and phomapy-
rones) and bright yellow (wasabidienone, phoma-
ligin) compounds, which belong structurally to
the cyclohexenediones (Pedras 1998a). Secondary
metabolites that are identical to wasabidienone E,
the major component of the yellow fraction, and
also to the two phomaligin isomers were isola ted
from Phorna wasabiae (Pedras 1998a). P. wasabiae
is the causative agent of blackleg disease in the
crucifer was abi (Eutrerna wasabi; Soga 1976). The
existence of yellow pigments in both NA-isolates
and P. wasabiae suggests that NA-strains of L.
rnaculans are related to P. wasabiae (Pedras et al.
1995). Like groups 4 and 5 of the NA-isolates of
P. Ungarn (Voigt et al. 2001), P. wasabiae farms
reddish-orange pigments turning to black after
3-4 weeks in liquid culture (Soga 1976). Thus,
P. wasabiae may be part of the NA-species
complex.
The general picture on the clear-cut differ-
ences between A- and NA-strains is repeated at
the level of proteins. Glucose phosphate iso-
me rase allozyme patterns correlate with patho-
type groups. All A-isolates are biochemically
characterized by the fast isozyme of glucose phos-
phate isomerase (Hall et al. 1993). In addition,
immunological assays based on monoclonal anti-
bodies distinguish between A- and NA-isolates
(Stace-Smith et al. 1993).
G. Nonaggressive Strains Are Related
to Phoma wasabiae
The 5.8S ribosomal RNA gene and the adjacent
transcribed spacers ITS 1 and ITS 2 from several
Leptosphaeria and Phorna species were com-
pared. Sequence data show that the NA-group is
more related to P. wasabiae than to the A-group
of P. Ungarn (Pedras 1998a). Moreover, patho-
genicity tests reveal similar disease symptoms of
 Disease Management of Phoma Infections
P. wasabiae and the NA-type of P. lingam on
was abi (Pedras 1998a). On Japanese horseradish
both produce similar foliar symptoms, whereas
the A-type of P. lingam causes a hypersensitive
response due to an incompatible inter action
(Pedras 1998a).
Unlike phomaligol, the yellow wasabidienone
is phytotoxic for both, wasabi and rapeseed
(Pedras 1998a). This explains penetration and col-
onization of foliar leaf tissue as the cause of foliar
leaf spots on oilseed rape (Johnson and Lewis
1994). Perhaps due to the lack of sirodesmins, the
NA-types of P. lingam and P. wasabiae are not
capable of forming stern canker in oilseed rape.
Thus, the NA-types of P. lingam and P. wasabiae
are more related to each other than the NA- to the
A-strains of P. lingam. It has been proposed that
the NA-strains of P. lingam should be reclassified
as one or more separate fungal species (Pedras
1998a; Voigt et al. 2001).
V. Development of Novel Pathotypes:
Phylogeny of Phorna Ungarn
Up to three strains were observed simultaneously
in individual sterns of Thlaspi arvense and B.
napus (Petrie et al. 1995). Coexistence of different
genotypes increases the potential for exchange of
genetic material, which may lead to the generation
of novel pathotypes. Taylor and Borgmann (1994)
provided evidence for transfer events of an A-
specific repetitive element (LMR1) to a Canadian
NA field isolate, which is more aggressive on
rapeseed than a similar isolate that lacks the
element. The offspring of this putative horizontal
gene transfer event may be ascribed to a new
pathotype of intermediate aggressivity on rape-
seed. Compared with approximately 80 copies
of LMR1 per haploid genome in A-strains, NA-
isolates contain less than five copies (Taylor et al.
1995). It is possible that regulatory factors con-
trolling pathogenicity are linked to the transferred
repetitive element (Taylor and Borgmann 1994).
The horizontal transfer of LMR1 to an NA-strain
correlates with host range expansion of this isolate
to include B. juncea (Taylor et al. 1995). The mech-
anism of gene transfer between sexually incom-
patible strains is still mysterious.
Increasing evidence suggests that the two
pathotype groups represent different species. Phy-
logenetic analyses of RFLP da ta (Koch et al. 1991)
199
and of ITS 1/2 sequences (Morales et al. 1993a)
indicate thatA- and NA-strains of P. lingam are not
monophyletic, and represent very likely re pro duc-
tively isolated groups. 5.8S, 18S and ITS sequence
data indicate that NA-strains are a sister lineage to
L. microscopica, L. nodorum and L. doliolum,
while the A-isolates do not match in either lineage
and represent an additional branch of the genus
Leptosphaeria (Morales et al. 1995). Thus, the
A/NA classification does not hold any longer;
strains formerly ascribed to one of the pathogenic-
ity groups appear to be members of different bio-
logical, phenetic and phylogenetic species.
Speciation of P. Ungam is still in progress.
Population genetic studies suggest high variability
in virulence patterns on extended sets of geno-
types of Brassica differentials, which may have the
potential to adapt to new host plant reservoirs
(Kuswinanti et al. 1999). Pathogenicity variation
and host range alterations are the first steps
towards the development of genetically isolated
populations and species.
VI. The Blackleg Complex:
Infection, Colonization
and Symptoms
Blackleg disease is monocyclic, with little or no
secondary spread by splash-borne asexual spores
(pycnidiospores) produced on primary leaf lesions
during a season (Hammond and Lewis 1986).
The discharge of sexual spores (ascospores) pro-
duced in fruiting bodies (pseudothecia) on stern
debris from the previous year represents the
major SOuree of primary inoeulum in the field
(Humpherson-Jones 1986; Hall 1992; West et al.
1999). Air-borne ascospores of L. maculans
adhere to the leaf surface and first infect the
leaves, predominantly via stomata (Hammond et
al. 1985). Leaf infection occurs at temperatures
from 8°C to 24°C and needs leaf-wetness dura-
tions between 8h and 72h (Biddulph et al. 1999).
After inoculation with ascospores in autumn, the
fungus initially colonizes the intercellular space
between the mesophyll cells as a biotroph and
causes primary lesions on leaves as a necrotroph
(Hammond et al. 1985; Hammond and Lewis
1987). The disease is in its leaf-spot phase.
The fungus pro duces pycnidiospores in asexual
fruiting bodies (pycnidia) in the dead tissue.
Pyenidiospores may inerease the disease pressure
200 K. Voigt and IW. Wästemeyer
by acting as secondary inoculum (Barbetti 1976),
spreading by rain-splash to other leaves and
neighboring plants. Prolonged moist weather
favors rapid spread and development of the
disease (Salisbury et al. 1995). The germinating
spores release phomalide, which diffuses into the
plant tissue and facilitates initial fungal penetra-
tion and colonization of the foliar rapeseed tissue
(Pedras 1998a; Pedras and Biesenthal 1998). The
host-selective phytotoxicity of phomalide deter-
mines the pathogenicity range on different hosts.
Plants which are insensitive to phomalide are
blackleg-resistant (Pedras 1998a; Pedras et al.
1999; Pedras and BiesenthaI2000).
During winter, the fungal pathogen is able to
grow systemicly from leaf spots down the petioles
mainly in xylem vessels or between cells of the
xylem parenchyma and cortex. The stern is
infected at a rate of 5 mrn/day at 18/12°C and
1.4 mm/day at 3°C, The intercellnlar systernic
growth phase is biotrophic and virtually symp-
tomless (Hammond et al. 1985). In addition to
spreading systemically from leaf to stern, L.
maculans sometimes directly invades sterns under
field conditions (Xi et al. 1991). The fungus finally
becomes necrotrophic, invades and kills cells of
the stern cortex, resulting in stern canker lesions
(Hammond et al. 1985; Salisbury et al. 1995).
In early summer, severe basal canker formation
around the hypocotyl girdles the stern base. The
secretion of sirodesmins reaches its maximum.
In contrast to phomalide, the sirodesmins are
non-specific toxins with antiviral, antifungal,
phyto- and zootoxic activity (Boudart 1978; Koch
et al. 1989). Due to plasmolysis caused by the
sirodesmins (Boudart 1978), the invading fungus
kills the lower stern and hypocotyl tissue. Cell-
wall-degrading enzymes, mainly cellulases, pectate
lyases and polygalacturonases, degrade the plant
tissue and are believed to be involved in the
process of necrosis (Easton and Rossall 1985;
Annis and Goodwin 1997; Sexton et al. 2000).
There is, however, no necessary correlation
between the level of cell wall-degrading enzymes
and aggressivity (Annis and Goodwin 1996).
The stern canker phase is symptomatically the
most devastating stage of the blackleg disease.
At the time of flowering, most cankers become
obvious, but leaf lesions are still the most common
symptom, whereas crown cankers predominate
at harvest (Hall et al. 1993; Wratten et al. 1995).
Stern cankering is the main cause of yield losses.
Prevalence, incidence and severity of stern cankers
are greater in winter than in spring oilseed rape.
Over the 4-year period 1986-1989, the annual
prevalence of fields affected by stern cankers
ranged from 60% to 100% in winter oilseed
rape and from 27 to 31 % in spring oilseed rape
(Hall et al. 1993).
Severity of stern canker formation is influ-
enced by temperature: Stern cankers, which first
became visible after 77 days at 18/12°C, were
more abundant and severe than those at 3°C,
which formed only after 175 days. Infected leaves
senesced prematurely only at higher tempera-
tures, around 18/12 °C (Hammond et al. 1985). The
worst affected plants lodge and die without pro-
ducing seed, less severely affected plants may
survive, but seed production is reduced and the
seed is generally of poor quality (Salisbury et al.
1995). Severe basal stern canker lesions originate
from infections on leaves at the rosette stage in
autumn, whereas lesions higher up the stern orig-
inate from infections on later leaves (Hammond
and Lewis 1986; Biddulph et al. 1999). The earlier
stern lesions occur, the greater are the yield losses
(Zhou et al. 1999). After harvest, the fungus
remains as a saprophyte on infected stubble, which
serves as the main source of inoculum for the crop
in the new growing season (Humpherson-Jones
1986).
The rapid metabolism of the Brassica phy-
toalexin brassinin by A-strains of L. maculans
results in plants that are more susceptible to
fungal colonization (Pedras 1995). A succession
of secondary invaders (species of Alternaria,
Verticillium and Fusarium as well as Cylindrosp-
orium concentricum, Sclerotinia sclerotiorum and
Rhizoctonia solani) colonizes the necrotic plant
tissue (Schleier et al. 1997). Regarding diagnostic
aspects of Phoma disease management, blackleg
has to be seen as a multi-disease complex, rather
than as the consequence of a single causative
agent. In addition, coinfection by A- and NA-types
of L. maculans (Mahuku et al. 1996 a,b) and
colonization with related Phoma spp. have been
observed (Krüger and Wittern 1985; Rouxel et al.
1995). A-isolates were dominant (>80%) at the
stern base, whereas NA-isolates were isolated with
high er frequencies from higher parts of the stern
and also from leaves (Thürwächter et al. 1999). On
ageing host tissue, when plant defence is reduced,
even non-aggressive isolates cause severe symp-
toms with heavy sporulation (Badawy et al. 1992).
One important selective advantage of fungal
infection cornplexes relies on the synergistic
 Disease Management of Phoma Infections
metabolism of components of plant defence com-
ponents. For example, the crucifer phytoalexin
camalexin is not metabolized by A- or NA-strains
of L. maculans, but the root rot pathogen Rhizoc-
tonia solani, usually a pathogen of false ftax
(Camelina sativa), is able to initiate the biotrans-
formation of camalexin by converting it into
hydroxycamalexin, which is further transformed
into even less toxic compounds by L. maculans
(Pedras 1998a). Although camalexin is less
effective insuppressing mycelial growth of L.
maculans, it inhibits spore germination and the
overall development of other fungal and bacterial
members of the blackleg complex (Pedras et al.
1998a).
VII. Targets for Plant Proteetion
A. Gene-for-Gene Interactions
Since the discovery of gene-for-gene relationships
between plant resistance and fungal avirulence
genes (Flor 1946), this concept has been exploited
at the classical and the molecular level for under-
standing plant-pathogen interactions (Joosten
et al. 1997).
Resistance genes encode components for the
recognition of pathogens and also building blocks
of defense responses (for review, see Rammond-
Kosack and Jones 1996). These include cuticle
development, callose deposition, lignification,
wo und barriers, tylosis (structural resistance), pR
variation, toxic compounds, enzyme inactivators
and phytoalexins (Parry 1990). Resistance genes
determine the nature and extent of the resistance.
Specific resistance against one or few physiolo-
gical races of a pathogen is determined mono- or
oligogenically, usually by two or three genes
(Manners 1993). General resistance affects all
races and is in most cases polygenie (Manners
1993; Shew and Shew 1994).
Avirulence genes function as targets for the
plant's resistance cascade. Genetic engineering
of host plants with avirulence genes from their
pathogens mimics pathogenic challenge and may
force the plant to keep a constant level of
resistance against the pathogen (De Wit 1992).
Transformation of plants with cloned avirulence-
resistance gene cassettes may result in plants with
increased resistance to pathogens (De Wit 1992;
Lauge and DeWit 1998).
201
Constitutive as well as induced defense under-
lies disease resistance of oilseed rape (Pedras
1998a). Since the turn of the nineteenth century,
plant breeders have used disease-resistant genes
(R genes) in sexual crosses for the control of plant
diseases (Staskawicz et al. 1995). Also, the trans-
fer of resistance traits by somatic hybridization
has stimulated genetic improvement of crops
(Glimelius et al. 1991). The molecular character-
ization of pathogen recognition and resistance
expression will lead to novel strategies for plant
breeding. Several R genes controlling resistance
to L. maculans in many B. napus cultivars have
been identified (Dion et al. 1995; Ferreira et al.
1995; Mayerhofer et al. 1997; Ansan-Melayah et al.
1998; Pilet et al. 1998 a,b; Dixelius 1999). RAPD
or RFLP markers linked to the resistance
phenotypes were mapped (Chevre et al. 1997;
Mayerhofer et al. 1997) by bulk segregant analy-
sis of segregating Fl-populations from a single
cross (Michelmore et al. 1991). If the parental
generation is sexually incompatible, somatic
hybridization via plant protoplast fusion helps to
generate hybrid plants (Glimelius et al. 1991). The
establishment of Agrobacterium tumefaciens-
mediated transformation of double haploid B.
napus lines (Kazan et al. 1997) also permits the
transfer of genes independent of genetic incom-
patibility barriers. Single major loci controlling
either cotyledon (LEMl; Ferreira et al. 1995)
or adult plant (LmFrl; Dion et al. 1995) resistance
to L. maculans were mapped using segregating
populations of Fl-derived double haploid lines
and RFLP markers.
A gene-for-gene relations hip was suggested
for B. napus and L. maculans with respect to the
R-gene/avirulence gene pair LEMl/alml
(Pongam et al. 1998). The single 10cus LEMI con-
tro1s cotyledon resistance. RAPD markers were
identified which are linked to an avirulence gene
in L. maculans controlling cultivar specificity
(AvrLml; Ansan-Melayah et al. 1995). Race-
specific resistance is conditioned by the match-
ing gene pairs RlmllAvrLml and Rlm21AvrLm2
(Ansan-Melayah et al. 1998).
Molecular markers 1inked to resistance phe-
notypes are useful for breeding black1eg-resistant
varieties by marker-assisted selection and may be
seen as the essential first step towards map-based
cloning of resistance genes (Mayerhofer et al.
1997). In particular, R genes that enable plants to
resist a range of pathogens and thus are invo1ved
in common resistance mechanisms are useful for
202 K. Voigt and IW. Wöstemeyer

breeding purposes (Staskawicz et al. 1995). Quan- tion to overcome the sirodesmin-inhibiting co m-
titative trait loci were also identified (Pilet et al. ponent of the plant's chemical defence. There are,
1998b), which may correspond to clusters of R however, no enzymes for degrading other Brassica
genes involved in multiple disease resistance phytoalexins.
against several fungi of the blackleg complex in During evolution, host plants have evolved
B. napus. several mechanisms to cope with the rapid adap-
tation of pathogens. Plant chitinases, which are
believed to play a critical role in plant defense
B. Chemical and Structnral Barriers of the Host against fungal attack, undergo rapid adaptive evo-
Intion by amino acid replacements in the active
In response to tissue invasion by fungi, the host site cleft (Bishop et al. 2000). The significance of
plant synthesizes an array of antimicrobial chem- chitinases for eliciting induced resistance is gener-
ical compounds, while the fungus pro duces phyto- ally accepted in the L. maculans-Brassica inter ac-
toxins that damage the plant tissue and facilitate tion (Thürwächter et al. 1995).
perthotrophic invasion. There are also mecha- The release of hydrogen cyanide from
nisms for detoxifying phytoalexins by L. maculans cyanogenic glucosides in over 2000 plant species
and for phytotoxins by the host. Phytoalexin is thought to provide a protective barrier against
detoxification and phytotoxin biosynthesis are infection by fungal microorganisms (Osbourn
coregulated in the Leptosphaeria-Brassica system 1996). A gene for a cyanide hydratase was cloned
(Pedras 1995). from an aggressive isolate of L. maculans (Sexton
As a result of infection, B. napus synthesizes and Howlett 2000). The gene is transcriptionally
an array of low-molecular-weight antimicrobial regulated in L. maculans-challenged cotyledons
metabolites, the phytoalexins brassinin, spiro- and can be induced by potassium cyanide.
brassinin, cyclobrassinin, dioxibrassinin, methoxy- Transcripts are detectable during infection of
brassinin, methoxyspirobrassinin, cyclobrassinone, cotyledons. The enzyme converts hydrogen
brassilexin and brassicanal, which diffuse from the cyanide to the less toxic compound formamide,
site of synthesis into water droplets on the leaf which can be utilized by L. maculans as a sole
surface, where they affect hyphal growth (Gross nitrogen source.
et al. 1994; Pedras 1998a; Pedras and Biesenthai Fungal attack is responded by many host
1998). Phytoalexin accumulation is associated reactions, including lignification, calcium accumu-
with plant resistance, although the genetic lation, cambium formation and callose deposition
information for phytoalexin synthesis is found at the perimeter of lesions and at the periphery of
in susceptible as well as in resistant plants lignified zones, which create mechanical barriers
(Kuc 1995). against the invading fungal mycelium (Hammond
Biotic elicitation is caused by fungal and Lewis 1987). There are substantial differences
sirodesmin toxins, which induce the accumulation in host reaction to the two pathogenicity groups
of brassinin phytoalexins (Pedras and Seguin- of L. maculans; only the lignification response
Swartz 1992). Pedras (1995) also discovered an was observed with A-isolates, whereas the whole
inhibitory effect of brassinin on the biosynthesis sequence of reactions was observed with the
of sirodesmins. Inhibition of phytotoxin pro duc- NA-group. Here reside probably the mechanistic
tion is beneficial to the plant as it slows down reasons for the differences in colonization
the fungal damage of plant cells. In vitro, aggres- patterns between the two pathogenicity groups.
sive isolates do not synthesize sirodesmins when
incubated with brassinin, whereas incubation
with structurally related phytoalexins (brassicanal C. Chemical Response
A, brassilexin and camalexin) does not affect of Leptosphaeria maculans
sirodesmin biosynthesis (Pedras et al. 1997).
Plants secreting brassinin are attacked by the CeU-wall-degrading enzymes (CWDEs) secreted
host-selective toxin phomalide, the synthesis of by L. maculans were found to be involved in
which is not affected by brassinin. Phomalide pathogenesis, but at a later stage of infection
facilitates fungal infection in brassinin-exposed during necrosis development (Easton and Rossall
areas (Pedras 1998b). Furthermore, L. maculans 1985). Hassan et al. (1991) measured elevated
has evolved mechanisms for brassinin detoxifica- activity levels for cellulases, a- and ß-glucanases
 Disease Management of Phoma Infections
and for polygalacturonases, some of which are
catabolically repressed by glucose and induced
by their polymerie substrates (Sexton et al. 2000).
Thus, CWDEs represent pathogenicity factors
that might be targets for plant proteetion. CWDE
inhibitors down-regulate the CWDE activity and
may be involved in the resistance of plants to
blackleg (Easton and Rossall 1985). The level of
polygalacturonase inhibitory activity in stern
extracts was significantly related to the resistance
of cultivars to L. maculans (Annis and Goodwin
1997).
The aggressivity of L. maculans isolates may
be determined by the host-selectivity of their
toxins (Pedras 1998a,b; Pedras et al. 1998b, 1999;
Pedras and Biesenthai 2000). Nevertheless,
the role of the non-specific sirodesmin toxins in
pathogenesis of L. maculans is still controversial.
Boudart (1978) found no linear correlation
between toxin formation and aggressivity. A
naturally occurring hypovirulent mutant of L.
maculans is able to form sirodesmins in amounts
higher than the general A-type, but is significantly
less necrotic. In addition, pathogenicity of three
sirodesmin low-producers was not abolished
after nitrosoguanidine mutagenesis. UV-induced
sirodesmin-deficient mutants from aggressive iso-
lates of L. maculans were reduced 100- to 1000-
fold in sirodesmin PL formation and had the same
ability as the wild-types to infect cotyledons of sus-
ceptible cultivars of B. napus (Sock and Hoppe
1999). On the stern basis, however, mutants caused
significantly smaller lesions. The phytotoxicity of
sirodesmins has no decisive role in pathogenesis
and apparently is not involved in virulence on
cotyledons; however, it plays a role as aggressivity
factor on the stern base of B. napus. Sirodesmin
formation can be suppressed by zinc ions, which
were found to protect B. napus plants against L.
maculans (Rouxel et al. 1990). The effect of zinc
may verify the role of sirodesmins in pathogene-
sis, although it cannot be exduded that zinc ions
exert multiple effects in the plants.
Aggressive isolates convert brassinin to the
significantly less toxie indole-3-carboxylic acid,
whereas the metabolism of brassinin by NA-
strains pro duces mainly indole-3-acetylamine
(Pedras 1995). Both routes affect the dithiocarba-
mate group of brassinin which is essential for its
antifungal activity. Thiocarbamates have been
used as pesticides and herbicides for more than
two decades; their detoxification is a survival
strategy used by diverse microorganisms (Pedras
203
1998b). Despite the dose biogenetic relation-
ship of Brassica phytoalexins, all phytoalexins are
transformed via different enzymatic processes at
different rates (Pedras and Khan 1996).
D. The Use of Toxin Resistance
in Plant-Breeding Programs
Since there is evidence for correlations between
toxin tolerance and pathogen resistance (Buiatti
and Ingram 1991), considerable efforts have been
directed towards breeding lines with resistance
against the toxins of L. maculans (Pedras 1998a).
Pathogen-resistant cultivars are less sensitive
to the non-selective toxins sirodesmin PL and
phomalirazine than susceptible cultivars (Pedras
and Seguin-Swartz 1992). Investigation of plant
reactions to host-selective toxins should allow the
elucidation of specific disease resistance mecha-
nisms (Pedras 1998a).
In breeding programs for blackleg resistance,
selecting for tolerance against the host-selective
phytotoxin phomalide may have a potential in
brassicas (Pedras and Biesenthai 1998, 2000).
Because disease-resistant tissue is affected to a
much lower extent than susceptible tissue, it is
assumed that phomalide may be converted enzy-
matically to less phytotoxic compounds predomi-
nantly by resistant tissue. The isomerization of
phomalide into its non-toxic isomer, isophoma-
lide, is discussed as a potential mechanism leading
to resistance (Pedras 1998a).
E. Canola Quality and Plant Protection
Phytoalexins are involved in disease control, but
the protective effect of phytoalexins is restricted
to the area of infection (Pedras and Seguin-Swartz
1992; Kuc 1995). Brassica phytoalexins are sulfur-
containing phytoalexins, which are characteristic
exdusively for cruciferous plants (Gross 1993;
Pedras 1998a). The sulfur-containing indole deriv-
atives are synthesized from the amino acids L-
tryptophan and L-methionine. This could be one
of the reasons why crucifers have high sulfur
requirements. These phytoalexins are elicited
abiotically by CuCl2 (Gross et al. 1994).
The modern "double-zero" varieties of canola
essentially lack erucic acid and glucosinolates and
meet the demands for rapeseed oil and meal
production for cattle feed (Parry 1990). Canola
204 K. Voigt and IW. Wästemeyer
quality standards require an erucic acid conte nt
below 2% in the extracted oil and aliphatic glu-
cosinolates below 30,umol/g of residual me al
(Rimmer and van den Berg 1992). However,
volatiles released from glucosinolates in B. juncea
and Wasabia japonica (syn. Eutrema wasabi) are
toxic to L. maculans (Sexton et al. 1999), and thus
are desirable in blackleg disease management.
Glucosinolates are sulfur-containing glucosides
exclusively found in crucifers. They are derived,
depending on the nature of their side chains,
from aliphatic, indolyl or aralkyl a-amino acids
(Osbourn 1996). Due to the obvious chemical sim-
ilarity between glucosinolates and Brassica phy-
toalexins, which have an indole ring and at least
one sulfur atom in common, it was suggested that
glucosinolates and phytoalexins share biosyn-
thetic steps (Pedras et al. 1997; Pedras 1998a). If
this biogenetic correlation can be substantiated,
the potential for indole glucosinolates should be
maintained in breeding programmes. The older
'zero' varieties with low levels of erucic acid and
full glucosinolate content meet these require-
ments. They are at least as productive and less sus-
ceptible to infections (Parry 1990). The breeding
of varieties with balanced glucosinolate contents
may meet the demands for both plant protection
and industrial use. A general approach is to cross
a resistant with a quality parent and to re-select
for canola quality traits combined with blackleg
resistance (Rimmer et al. 1995).
B. juncea (Indian mustard) expresses resis-
tance by rapid necroses at the stomatal pore and
in the mesophylllayer, and thus arrests fungal pen-
etration and growth (Chen and Howlett 1996).
There is some potential in developing rapeseed-
quality lines of B. juncea, ideally double-zero
cultivars retaining blackleg resistance due to the
hypersensitive reaction already mentioned
(Pedras et al. 1998b). However, L. maculans
strains have been isolated which are pathogenic to
brown and white mustard (B. juncea and Sinapis
alba) and do not attack B. napus (Pedras et al.
1998b, 1999).
F. Phytoalexin Detoxification
Controlling pathogenic fungi could involve the
inhibition of fungal enzymes involved in detoxifi-
cation of phytoalexins (Pedras 1998a,b). Enzyme-
mediated transformations are usually simple
chemical conversions: oxidations, reductions or
one-carbon degradations mediated by P-450
enzymes. The phytoalexin brassinin plays a key
role in the plant defense of crucifers. A- and
NA-isolates of L. maculans detoxify brassinin by
oxidation (Pedras 1998a,b). This transformation
occurs significantly faster in A-isolates than in
NA-isolates (48h vs. 5-6 days). Also, biotrans-
formation of brassicanal via aldehyde reduction
is ten times slower in NA- than in A-isolates.
Since brassinin is the biogenetic precursor of
several other Brassica phytoalexins, its rapid
detoxification deprives the plant of other impor-
tant phytoalexins, and renders the plant more
susceptible to further fungal colonization (Pedras
1998a).
G. Evaluation of Blackleg-Resistant Cultivars
Blackleg resistance in selected hybrids has been
evaluated by an array of screening methods on
cotyledons, leaves and sterns (Rimmer and van
den Berg 1992; Rimmer et al. 1995). Because
glasshouse screening for resistance has limited
success, alm ost all screening is done in the field in
designated blackleg nurseries or on farms using
infected stubble from the previous year's crop for
building up infection pressure (Wratten et al.
1995). The susceptible cultivar Westar is usually
sown every tenth row as an indicator of disease
pressure and to provide a reliable source of pyc-
nidiospore inoculum. Selection is usuallY per-
formed on a single plant basis, screening for
absence of basal stern canker as the main criterion.
Selection begins in the F z generation or later
generation bulks. In vitro selection systems for
blackleg resistance in B. napus, utilizing the
culture filtrates of aggressive L. maculans strains,
help to produce blackleg-resistant embryogenic
cultures (Sacristan 1982). Unfortunately, in vitro
response is not a reliable indicator for adult plant
resistance behavior. Both are probably deter-
mined by different loci (Sacristan 1982; Snowdon
et al. 2000). Susceptible cotyledon reactions are
often followed by resistant stern reactions in adult
plants. Resistant cotyledons, however, are always
associated with resistance in sterns (Kutcher et al.
1993). Microspore and protoplast cultures of cul-
tivars are suitable test systems for the assessment
of toxin sensitivity before the selected lines are
evaluated in the field (Pedras and Seguin-Swartz
 Disease Management of Phoma Infections
1992). Tissue cultures of thin cell layer explants
from soil-grown plants or in vitro grown shoot
cultures allow differentiation of resistant and sus-
ceptible cultivars. Taken together, there is a strong
basis for in vitro breeding programmes to select
lines of oilseed rape with novel resistance traits to
L. maculans (Gretenkort and Ingram 1993).
VIII. Diagnosis of Blackleg Disease
Prevention of L. maculans requires sensitive tests
at the seed level (Wood and Barbetti 1977; McGee
1995). For monitoring and understanding the
epidemiology of this pathogen, it is desirable to
detect L. maculans in the host plant and to differ-
entiate between the pathogenicity groups.
A. Conventional Disease Assessment
Conventional diagnostie approaches take place in
early autumn and are based either on visual symp-
toms (Phoma leaf spotting) or on isolation and
cultivation of the blackleg fungus (Krüger and
Wittern 1985; West et al. 1999). The methods
currently employed for differentiating between
A- and NA-isolates are laborious and time-
consuming and require individual plating of seeds,
plant segments or stubble on fungal medium, fol-
lowed by induction of fungal sporulation and iso-
lation of pycnidia. Single-spore isolates are
subsequently tested for (1) growth rate on potato
dextrose agar (NA-isolates grow faster), (2)
pigment production on Czapek-Dox medium sup-
plemented with yeast extract (only NA-isolates
produce yellow to blackish pigments), (3) conidial
germination (NA-isolates produce longer germ
tubes in the first 40 to 44h of growth), (4) testing
aggressivity on cotyledons, and sometimes (5)
sirodesmin toxin production (A-isolates form
sirodesmins, NA-isolates do not). The Interna-
tional Seed Testing Association (ISTA) recom-
mends the 2,4-D blotter method for examining
seeds for pycnidia after 10-11 days of incubation
at 20 C on bl otters wetted with a 0.2% 2,4-
G certify Phoma-free seed, and therefore help con-
dichlorophenoxyacetic acid solution (Andreoli
and Maguire 1995). Alternatively, the use of
absicic acid at 100mg/l was proposed (Andreoli
and Maguire 1995). This seed assay, however, does
not differentiate between A- and NA-isolates
(Taylor 1993).
205
B. Molecular Prob es and Assays
Several molecular techniques were developed to
identify L. maculans and its pathogenicity groups.
Phenotypic and physiological strain variation
between A- and NA-strains (Koch et al. 1989) was
confirmed by biochemical and molecular tools
(Hassan et al. 1991). The electrophoretic isozyme
pattern of glucose phosphate isomerase was
revealed directly with material from leaf lesions
and proved a useful and reliable method to iden-
tify L. maculans and its pathogenicity groups in
large sampie sizes (Brun et al. 1997). Primers were
designed which distinguish A- and NA-strains in
random amplified polymorphie DNA (RAPD)
assays (Schäfer and Wöstemeyer 1992; 1994;
Wöstemeyer et al. 1992). Diagnostic RAPD bands
were sequenced and conventional PCR primer
pairs were derived, which unequivocally discrimi-
nate the two major pathogenicity groups (Voigt
and Wöstemeyer 1995; Voigt et al. 1997, 1998).
Based on the ITS 1 region different primer pairs
were synthesized, which specifically amplify DNA
from either the A- or the NA-strains (Xue et al.
1992).
A repetitive DNA element of 5238bp and a
copy number of around 80 per haploid genome
were identified on all chromosomes of the
A-group of L. maculans. It was exploited as a
highly sensitive diagnostic marker (Taylor and
Borgmann 1994). A sensitive PCR assay was
developed, which detects 2 contaminated in 1000
seeds (Taylor 1993).
PCR methods are less laborious and require
substantially less time in comparison with the 11
to 22 days for the methods which are recom-
mended by the ISTA (Andreoli and Maguire
1995). They do not require cultivation of the fungi
and allow detection in infected tissue one day
after inoculation. PCR methods require minimum
efforts for DNA-extraction and can be routinely
used for large-scale diagnosis of field isolates.
Plant material colonized by mycelium (Voigt et al.
1998) or pycnidia from leaf spots (Balesdent et al.
1998) are appropriate for analyses. PCR methods
are suited to test pods and seeds, can be used to
siderably to reduce the risk of epidemics. In ad-
dition, a competitive polymerase chain re action
technique was developed to quantify L. maculans
during disease development in leaves (Mahuku et
al. 1995). The assay is rapid, accurate, and detects
206 K. Voigt and J.w. Wöstemeyer
L. maculans DNA in the range around lOfg.
There have also been molecular attempts to assess
the species-spectrum of fungal pathogens in the
blackleg infection complex. Fungi of the taxa
Alternaria brassicae, A. brassicicola, A. raphanii,
Cylindrosporium concentricum, Fusarium monili-
forme, Pythium sp., Rhizoctonia solani, Sclerotinia
sclerotiorum, Verticillium dahliae, and V. latericium
can be distinguished (Schleier et al. 1997; Voigt et
al. 1997).
IX. Management of Blackleg Disease
The spread of blackleg was favored by commer-
cial pressures to increase uniformity and produc-
tivity of rape seed cultivation (Juska et al. 1997).
Epidemical blackleg outbreaks cannot be stopped
by chemical or biological me ans. Blackleg disease
must be controlled primarily by preventive
measures (Pedras 1998a). Knowledge of the epi-
demiology of the causative agent of blackleg is
fundamental for the development of disease
control practices (Hall 1992). The use of different
plant varieties and multilines has increased
genetic variablity and added considerably to plant
health in the agro-ecosystem.
A. CuItural Control
Cultural practices, which rendered the conditions
unfavorable for growth and spreading of the
pathogen, were the first methods to control soil-
borne pathogens (Sumner 1994). Controlled cuIti-
vati on parameters comprise seed health, time and
depth of seeding, crop rotation, tillage, cultivar
selection, sanitation, plant nutrition and fiooding.
These cultural measures aimat reduction of inocu-
lum. Rapeseed sterns with basal cankers, in par-
ticular, produce ascospores earlier and in greater
numbers than sterns with superficial lesions
(Petrie 1995). Rapeseed debris serves as inoculum
for at least three seasons (36months). The quick
removal of contaminated stubble immediately
after harvest by burning and deep plowing fol-
lowed by appropriate tillage reduces dispersal and
carry-over of primary inoculum (Krüger and
Wittern 1985; Parry 1990; Gugel and Petrie 1992).
In a field experiment, fragmentation of stern
debris by tillage and burial decreased survival of
L. maculans from 85 % before burial in July to
15 % in September and 3 % in December. The
decrease in L. maculans corresponds with an
increase of Trichoderma spp. (Baird et al. 1999).
Ascospores from crowns and taproots, which
deteriorate slowly under dry surface soil condi-
tions, continue to be discharged for 3-5 years. The
mean 3.3-year length of crop rotation in rapeseed
cultivation is substantially exceeded (Petrie 1995).
Burial of diseased crop debris prornotes the
decay of infested host material, whereas reduced
plowing conserves soil moisture, reduces soil
erosion, lowers labor and fuel costs, but maximizes
blackleg disease by spreading air-borne inoculum
(Kharbanda and Tewari 1996).
The date of sowing correlates with disease
development. Seeding before (Europe) or after
(Australia) the maximum discharge of ascospores
re duces crop susceptibility by the development of
well-established plants. Ascospores are dispersed
by wind up to 8 km (McGee 1977; West et al. 1999).
Successful disease management pro grams must
therefore also include neighboring fields.
Compared with air borne ascospore infections,
seed infection appears to be of lower importance
in causing epidemics. However, seed-borne infec-
tions introduce the disease to new areas (McGee
1977; Salisbury et al. 1995). The management of
seed health by modern seed technologies regu-
lates transmission of seed-borne diseases, and is
necessary to minimize the introduction of black-
leg into previously uninfested regions (McGee
1995; Kharbanda and Tewari 1996). A seed lot
with 1% contaminated seeds gives rise to cankers
in 3.3 % of the plants, to widespread distribution
of diseased plants in the field, and to yield losses
of 1-2% (Hall et al. 1996). Certificates stating
that seeds are free from blackleg are mandatory
for certified seed production and seed importa-
tion (Gabrielson 1983). The production of uncon-
taminated propagation material can be combined
with short hot water treatments, which are used
to free cabbage seed from L. maculans without
losing germination activity of the seeds (Agrios
1997).
Rotations of 3.3-4 years between Brassica
crops will not only control blackleg, but will also
keep other crucifer diseases at reasonable levels
(Gugel and Petrie 1992; Kharbanda and Tewari
1996). Since B. alba, B. arvensis, B. campestris, B.
kaber, B. tournefortii, Descurainia spp., Matthiola
incana, Raphanus raphanistrum, Sinapis arvensis,
Sisymbrium spp. and Thlaspi arvense are impor-
tant weed hosts of aggressive rapeseed pathogens
 Disease Management of Phoma Infections
(Gabrielson 1983; Petrie et a1. 1995), isolation of
oilseed rape from other brassicas and eradicating
alternate hosts and volunteer plants between the
cropping seasons will also reduce infection pres-
sure (Parry 1990; Salisbury et a1. 1995). For the
same reason, separation of spring and winter rape-
seed crops is essential (Rempel and Hall 1993).
Although important, cultural practices alone do
not provide sufficient disease control, particularly
as rapeseed cultivation areas are increasing.
B. Chemical Control
The strategie use of chemieals supplements cul-
tural practices in areas prone to severe blackleg
infestation (Salisbury et a1. 1995). Several fungi-
eides for seed treatment and foliar spraying were
evaluated in laboratory, growth chamber and field
tests. The details of their development and mode
of employment have been reviewed by Manners
(1993) and Morton (1994).
Fungicides need to be applied before the
pathogen propagules enter and establish in the
host. Fungicidal seed treatment protects against
the establishment of pycnidial seed infestations
with consistent results (Parry 1990), but fails to
control the disease caused by natural infection via
extended ascospore exposItIons (Kharbanda
1992). Treatment with foliar sprays in early spring
gives inconsistent results and does not show the
desirable efficiency (Parry 1990; Pedras 1998a).
Fungieides do not control blackleg disease on ce
the pathogen has reached the stern; fungicidal
management relies on leaf proteetion (Gladders
et a1. 1998). Effective foliar fungieide treatment
depends on early use, immediately after the first
leaf spots occur, several months before the symp-
toms are observed on sterns (West et a1. 1999).
Autumn applications of fungieides are more effec-
tive than spring applications. On the other hand,
autumn fungieide applications are effective only
for a limited period, due to fungieide degradation,
dilution by leaf expansion and the appearance
of untreated leaves. Seconds sprays might be
required but need careful analysis of costs in rela-
tion to potential yield response. Based on current
prices for oilseed rape and fungi eides, second
sprays are uneconomical in most cases. With
respect to yield/loss relationships, disease man-
agement must focus on cost-effective control
strategies that minimize the use of pesticides. The
systemic fungieides prochloraz, an ergosterol
207
biosynthesis inhibitor, and thiabendazole, a benz-
imidazole derivative, were used in stern canker
control as foliar spray and seed dressing (Parry
1990). Triazole fungieides applied as foliar sprays
can also reduce blackleg and improve yield. Split
applications, spraying on foliage at the late rosette
stage and on the main raceme at the stage of flow-
ering, were most effective and reduced disease
incidence by 9% and severity by approximately
23 % (Rempel and Hall 1995). Postharvest fungi-
eid al treatments of stubbles reduce the amount of
primary inoculum by suppressing the sexual stage
of L. maculans on straw (Humpherson-Jones and
Burchill1982).
Apart from Brassica spp. as host plants, A-
and NA-isolates were found in symptomless
cruciferous weeds, Thlaspi arvense, Descurainia
sophia and Capsella bursa-pastoris, acting as a
reservoir of inoculum (Petrie et a1. 1995). ehen
and Seguin-Swartz (1999) were able to reisolate L.
maculans from necrotic cotyledons, leaf and stern
tissue 10-40 days post-inoculation of the wild cru-
eifers Arabidopsis thaliana, Dipiotaxis muralis,
D. tenuifolia, Raphanus raphanistrum and Sisym-
brium loeselii. Since fungal inoculum from weeds
may playa role in disease initiation (Hall 1992),
the application of herbicides is of practical use to
prevent cross-infections. Because insects transmit
the disease to healthy crops (Krostitz 1994), insec-
ticide treatments mayaiso lower the spread of the
disease.
C. Biological Control
Biological control of plant pathogens implies the
creation of an environment that encourages the
development of antagonistic microorganisms in
the soi1. An intact soil ecosystem contains a micro-
bi al community, which is able to protect plants
from stress factors. In contrast to natural environ-
ments, agro-ecosystems display reduced biodi-
versity, a phenomenon caused by the shift in
plant diversity towards monocultures. Introducing
single microorganisms with proven activity is the
most popular strategy for biological contro1. The
establishment of microbial species in soil adds to
biological complexity and prornotes the stability
of the soil ecosystem.
Biocontrol agents may work through (1)
antibiosis by formation of antifungal antibiotics,
hydrogen cyanide and other volatiles, (2) para-
sitism on pathogens and excretion of lytic
208 K. Voigt and J.w. Wöstemeyer
enzymes, (3) competltlon for nutrients, e.g., by
secretion of iron-binding siderophore molecules, a
mechanism described for fluorescent pseudomon-
ads, (4) physical exclusion of the pathogen by co m-
petition for infection sites and space, (5) induction
of defense in the host plant, or (vi) a combination
of several mechanisms (for reviews, see Fravel and
Engelkes 1994; Handelsman and Stabb 1996;
Buchenauer 1998; Vilich and Sikora 1998). The
progression of Phoma diseases can be retarded by
microbial eradication of crop debris (Köhl and
Fokkema 1998). First results have been achieved
in microbial control of blackleg disease by the
antagonistic bacteria Erwinia herbicola, a bac-
terium isolated from the phyllosphere of B. napus
that was found to be highly antagonistic to L. mac-
ulans. The phyllobacterium inhibits the germina-
tion of fungal spores and re duces their germ tube
length by the excretion of a thermolabile antifun-
gal substance when applied to seedlings prior to
inoculation by the fungus (Chakraborty et al.
1994). Inoculation with less pathogenic races of
the same or related fungal species, e.g .. with NA-
isolates of L. maculans, can also control blackleg
disease by triggering defence reactions against
naturally occurring A-isolates (Thürwächter et al.
1995). Lignification, cambium formation and
cellulose deposition were induced after stern
penetration by NA-isolates, thus providing mech-
anical barriers against infection with A-isolates
(Hammond and Lewis 1987). Mahuku et al.
(1996a) observed a significant reduction in lesion
size after hyperinfection with A-isolates in previ-
ously NA-infested plants. Coinoculation with NA-
strains suppresses growth of A-strains and induces
general plant defense mechanisms.
Many biological agents are able to trigger sys-
temic resistance in plants that is phenotypically
similar to pathogen-induced systemic acquired
resistance (SAR) and non-pathogenic microor-
ganism-mediated induced systemic resistance
(ISR). Both depend on the plant-signal molecules
salicylic acid or jasmonic acid (van Loon et al.
1998; van Wees et al. 2000). Also, dichloroisonico-
tinic acid (INA) and certain benzothiazoles, which
have been used as fungicides, are known to act as
plant defence activators in SAR reactions (Agrios
1997). SAR and ISR pathways provide an attrac-
tive tool for the improvement of disease control
(van Wees et al. 2000). Much information has been
collected on determinants and mechanisms of ISR
and SAR (Delaney et al. 1994; Buchenauer 1998;
Mauch-Mani and Metraux 1998; van Loon et al.
1998; van Wees et al. 2000).
D. Breeding for Resistance
The most successful improvement of host resis-
tance to pathogens was achieved by breeding for
resistance. Several blackleg-resistant cultivars
are now available. When these are grown with
appropriate crop rotation, losses by blackleg are
minimal (Salisbury et al. 1995). However, L.
maculans is known to be highly variable in
virulence pattern (Kuswinanti et al. 1999). Con-
tinued improvements in blackleg resistance have
been made by hybrid breeding programs.
Intraspecific resistance in B. napus (e.g., in the
French cultivar Jet Neuf) is mostly partial, non-
specific and mainly restricted to adult plant resis-
tance, especially to reduced disease severity and
canker formation. Two types of resistance in B.
napus were identified (Roussel et al. 1999). The
first type, which occurs at the seedling stage, is
thought to be mono- or oligogenic (Mengistu et al.
1991; Rimmer and van den Berg 1992; Ansan-
Melayah et al. 1995), whereas the second type is
partial, polygenic and only expressed at the collar
level; cotyledons and leaves remain susceptible
(Ferreira et al. 1995; Pilet et al. 1998a). With
respect to the polygenic nature of adult plant resis-
tance to blackleg in B. napus, good resistance
levels are required in both parents. Good starting
genotypes are Japanese spring and French winter
lines (Salisbury et al. 1995). However, the resis-
tance to L. maculans, expressed by most commer-
cial cultivars of B. napus, does not totally prevent
significant crop losses (Roussel et al. 1999).
Enrichment of resistance genes has become a
major challenge in oilseed rape breeding pro-
grammes. Due to limitations in the gene pool of
B. napus cultivars, other sources of resistance
genes need to be found. A promising possibility is
the introgression of 'allen' resistance loci beyond
species and genus barriers.
The genomes of Brassica species are related.
They represent amphidiploids resulting from
crosses between the primary diploid species B.
rapa (AA,2n = 20), B. nigra (BB,2n = 16) and B.
oleracea (CC, 2n = 18) (Downey and Röbbelen
1989). B. napus (AACC, 2n = 38) can be synthe-
sized artificially from crosses between B. rapa
and B. oleracea, B. carinata (BBCC, 2n = 34) from
crosses between B. oleracea and B. nigra, B. juncea
(AABB,2n = 36) from crosses between B. nigra
and B. rapa. This relationship among Brassica
species has enabled plant breeders to create
synthetic amphidiploids and to transfer blackleg
resistance between species (Rimmer and van den
 Disease Management of Phoma Infections
Berg 1992). Attempts to expand the gene pool
were initiated by transfering the resistance genes
carried by the "B" genome of the mustard species
B. carinata, B. juncea and B. nigra into B. napus
(Delourme et al. 1995). L. maculans resistance is
conserved in a triplicate region of the Brassica "B"
genome (Dixelius and Wahlberg 1999). Therefore,
Brassica species with a "B" genome function are
excellent donors of blackleg resistance (Sacristiin
and Gerdemann 1986). Most work has been
focused on the "B" genome species B. juncea
(AABB, 2n = 36), B. nigra (BB, 2n = 16) and
B. carinata (BBCC, 2n = 34), but wild accessions
of the "A" genome species B. rapa (or its synonym
B. campestris, turnip; AA, 2n = 20) and the "C"
genome species B. oleracea (CC, 2n=18), and also
wild cruciferous weeds like Dipiotaxis muralis and
D. tenuifolia, are promising new donors of resis-
tance loci (for reviews, see Downey and Räbbelen
1989; Salisbury et al. 1995; Wratten et al. 1995).
Chen and Seguin-Swartz (1999) found resistance
to L. maculans in the wild crucifers Arabi-
dopsis thaliana, Dipiotaxis muralis, D. tenuifolia,
Raphanus raphanistrum and Sisymbrium loeselii,
which expressed hypersensitive responses and
lignin deposition in tissues challenged with L.
maculans. Rimmer et al. (1995) reported blackleg-
resistant genes in white mustard (Sinapis alba).
Also, Sinapis arvensis has proven valuable for
introducing blackleg resistance to oilseed rape
(Snowdon et al. 2000). Cotyledon and adult plant
resistance were associated with the presence of an
acrocentric additional chromosome in S. arvensis.
The Brassica mustards B. juncea (Indian mustard)
and B. carinata (Ethiopian mustard) provide
excellent sources of blackleg resistance as shown
by interspecific crosses with B. napus (Sacristan
and Gerdemann 1986).
However, some caution is required with this
approach (Taylor et al. 1995; Wratten et al. 1995;
Pedras et al. 1998b, 1999; Purwantara et al. 1998;
Sexton et al. 1999), as several pathotypes of L.
maculans can attack B. juncea and have been
found in the Australian and Canadian fungal pop-
ulations. Moreover, the apparent lack of symp-
toms on cotyledons and leaves is sometimes
followed by colonization of root or basal stern
tissue (Rimmer et al. 1995). Purwantara et al.
(1998) suggest the use of B. carinata as a source
of blackleg resistance. Resistance carried by the
"B" genome is total and is characterized by a
hypersensitive response at the cotyledon stage
(Delourme et al. 1995). Genetic studies have
shown that this resistance is mono- or oligogenic.
209
Knowledge of the chromosomal localization of
the resistance loci allows breeding of mono- or
oligosomic addition lines, which contain the 38
chromosomes of the AACC genome of B. napus
and one or few chromosomes of the "B" genome
obtained by interspecific crosses with B. carinata
(BBCC,2n = 34), B. juncea (AABB, 2n = 36) or B.
nigra (BB, 2n = 16) (Delourme et al. 1995). At
least three different B. nigra chromosomes were
found to contribute to blackleg resistance in B.
napus-B. nigra addition lines (Zhu et al. 1993). In
contrast, the B. juncea resistance is monogenie
(Chevre et al. 1997). Inheritance studies of black-
leg resistance originating from B. juncea and B.
nigra in ne ar isogenic lines of B. napus provided
evidence for a single dominant allele, controlling
B. juncea-type resistance in B. napojuncea, and
two independent dominant loci controlling B.
nigra-type blackleg resistance in B. naponigra
(Dixelius 1999). In order to transfer interspecific
resistance to winter oilseed rape varieties, selec-
tion schemes combining selfing and backeross
were developed which led to the selection of
stable B. napus-B. juncea recombinant lines, car-
rying the monogenie blackleg resistance from B.
juncea (Chevre et al. 1997). This resistance was
attributed to the resistance gene flm1 from the B.
juncea 'B' genome (Somda et al. 1999). Recombi-
nant B. napus lines carrying the Jlm1 gene of B.
juncea and addition lines bearing the B. nigra
chromosome B4 (AACC+B4, 2n = 38+1) have a
high degree of collar resistance and display a
hypersensitive re action on both, cotyledons and
leaves (Roussel et al. 1999). The B. napus-B.
juncea hybrid lines bearing flm1 revealed lower
expression of resistance associated with a delay in
plant responses. Resistance towards A-strains was
variable and no resistance towards NA-strains
was observed (Roussel et al. 1999; Somda et al.
1999). Some resistance genes provide protection
against fundamental characteristics common to L.
maculans and the completely unrelated oomycete
Peronospora parasitica (Mitchell-Olds et al. 1995).
Multiple disease resistances may protect against
a broader range of pathogen races and contribute
to a durable defence that is not easily circum-
vented by rapidly evolving fungal pathogens
(Mitchell-Olds et al. 1995). However, all Brassica
cultivars differ in efficiency and durability of resis-
tance, and most of the cultivated varieties are too
susceptible to blackleg. Almost all spring cultivars
are not blackleg-resistant (Pedras 1998a).
The introduction of genetic disease resistance
by conventional plant breeding requires much
210 K. Voigt and IW. Wöstemeyer
time and effort. New methods for a more efficient
transfer of fungal disease resistance, somatic
hybridization and genetic engineering need to be
included in improved breeding programs.
E. Forecasting Blackleg Epidemics
Forecasting of stern canker epidemics improves
disease control, optimizes fungicide use and
re duces the risk of crop damage. Although the
disease is monocyclic, the period of ascospore
release is extended over several months. In
Australia, this release is highest during May-
August, which coincides with the sowing period
(Salisbury et al. 1995). In Canada, the incidence of
mature asci rises slowly during autumn from
17-26% in mid-September, 1month after harvest
of the current season's spring rapeseed, to 35-49%
in mid-November (Rempel and Hall 1993). In
Germany, maximum discharge of ascospores
starts in September with 60-70% of all released
ascospores (Schramm and Hoffmann 1991) and
reaches a second peak 1 or 2 months later
(Thürwächter et al. 1999). High infection levels in
autumn are the consequence of ascospores dis-
charged during that time. The earlier ascospores
germinate and disease manifestation occurs,
the more basal are the stern canker lesions
(Hammond and Lewis 1986). Since early infec-
tions cause the greatest yield losses, the risk of
severe stern canker epidemics needs to be forecast
in autumn, when the pathogen is still in the leaves.
In the leaf-spotting phase plants can be success-
fully treated with foliar fungicides. If applied later,
when the mycelium has already spread through
the stern, fungicides have limited curative activity
(West et al. 2000). Stern cankers develop by infec-
tion of young plants from the cotyledon to the
eight-Ieaf stage (Hall 1992). The first releases
of ascospores can be predicted by monitoring
weather conditions and maturation of pseudothe-
cia. The amount of ascospore discharge is influ-
enced by rain and temperature (Schramm and
Hoffmann 1991). The release of ascospores from
the pseudothecia reaches its maximum a few
hours after rainfalls of >25mm (McGee 1977;
Krüger and Wittern 1985). The accuracy of risk
assessment is improved by updating the forecasts
during the winter, by including factors relating
to the intensity and duration of the Phoma leaf-
spotting phase. Weekly observation of Phoma
leaf spotting in winter oilseed rape allows a fore-
cast of the the severity of stern canker epidemics
and guides the timing of fungicidal treatments
(West et al. 1999). Lesion size is associated with
the quantity of L. maculans in the first 12 days
after inoculation of a susceptible cultivar. The
quantity of the fungus increases during the first
12 days after inoculation and then declines in coin-
cidence with sporulation, rapid necrosis and the
onset of leaf senescence (Mahuku et al. 1995),
while the fungus invades the petiole and stern
tissue.
There are several reasons for not using the
degree of Phoma leaf spotting as an exclusive
parameter for the prediction of disease severity.
(1) Visible leaf spot infections do not correlate
with the degree of latent infections in the crown
region (Schramm and Hoffmann 1991; Petrie et al.
1995); (2) a significant correlation was found
between latent infections in the crown region in
autumn and disease severity at the stern base at
harvest time (Schramm and Hoffmann 1991).
Resistant plants usually suppress the expression of
the disease by prolongation of the latent phase
(Xi et al. 1991); (3) sometimes L. maculans
directly invades sterns (Xi et al. 1991), resulting in
stern lesions prior to leaf spots; and (4) suscepti-
ble cotyledon reactions are not necessarily associ-
ated with susceptible stern reactions (Kutcher et
al. 1993), resulting in an overestimation of disease
severity. Taken together, the incidence of both leaf
and stern infections is necessary to assess disease
severity. Also, quantification of latent incidences
may be of importance. For this purpose, rapeseed
plants need to be transferred at various times from
the field into the laboratory and examined by
using an incubation method which shortens visible
manifestation of the disease (Schramm and
Hoffmann 1991). In the long run, further factors
(e.g., climatic parameters, seed- and soil-borne
inoculum, cultural practices) have to be included
in models for forecasting the impact of blackleg
on oilseed rape.
x. Conclusions
Phoma lingam or its sexual form, the loculoas-
comycete Leptosphaeria maculans, is known as
the causative agent of blackleg disease in oilseed
rape and vegetable brassicas. The pathogen is dis-
tributed worldwide over all growing areas. Phoma
cankers in the lower stern and hypocotyl regions
 Disease Management of Phorna Infections
are often coinfected by several other fungi and
bacteria. Therefore, disease management, espe-
cially with respect to reducing infection pressure
in soils, requires coping with a bunch of different
organisms, the blackleg complex. The natural
reservoir of these fungi are many different weed
crucifers, which must therefore be included into
management considerations.
L. maculans has a differentiated pathotype
structure, which is structured into two major
pathogenicity groups, the aggressive (A) and
the non-aggressive (NA) group. With respect to
physiological traits (sirodesmin secretion, pigment
formation) and according to genetic characters
(RAPD, microsatellite and RFLP patterns, elec-
trophoretic karyotype, rDNA sequences), the A-
group should be regarded as L. maculans sensu
stricto, whereas the NA-isolates probably repre-
sent at least one and possibly more than one
different species.
Knowledge of genetic variability in different
genotypes of L. maculans and related species and
of the potential to form sexual and somatic
hybrids between different pathotypes is essential
for an assessment of novel recombinants that
might change host range and break resistances
of Brassica cultivars. Therefore, and due to the
observation that L. maculans populations seem to
adapt quickly to altered cultivar or environment al
conditions, breeding for resistance, ideally based
on multilocus approaches, is required in Brassica
agriculture.
Effective disease management requires an
integrated approach of breeding, Phoma fore-
casting, genotype monitoring, weed and insect
control, reasonable fungicide spraying regimes,
crop rotations of 4-5 years, and soil and especially
stubble treatment that minimizes survival of
ascospores.
Consequent exploitation of additional funda-
mental research is urgently required for improve-
ments in Phoma control and even for maintaining
the status quo, due to the rapid coevolution of
PhomalBrassica populations. Understanding the
genetics of fungi from the blackleg complex is
still rudimentary, and much more must be learnt
about the chemical cross-talk between plant and
pathogen. Detoxification of the phytoalexin
brassinin by L. maculans and phomalide isomer-
ization by the plant are just examples where
breeding for resistance could find appropriate
starting points. Much more can and should be
revealed.
211
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Zhu JS, Struss D, Röbbelen G (1993) Studies on resistance
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12 Management of Fusarium Diseases
KERSTIN VOIGT
CONTENTS
I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . .
11. The Genus Fusarium: Plant Pathogenic
Fungi Explore a Wide Host Range . . . . . . . . .
A. Diseases Caused by Fusarium on
Agricultural Crops . . . . . . . . . . . . . . . . . . . . . .
1. Development of Fusarium Diseases ........
2. Disease Symptoms and Host Range . . . . . . . .
3. Epidemiology and Distribution. . . . . . . . . . . .
B. Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Visual Disease Assessment . . . . . . . . . . . . .
2. Molecular Probes and Assays are Powerful
Tools for the Identification of Fusarium
Diseases ...........................
C. Origin and Phylogeny of Fusarium Species
Complexes . . . . . . . . . . . . . . . . . . . . . . . . . . . .
IH. Management of Fusarium Diseases . . . . . . . . .
A. Cultural Methods ......................
B. Chemical Methods . . . . . . . . . . . . . . . . . . . . . .
C. Biological Control Strategies .............
1. Fusarium Against Fusarium ............
2. More Biocontrol Agents . . . . . . . . . . . . . . .
D. Breeding for Resistance .................
E. Forecasting Fusarium Diseases ............
IV. Targets for Plant Protection ..............
A. Defence Mechanisms of the Host Plant .....
1. Plant Chitinases .....................
2. Fungitoxic Exudates and Phenolic
Compounds . . . . . . . . . . . . . . . . . . . . . . . . .
3. Resistance Genes ....................
B. Chemical Weapons of Fusaria Function
as Pathogenicity Factors .................
1. Plant-Cell-Wall-Degrading Enzymes .....
2. Suppressors of Plant Defence Response
Reactions ..........................
3. Mycotoxins .........................
4. Growth Regulators . . . . . . . . . . . . . . . . . . .
V. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Institut für Mikrobiologie, Pilz-Referenz-Zentrum,
Friedrich-Schiller-Universität, Neugasse 24, 07743 Jena,
Germany
I. Introduction
217
218 Species of the genus Fusarium (Fig. 12.1) rank
among the most common pathogens causing
220 seed- and soilborne diseases on agricultural
221
222 crops worldwide. Among 59 species, representing
223 35 fungal genera, fusaria have been the most fre-
223 quently detected pathogens on wheat seed from
223
Ontario, Canada (Clear and Patrick 1993). The
frequency of Fusarium spp. is increasing. Many
223 tend to be 'opportunistic' pathogens, causing dis-
eases which are most damaging on plants that are
224
225
debilitated or otherwise predisposed to infection.
225 They are often encountered as members of inter-
227 acting pathogen complexes, which include other
228 genera of facultatively parasitic soilborne fungi,
230
230 such as Phoma, Rhizoctonia, Pythium, Verticillium,
231 Alternaria, and others (Schleier et al. 1997). Up to
232 17 causal organisms of the genus Fusarium are
232 associated with Fusarium ear or head blight (scab)
233
233 of small grain cereals (Parry et al. 1995). They are
primarily soilborne pathogens, surviving long
233 periods as resistant chlamydospores or thiekened
233 hyphae on organic debris in soil (Benson 1994),
234 and many exhibit wide host ranges. Therefore,
234 management of Fusarium diseases remains a dif-
ficult task. Disease control measures involve prin-
235
ciples of disease avoidance, pathogen exclusion,
235
236 pathogen eradication and disease resistance in
236 agro-ecosystems. The methods implement differ-
237 ent strategies, depending on eultural, chemical,
biologie al measures and control via breeding for
resistance and fore casting epidemics, which will be
reviewed in more detail in relation to the biology
of the fungus.
While many reports discuss aspects of the
management of single Fusarium diseases within
specific growing areas in more detail (Häni 1981;
Nelson et al. 1981; Parry et al. 1995; Gilbert and
Tekauz 2000; Tekauz et al. 2000), this review rep-
resents an updated review of all Fusarium diseases
which are globally of agricultural relevanee. For
The Mycota XI
Agricultural Applications
Kempken (Ed.)
(?) '\nrlnp-pr-Vprh'l(J Rprlin HpirlplhPTO ?OO?
218
Fig. 12.1. Colony morphologies of six Fusarium spp. from wheat, maize and rice grown on potato dextrose agar for 7
days at room temperature
reviews dealing with disease management of plant
pathogens not belonging to the genus Fusarium,
see the appropriate chapters of this volume.
11. The Genus Fusarium:
Plant Pathogenic Fungi Explore
a Wide Host Range
Species of the genus Fusarium are typical soil
microorganisms. Most of these exist as integral
members of plant-fungus communities that may
be in part a result of interspecific competition for
a limited number of niches in the soil environ-
ment. Following their plant associates, fusaria
became distributed worldwide, ranging from trop-
ical and temperate areas to zones with extreme cli-
matic conditions, such as deserts, alpine and arctic
regions (Stoner 1981). Phytopathogenic species of
the genus Fusarium are characterized by an
extensive host range. Fusarium spp. have been
reported on all cereal crops in western Europe
(Cassini 1981) and in North America (Cook
K. Voigt
1981a; Tekauz et al. 2000), on wheat, barley and
cotton in China (Cook 1981 b), on rice plants in
Japan, Taiwan and Thailand (Sun and Snyder
1981), on all important crops of the tropics (Stover
1981; Waller and Brayford 1990), and on timber
trees in forest nurseries worldwide (Bloomberg
1981). Host plants comprise all plant families of
economical importance (Table 12.1). Fusaria dis-
perse through infected seeds over long distances
into areas with noninfested soils and cause serious
economicallosses in both yield and grade of agri-
cultural crops. Over 60 Fusarium spp. have been
reported in raw food and feed materials since 1969
(DeNijs et al. 1996). These fungi form a large
variety of mycotoxins (D'Mello et al. 1997), spoil-
ing the quality of agricultural crops for animal and
human consumption. For example, Fusarium-
damaged kernels of wheat are characterized by
high contents of the trichothecene deoxyni-
valenol, a mycotoxin that is produced by F
graminearum, the primary etiological agent of
Fusarium head blight (Dexter et al. 1996). The
deoxynivalenol levels are retained during pro-
cessing and cooking (Nowicki et al. 1988) and can
also persist in malt and beer (Prom et al. 1999).
 Management of Fusarium Diseases 219

Table 12.1. Diseases caused by species of the genus Fusarium. (After: Armstrong and Armstrong 1981; Louvet and Toutain
1981; Stover 1981; Sun and Snyder 1981; Parry 1990; Waller and Brayford 1990; Fravel and Engelkes 1994; Morton 1994;
Schänbeck et al. 1994; Sumner 1994; Jeger et al. 1996; Agrios 1997; Schisler et al. 1998; Secor and Gudmestad 1999; Tekauz
et al. 2000)

Host range Disease Host plant Causal agent

Vascular wilts Formae speciales of F. oxysporum

Starch sources Panama disease Banana (Musa sp.) F.oxysporum f. sp. cubense
Sweet potato (Ipomoea sp.) F.oxysporum f. sp. batatas
Potato (Solanum sp.) F.oxysporum f. sp. solani
Legurnes as protein Broad bean (Vicia sp.) F.oxysporum f. sp. fabae
sources Bean (Phaseolus sp.) F.oxysporum f. sp. phaseoli
Pca (Pisum sp.) F.oxysporum f. sp. pisi
Alfalfa (Medicago sp.) F.oxysporum f. sp. medicaginis
F.oxysporum f. sp. glycines
Soybean (Glycine sp.) F.oxysporum f. sp. lentis
Lentil (Lens sp.) F.oxysporum f. sp. trifolii
Clover (Trifolium sp.)
Oil sources Castor bean (Ricinus sp.) F. oxysporum f. sp. ricin i
Saffiower (Carthamus sp.) F. oxysporum f. sp. carthami
Oil palm (Elaeis sp.) F. oxysporum f. sp. elaeidis
Sesame (Sesamum sp.) F. oxysporum f. sp. sesam i
Sources of plant fiber Hemp (Cannabis sp.) F. oxysporum f. sp. cannabis
Flax (Linum sp.) F. oxysporum f. sp. lini
Cotton (Gossypium sp.) F. oxysporum f. sp. vasinfectum
Ornamental plants and Chrysanthemum F. oxysporum f. sp. chrysanthemi
fiowering bulb crops China aster (Callistephus sp.) F. oxysporum f. sp. callistephi
Carnation (Dianthus sp.) F. oxysporum f. sp. dianthi
Orchid (Cattleya sp.) F. oxysporum f. sp. cattleyae
Transvaal daisy (Gerbera sp.) F. oxysporum f. sp. gerberae
Lily (Lilium sp.) F. oxysporum f. sp. lilii
Tulip (Tulipa sp.) F. oxysporum f. sp. tulipae
Vegetables Asparagus (Asparagus sp.) F. oxysporum f. sp. asparagii
Beet (Beta sp.) F. oxysporum f. sp. betae
Cabbage (Brassica sp.) F. oxysporum f. sp. conglutinans
Tomato (Lycopersicon sp.) F. oxysporum f. sp. lycopersici
Radish (Raphanus sp.) F. oxysporum f. sp. raphani
Cucumber (Cucumis sp.) F. oxysporum f. sp. cucumerinum
Spinach (Spinacia sp.) F. oxysporum f. sp. spinaciae
Fruits Bayoud disease Muskmelon (Cucumis sp.) F. oxysporum f. sp. melonis
Watermelon (Citrullus sp.) F. oxysporum f. sp. niveum
Strawberry (Fragaria sp.) F. oxysporum f. sp. fragariae
Guava (Psidium sp.) F. oxysporum f. sp. psidii
Date palm (Phoenix sp.) F. oxysporum f. sp. albedinis
Herbs Dill (Anethum sp.) F. oxysporum f. sp. anethi
Basil (Ocimum sp.) F. oxysporum f. sp. basilicum
Coriander (Coriandrum sp.) F. oxysporum f. sp. coriandrii
Luxury foods Tobacco (Nicotiana sp.) F. oxysporum f. sp. nicotianae
Coffee (Coffea sp.) F. oxysporum f. sp. coffeae
Vanilla (Vanilla sp.) F. oxysporum f. sp. vanillae
Ginger (Zingiber sp.) F. oxysporum f. sp. zingiberi
Timber tree crops Pine (Pinus sp.) F. oxysporum f. sp. pini
Oak (Quercus sp.) F. oxysporum f. sp. querci
Rot diseases
Basal rot Onion (Allium sp.) F. oxysporum f. sp. cepae
Crown and root rot Tomato (Lycopersicon sp.) F. oxysporum f. sp.
radicis-lycopersici
Stern rot Cereals and grasses F. graminearum a
F. culmorum
F. avenaceum b
220 K. Voigt

Table U.1. Continued

Host range Disease Host plant Causal agent

Top distortion Sugarcane F. moniliforme

(pokhah boeng)
and stern rot
Foot (stern base), Cereals and grasses F. graminearum a
Crown and root rot F. culmorum
F. avenaceum b
Cucurbits, squash F. solani f. sp. cucurbitae
Bean (Phaseolus sp.) F. solani f. sp. phaseoli
Pea (Pisum sp.) F. solani f. sp. pisi
Dry rot Potato (Solanum sp.) F. sambucinum e
F. sulphureum
Stalk and ear rot Maize F. graminearum a
F. culmorum
F. moniliformec
Head blight (scab, Wheat, barleyand F. graminearum a
ear blight) other cereals F. crookwellense
F. avenaceum b
F. moniliformec
F. poae
F. sporotrichioides
F. solani
Seedling blight and Non grain crops
darnping off and cereals
Snow rnould Cereal grains and grasses F. nivaled
Bakanae disease Rice F. moniliformec
(Hypertrophy)

aAscornycetous stage: Gibberella zeae (Booth 1981).

bAscornycetous stage: Gibberella avenacea (Booth 1981).
CAscornycetous stage: Gibberella fujikuroi (Booth 1981).
dAscornycetous stage: Monographella/Calonectria nivalis (Booth 1981).
eAscornycetous stage: Gibberella pulicaris (Booth 1981).

Averaged over two years, F. graminearum reduced
grain yield by 32%, at a disease severity of 28%
(Wong et al. 1992). Fusarium damage has a nega-
tive effect on kerne 1 weight, semolina yield and
glutenin proportion (Dexter et al. 1997). The loss
of dough functionality and consistency as weIl as
loaf volume potential has been attributed to
fungal proteases hydrolyzing wheat storage pro-
teins in Fusarium-damaged kernels (Nightingale
et al. 1999).
A. Diseases Caused by Fusarium
on Agricultural Crops
The ubiquitous distribution of Fusarium species
predisposes a wide range of plant disease types
(Table 12.1). The table is not designed to be
exhaustive, but rather illustrative of certain
distinguishing features in the characterization of
Fusarium diseases. Many other aspects of fusaria
and their diseases, management and their interac-
tion with the host plant have also been considered
in several text books (Parry 1990; Manners 1993;
Agrios 1997).
Fusarium wilts, caused by F. oxysporum, and
also Fusarium root rots, caused by more or less
specific strains of F. oxysporum, are among the
most severe plant diseases in the world. F. oxys-
porum strains represent a high degree of host
specificity which results in the concept of formae
speciales that are strictly adapted to a plant
species, and races that are adapted to cultivars of
the host plant (Alabouvette et al. 1998). More
than 100 formae speciales have been described
(Armstrong andArmstrong 1981). Fusarium wilts
affect many plant species belonging to aIl botani-
cal families with the exception of Graminaceae
(Alabouvette et al. 1998). The vascular wilts
end anger vegetables and fiowers, herbaceous
perennial ornamentals, field crops (e.g., cotton and
tobacco), plantation crops (e.g., banana, coffee
and sugarcane) and silk trees (Agrios 1997). Most
of the wilt-causing fusaria belong to the species
 Management of Fusarium Diseases
Fusarium oxysporum. Different host plants are
attacked by special forms (formae speciales; f. sp.)
or races of the fungus. The fungus that attacks
tomato is designated as F oxysporum f. sp. lycop-
ersici; cucurbits, F oxysporum f. sp. conglutinans;
banana, F oxysporum f. sp. cubense; cotton, F
oxysporum f. sp. vasinfectum; carnation, F oxys-
porum f. sp. dianthii, and so on.
Other fusaria are less systemic and attack pri-
marily the roots and lower sterns of cereals and
grasses (root and stern rots). Fusarium foot rot is
caused by F nivale, F culmorum, F avenaceum
(Gibberella avenacea), and F graminearum (G.
zeae) (Parry 1990). Stalk rot and ear rot diseases
of corn (F graminearum) are distributed world-
wide and cause serious losses that have increased
up to 30% (Agrios 1997). F graminearum and the
related F culmorum, F crookwellense and F
moniliforme cause seedling blight and head blight
(scab) of small grains (Agrios 1997). Yield losses
can be as high as 50%.
Fusarium head blight results in severe damage
to wheat and other cereals and has reached epi-
demie proportions in the world, especially in areas
with high temperature and high relative humidity
during the heading and blossoming period.
Fusarium head blight is currently the most signifi-
cant disease of barley in parts of western Canada
(Tekauz et al. 2000). Outbreaks of severe epi-
demics were experienced in 1993, 1994 and
1996-1998. Although barley may not have been a
main host for Fusarium, a shift from wheat to
barley is believed to have resulted from funda-
mental changes in pathogen population and envi-
ronmental conditions (Tekauz et al. 2000). Also, in
central Europe, disease has shifted from wheat to
rye. This implies a high degree of genetic diversity
of aggressiveness in individual field populations of
F graminearum and also F culmorum, the fungus
dominating the northern, maritime regions in
Europe (Vaneeuwijk et al. 1995; Miedaner and
Schilling 1996; Miedaner et al. 2000).
Fusarium solani and some formae speciales of
F oxysporum affect many different kinds of non-
grain crops (e.g., legurnes ) and cause rotting of
seeds and seedlings (damping-off), rotting of
roots, lower sterns, corms, bulbs and tubers, as well
as vascular wilt diseases.
Fusaria are also typical postharvest
pathogens. They cause postharvest deeays on root
erops, tubers and bulbs of vegetables and orna-
me nt als by the development of pink or yellow
moulds. During a few months storage the fungi
221
have time to diseolor the seeds of legurnes and
grains, kill their ovules, weaken or kill the
embryos, cause shriveling of seeds and produce
myeotoxins whieh are toxie to human and animals.
A pathogen of partieular concern in potato tuber
storages is F sambucinum (aseomycetous stage:
Gibberella pulicaris), whieh primarily eauses dry
rot in potatoes worldwide (Schisler et al. 1998;
Seeor and Gudmestad 1999). Annual erop losses
can re ach 25% (Schisler et al. 1998).
Many other aspeets of fusaria and their dis-
eases, importance and disease management have
also been considered in several textbooks (Parry
1990; Manners 1993; Agrios 1997).
1. Development of Fusarium Diseases
Blight and rot diseases of corn are favored by dry
weather early in the season and wet weather near
or after silking. Also, high plant density, high nitro-
gen and low potassium in the plant and early
maturity of hybrids make them more susceptible
to the diseases (Rowaished 1981). Asexual as well
as sexual spores may provide primary inoculum
for plant infection. In the case of ear or head blight
(seab) of wheat caused mainly by F graminearum
and its teleomorph G. zeae (Table 12.1), macro-
conidia and aseospores - both expressing supple-
mentary optimum temperature ranges for spore
produetion - initiate the disease cycle (Gilbert and
Tekauz 2000). Microscopy of inoculated wheat
glumes revealed that the fungus appeared to
penetrate through stomata, exhibited subcuticular
growth along stomatal rows, colonized glume
parenehyma cells, and sporulated within 48 to 76
h after inoculation (Pritseh et al. 2000). Infection
of small grains such as wheat ears was shown to
occur mainly during anthesis, and it has been
postulated that fungal growth stimulants may be
present in the anthers (Parry et al. 1995). Possible
sources of spore inoculum are crop debris, alter-
nate hosts and Fusarium seedling blight and foot
rot of cereals (Parry et al. 1995). Environmental
eonditions like moisture, solar radiation intensity,
photoperiod, temperature, wind, and inseet
vectors favor spore formation and dispers al per-
mitting loeal buildups of inoculum and develop-
ment of the diseases to epidemie levels
(BenYephet and Shtienberg 1994, 1997; Scherm
and Yang 1999; Clear and Patrick 2000; Gilbert
and Tekauz 2000). The mode of dispersal of inocu-
lum seems to be eomplex: contaminated arthro-
pod veetors, systemic fungal growth through
222
plants, and wind and rain-splash dispersal of
spores have been proposed (Parry et a1. 1995).
Long-distance dispers al is known to be human-
made through the introduction of infected seed
into previously noninfested areas (Clear and
Patrick 2000).
2. Disease Symptoms and Host Range
Vascular wilt diseases are the most invasive dis-
eases caused by Fusarium spp. All vascular wilts
have certain characteristics in common: The
infected plant parts lose turgidity, become flaccid
and light er green to greenish yellow, droop, and
finally wilt, turn yellow, then brown, and die.
Wilted leaves may be flat or curled. Young, tender
shoots also wilt and die. In the xylem vessels of
infected sterns and roots, mycelium and spores of
the causal fungus are present. The vessels are
clogged with mycelium, spores, polysaccharides
produced by the fungus and with pectichemicel-
lulotic plugs produced by the host. The flow of
water through such tissues is sometimes as low as
2 % of that through healthy tissues. The osmotic
properties of infected leaves resemble those of
drought-hardened plants. Toxins (fusaric acid,
lycomarasmin) secreted in the vessels by the wilt-
causing fungus are carried to the leaves, where
they cause reduced chlorophyll synthesis along the
veins and reduced photosynthesis. The alteration
in permeability of the leaf cell membranes leads
to substantialleakage of salts. Diminished ability
to control water loss through transpiration (water
economy) and the disturbance of host metabolism
result in leaf epinasty, wilting, interveinal necrosis,
and death. For example, Fusarium wilt of bananas,
or Panama disease, a name given around the turn
of the century when the disease became notorious
in the central American region, is characterized
initially by premature yellowing of the older
leaves with progressive necrosis and collapse or
chlorotic streaks and brown flecks of the inner
surface of the leaf petioles (Jeger et a1. 1996).
Typically, a purple-brown discoloration develops
in the vascular strands in the rhizome and
pseudostem from which the pathogen can readily
be isolated.
Root, foot aud stern rot is a group of diseases
which are quite variable pathologically. Many are
disease complexes in which other pathogens are
involved (Waller and Brayford 1990). Rot diseases
appear on affected plant organs as water-soaked
areas that later turn reddish, brown to black. Plant
growth is retarded. The roots and sterns are killed.
K. Voigt
In stalk rot of cereals, lower internodes
become soft and appear tan or brown on the
outside, while internally they may appear pink or
reddish. The pith disintegrates, leaving only the
vascular bundles intact. The rot mayaiso affect
roots and lead to a dull-gray appearance of the
leaves, premature death and stalk breakage.
In ear rots of corn, ears develop a pinkish or
reddish mould that often begins at the tip of the
ear. If infection occurs early, the ears may rot com-
pletely and the pinkish mould grows between the
ears and the tightly adhering husks.
Seedliug blight of corn and small grains
appears as a brown cortical rot and blight with
dark-colored lesions either before or after emer-
gence of the seedling above the soil line. The
seedlings become dwarfed and chlorotic and later
die. The fungal agent may be carried on or in
infected seed, or it may attack seed and seedling
from the soi1.
Head blight (scab) infected spikelets first
appear water soaked, then lose their chlorophyll
and become straw-colored. In warm, humid
weather, pinkish-red mycelium and conidia
develop and the infection spreads through the
entire head. Infected kerneis be co me shriveled
and discolored with a white, pink or light-brown
scaly appearance as a result of mycelial outgrowth
from the pericarp. A comparative assessment of
disease symptoms in wheat and barley is reviewed
by Tekauz et a1. (2000). Among 13 Fusarium spp.,
F graminearum infected the most seeds and
sampies of Ontario-grown wheats (Clear and
Patrick 1990). F graminearum was also the pre-
dominant out of 11 species of Fusarium on barley
and oat grown in Manitoba, Canada (Clear et a1.
1996). Unlike other scabs that are the result of
cork formation und er the epidermis due to fungal
attack, the scabby appearance of F graminearum
infected wheat is due to superficial accumulations
of fungal growth on the ears. Scabby kerneis are
white in color, light in weight and shriveled on
their surface. Their presence is a degrading factor
known as "tornbstone" kerneis and is a good indi-
cation that Fusarium head blight has occurred in
the crop (Clear and Patrick 1990).
Dry rots of bulbs, corms and tubers can occur
in the field and in storage. They are common on
plants such as onion, lily, gladiolus and potatoes.
The rot often starts at wounds or through the cuts
formed on such tissues during harvest. It is gener-
ally dry and firm. Tubers usually develop small
brown patches that soon enlarge, become sunken
and show concentric wrinkles that contain cavities
 Management of Fusarium Diseases
lined with white mycelium. Parts of the tubers or
the entire tubers are destroyed and become hard
and mummified.
There are many other diseases caused by
fusaria which will not be described in depth. The
reader who is particularly interested in Fusarium
diseases and their symptoms may consult other
reports (Nelson et al. 1981; Waller and Brayford
1990; Secor and Gudmestad 1999; Gilbert and
Tekauz 2000; Tekauz et al. 2000).
3. Epidemiology and Distribution
The development of disease epidemics depends
on environment al factors. There are often corre-
lations between disease levels and prevailing
temperature levels. In many cases the optimum
temperature for disease development is dose to
that for pathogen growth. The optimum tempera-
ture for the development of F oxysporum f. sp.
Uni, the cause of flax wilt, is 24°C, which is also the
optimum for the development of wilt (Manners
1993). However, the communication between host
and pathogen with respect to disease development
is modulated by environmental factors. Thus, the
same pathogen may have different disease optima
on different hosts. G. zeae (F graminearum), the
cause of head blight or scab of cereals, grows opti-
mally between 25 and 29°C in culture. Under stan-
dard conditions wheat is little affected at low
temperatures (below 16°C) but be comes devas-
tatingly blighted at 25°C, whereas maize is pre-
dominantly affected at low temperatures (around
8°C; Manners 1993). Snow mold of cereals caused
by F nivale occurs under snow-cool damp condi-
tions at temperatures of 5°C or less (Parry 1990).
B. Diagnosis
1. Visual Disease Assessment
Although Fusarium spp. have been recovered
from asymptomatic seeds exhibiting no detectable
levels of mycotoxin contaminations (Clear et al.
1989; Desjardins et al. 2000b), reddish to pink seed
discolorations indieate infection with Fusarium
spp. and high levels of mycotoxins, among them
deoxynivalenol (Clear et al. 1989). Deoxyni-
valenol was localized immunocytochemically in
pericarp tissues, pigment strands, aleurone cells
and starchy endosperm in Fusarium-infected
wheat spikes and kerneIs (Kang and Buchenauer
1999). Of the quality factors. meal color is always
223
affected deleteriously (Clear et a1. 1989; Dexter
et al. 1997). Seedling inoculation assays indicate
three levels of wilt and disease capacities among
isolates of F oxysporum f. sp. radicislycopersici by
means of their cell-free culture filtrates in 20 repli-
cates per treatment on tomato: rate I: 85-100%
wilting in 30min. rate II: 80-90% wilting in 60min
and rate III: 75-90% wilting within 90min
(Madhosingh 1995).
Methods for visual assessment of Fusarium
ear blight disease of wheat caused by F culmorum
and F poae were reported by Doohan et a1.
(1999a).
2. Molecular Probes and Assays are Powerful
Tools for the Identification of Fusarium
Diseases
A large arsenal of molecular tools for generat-
ing molecular-level polymorphisms, such as
DNA-DNA hybridizations, isoenzyme profiling,
restrietion fragment length polymorphism
(RFLP), DNA sequence analyses, electrophoretic
karyotyping, polymerase chain reaction (PCR) or
amplified fragment length polymorphism (AFLP),
has been exploited for the molecular diagnosis of
Fusarium spp. (reviewed by Waalwijk et a1. 1997).
These tools complement morphological diagnosis.
RFLP has been used to identify specialized races of
F oxysporum and to establish correlations with
specific fungal virulence genes. RFLPs also identify
vegetative compatibility groups (VCGs) of differ-
ent races of F oxysporum (Rosewich et al. 1999).
Polymerase chain reaction (PCR) techniques
have been used successfully for the diagnosis of
Fusarium spp. (Voigt et al. 1995; Schilling et al.
1996; Möller et a1. 1997; Doohan et al. 1999b).
Voigt et a1. (1995) obtained species-specific
RAPD patterns for F avenaceum, F moniliforme,
F culmorum, F graminearum and F decemcellu-
lare, which provide a putative source of taxon-
specific DNA fragments. Species-specific PCR
assays exist for F avenaceum, F culmorum and F
graminearum; corresponding primers were
derived from the nudear ribosomal internal tran-
scribed spacer regions (F avenaceum) or from
randomly amplified polymorphie DNA (RAPD)
fragments (F culmorum, F graminearum; Schilling
et al. 1996). RAPD fragments specific for individ-
ual anamorphs of the G. fujikuroi complex were
identified which serve as sources of prim er pairs
which are specific for F proliferatum (mating
population [MP] D of G. fujikuroi) and specific for
F moniliforme [MP A), F proliferatum [MP D)
224 K. Voigt
and F. nygamai [MP G] together (Möller et al.
1997). Assessing the genetic variability of the G.
jujikuroi complex by RAPDs, it has been reported
that substantial differences in DNA patterns are
correlated with MP classifications (Voigt et al.
1995). Also, genes contributing to the secondary
metabolism of fusaria were used to develop
species-specific PCR assays. Doohan et al. (1999b)
applied primers specific for trichothecene
mycotoxin biosynthesis to detect trichothecene-
producing Fusarium species in infected plant
material, a method which is of high potential use
in assessing food and feed quality.
Molecular tools have drastically promoted the
identification and diagnosis of Fusarium diseases,
and are applicable to study host-pathogen rela-
tionships. Quantitative PCR analysis has been
used to monitor F. culmorum and F. poae ear
blight of wheat and its fungicidal control in
glasshouse trials (Doohan et al. 1999a). Fungal
transformants expressing ß-D-glucuronidase
(GUS) activity have been used to detect infection
and quantify hyphal biom ass in infected plant
residues (Oliver et al. 1993). Transformation of F.
culmorum with the GUS reporter gene has been
used to evaluate fungicide efficiency against foot
rot disease of wheat (Doohan et al. 1998). As a
prerequisite of monitoring GUS activity, efficient
transformation systems of Fusarium are required
(e.g., F. moniliforme: Leslie and Dickman 1991; F.
oxysporum f. sp. lycopersici: Garcia-Pedrajas and
Roncero 1996).
At the biochemicallevel fusaria betray their
existence in residues of the host plant. They form
volatile compounds during spoilage of food and
animal feeds (Schnürer et al. 1999). Sesquiter-
penes, for example, are good indicators of myco-
toxin biosynthesis, because most of them are
sesquiterpenoid epoxides (Desjardins et al. 1993).
Recent developments in sensor technology have
led to the construction of volatile compound
mappers, which can be used to predict levels of
fungal colony-forming units in grain, the degree of
mycotoxin contamination, and the fungal species
involved in pathogenesis (Schnürer et al. 1999).
C. Origin and Phylogeny of Fusarium
Species Complexes
Fusarium spp. in association with their hosts are
known to migrate over long distances (Clear and
Patrick 2000). Results from both VCG and RFLP
analyses strongly support the inference that a
group of F. oxysporum f. sp. radicis-lycopersici, the
causative agent of crown and root rot disease of
tomato, constitutes a founder population that
resulted from intercontinental migration from
Florida to Europe (Rosewich et al. 1999). Such
migrations influence adaptation. In reciprocal
cross-infection experiments, parasites were found
to be significantly more adapted to their local host
populations than to hosts from distant populations
(Gandon et al. 1996). Based on metapopulation
models it has been proposed that host-parasite
migration rates exert apredominant effect on
coevolutionary adaptation (Gandon et al. 1996).
In particular, the pathogens are more likely to be
adapted to their local host population than to
allopatric hosts when the pathogen migration rate
is larger than the host migration rate. The oppo-
site should be observed whenever hosts migrate
more than pathogens.
As investigated for F. oxysporum f. sp. lycop-
ersici, VCGs are reliable indicators of evolutionary
origin and population structure (Elias et al. 1993).
Isolates within each VCG are clonal derivatives of
a common ancestor, and formae speciales arose
independently within each VCG (Katan 1999;
Katan and Di Primo 1999). Fusaria are usually het-
erokaryotic, which allows for beneficial effects of
complementation or heterosis (Leslie 1993). Vege-
tative and sexual heterokaryons are distinct from
each other and cannot be correlated. Vegetative
compatibility refers to VCGs, whereas sexual com-
patibility is governed by mating types which repre-
sent a dimictic system among strains of one
biological species or mating population (MP). The
taxon G. jujikuroi comprises at least seven mating
populations (MPs) that obviously represent differ-
ent biological species (A-G; Leslie 1995). The
number of additional MPs is increasing. Recently,
MP H has been identified among strains of F. sub-
glutin ans that cause pitch canker disease of pines
in South Africa, California and Florida (Britz et
al. 1999). Desjardins et al. (2000a) identified
Mexican/Central American F. subglutinans from
maize and its ancestral relative teosinte, which are
closely related to MP H from pine but may repre-
sent a new and distinct MP within the G. jujikuroi
complex. Differentiating these MPs and their
formae speciales usually requires mating and
sometimes pathogenicity tests, which are often
time-consuming and inconclusive. PCR-RFLP
based on intron and exon sequence data of the
histone H3 gene reliably distinguishes F. subgluti-
 Management of Fusarium Diseases
nans f. sp. pini trom the other biological species in
the G. jujikuroi species complex (Steenkamp et al.
1999). Also, RAPD patterns tend to be correlated
with MP classification, as shown for MPs A-F
(Voigt et al. 1995). Using pulsed field gel elec-
trophoresis, a haploid number of 12 chromosomes
was resolved in the MPs A -F (Xue et al. 1995), rep-
resenting an average haploid chromosome number
compared with other Fusarium spp. (Zolan 1995).
Each MP has a distinctive MP-specific karyotype.
More detailed considerations about the genomic
organization and the genetic status of the G.
jujikuroi species complex have been published by
Leslie (1999). All sexually fertile strains in the G.
jujikuroi species complex are heterothallic, with
individual mating types conferred by the broadly
conserved ascomycete idiomorphs MAT-l and
MAT-2. Steenkamp et al. (2000) obtained PCR
fragments which cosegregate with mating type, as
defined by sexual cross-fertility. MAT allele
sequences are useful indicators of phylogenetic
relatedness among species of the G. jujikuroi
complex, because mating-type genes as prerequi-
sites for sexual reproduction reflect evolution
directly at the level of sexual recombination and
speciation.
The G. jujikuroi complex represents a good
example of testing compatibility between species
diagnosed using the biological species concept
(BSC) and the phylogenetic species concept (PSC;
Taylor et al. 1999). BSC has been plotted in con-
cordance to PSC and nine biological species have
been defined using PSC (O'Donnell et al. 1998).
PSC proposes a more natural classification by sup-
plementing tradition al sectional and species-level
taxonomie schemes and identifies also morpho-
logically cryptic species. Knowing the identity of
species and populations and their reproductive
modes improves pathogen control and etiological
predietions (Taylor et al. 1999).
Reproductive isolation has also been shown in
F graminearum. Allelic genealogies constructed
from multiple single-copy nuclear genes re-
covered seven biogeographically structured
lineages, suggesting that they represent phyloge-
netically distinct species among F graminearum
(O'Donnell et al. 2000). Attempts to cross its
teleomorphic stage, G. zeae, with G. acuminata
(F acuminatum) and G. avenacea (F avenaceum),
as weIl as six other re la ted Fusarium spp., were not
successful (Bowden and Leslie 1999).
The genomes of fusaria are subject to reorga-
nization and prone to rearrangements. Rearrange-
225
ment via retrotransposons appears to be stress-
induced in F oxysporum (Anaya and Roncero
1996). Active families of transposable elements,
some of which are confined to Fusarium spp., have
been suggested to contribute to the genetic vari-
ability among species and races of fusaria
(G6mez-G6mez et al. 1999; Hua-Van et al. 2000;
Mes et al. 2000).
111. Management of Fusarium Diseases
The future of many agriculturally important crops
is dependent on the development of effective
methods of contral against Fusarium diseases.
Many of these diseases affect crops grown in
developing countries, causing starvation and
poverty. A primary goal in efficient disease man-
agement is to stop the geographic progression of
Fusarium diseases. Despite windborne introduc-
tion, long-distance dispers al of Fusarium inocula
is known to work mainly through the introduction
of infected seed into previously noninfested areas
(Clear and Patrick 2000). Fusaria survive in soils
as spores (micro- and macroconidia, chlamy-
dospores, ascospores) or thiekened hyphae associ-
ated with plant debris (Benson 1994). The spores
are adapted to long-term survival and are easily
spread by air, equipment, water and contact with
distributive vectors, making successful disease
control complicated. Nelson et al. (1981) summa-
rized many aspects of disease management for a
wide variety of Fusarium diseases spanning all
agriculturally important crops. The application of
single measures has not been satisfactory. A com-
bination of several disease control methods is
required for efficient integrated disease manage-
ment. The challenge of an integrated control straf-
egy is to balance all the components correctly in a
program that is flexible enough to allow preven-
tion of unexpected disease epidemics. This balance
may vary between fields and years and needs to be
revised and updated regularly.
A. Cultural Methods
Cultural contral strategies aim at the reduction
of primary inoculum and at the reduction of
secondary infection cycles. They reduce inoculum
density and infection pressure by conidia and
ascospores and also try to reduce the susceptibil-
226 K. Voigt
ity of target plants. Fusarium root and stern rots
become more severe when plants exposed to the
pathogen are stressed by low temperature, by
intermittent drought or excessive soil water, by
herbicides, by soil compaction and by subsurface
tillage pans, which restrict root growth. Control of
Fusarium rots in the greenhouse is obtained
through soil sterilization (60-75°C heat) and the
use of a healthy propagative stock (Sumner 1994).
Control of Fusarium diseases in the field
depends on a lower plant density, a balanced nitro-
gen, phosphate and potassium fertilization, and
the use of resistant plant varieties (Sumner 1994).
The form of nitrogen used is critical in control-
ling soil-borne pathogens (Sumner 1994). For
example, the degree of root infection increases
with higher levels of nitrogen, but the increase was
higher in plants treated with NH/ than in those
receiving N0 3- as nitrogen source (Rowaished
1981). The plant's nitrogen metabolism and
disease susceptibility seem to be related. Nitrate
increases and ammonia decreases nitrate reduc-
tase activity in both, light and dark-grown
seedlings. The activity of nitrate reductase is also
affected by several phytohormones (Sood et al.
2000). However, phytohormones are secreted by
several fungi induding F. moniliforme, one species
of the perithecial G. [ujikuroi complex (Tudzynski
1997). The yield of phytohormone formation
strongly depends on nitrogen concentration.
Nitrogen exhaustion represses fungal growth,
but increases the formation of plant growth-
promoting gibberellic acids (Tudzynski 1999).
These and other hormones counteract limitation
of plant growth and ensure longevity of the host
plant for the fungus in its latent phase. Thus, bal-
anced nitrogen fertilization helps to reduce fungal
activity and disease manifestation.
The application of sodium chloride or chlo-
ride fertilizers, an old practice, has been shown to
suppress Fusarium crown and root rot diseases
of asparagus and winter wheat (EImer 1992).
The uptake of Cl- is correlated with disease
suppressIOn.
Crop rotations, tillage and seed bed prepara-
tion are methods to eliminate inoculum residing
in overwintering straw and stubble, and, thus, to
ensure improved plant health. Loosening com-
pacted soil with subsoiler chisels before planting
in sandy loam soil has been the most dependable
method of reducing Fusarium root rot of beans
and peas (Sumner 1994). The beneficial effect
results from larger rooting depth and root gener-
ation that reduces plant stress. Optimal field
drainage reduces puddling and high humidity con-
ducive to rot diseases. Staggered seeding and the
planting of cultivars of differing maturities
prevent wholesale infection.
Crop rotation was probably the first method
of disease control (Sumner 1994). However, in the
case of unspecialized Fusarium spp. attacking a
wide range of host plant types, crop rotation is not
successful (Waller and Brayford 1990). In con-
trast, the formae speciales of F. oxysporum or F.
solani, causing host-specific diseases, are generally
better controlled by cultural measures. The spe-
cialization of F. solani f. sp. phaseoli on bean and
F. solani f. sp. pisi on English pea allows related
crops to follow each other with little or no loss
compared with continuous cropping. Sugar beet,
corn or spring wheat in rotation decreases basal
root rot of onion caused by F. oxysporum f. sp.
cepae. Isolates of the pathogen from onion in rota-
tion were less virulent than isolates from onion in
continuous culture (Sumner 1994). Rotation with
nonsusceptible crops, ensuring good soil drainage
and using other disease-free or fungicide-treated
propagative stock may help to reduce losses. The
tolerance level for Fusarium wilt in certified seed
potatoes ranges between 1 and 5 % of the total
stock (Agrios 1997). Research on inoculum
thresholds for seed-borne pathogens is of funda-
mental importance to effectively control world-
wide seed transmission offungal pathogens. Using
seed free of F. solani f. sp. cucurbitae eliminates
root and stern rot caused by the pathogen in
squash, because the fungus does not survive in soil
(Sumner 1994). Rotation of fusarial wilt of banana
(caused F. oxysporum f. sp. cubense) has been
reduced from around 40% to around 5% in
Taiwan by a 2-3year break during which rice is
grown (Manners 1993). Rotations away from
cereals reduce levels of Fusarium head blight, a
disease which is less specialized than Fusarium
wilt diseases but specialized enough to attack
exdusively grain crops like wheat, barley, rye and
corn (Miedaner and Schilling 1996; Gilbert and
Tekauz 2000; Miedaner et al. 2000).
The date of sowing is mainly related to the
development of epidemics. For example, advanc-
ing the sowing date for chickpea crops in southern
Spain from early spring to early winter can slow
down the development of Fusarium wilt epi-
demics, delay the epidemic onset, and minimize
the final impact of the disease (N avas-Cortes et al.
1998). Sowing in early winter allows for an
 Management of Fusarium Diseases
increase in residual soil moisture and a decrease
in temperature, factors which would be contrary in
spring and, thus, conducive to Fusarium wilt.
Flooding fields for long periods or dry fallow-
ing also reduce the number of soil-borne fusaria
by inducing starvation, lack of oxygen or desic-
cation creating unfavorable conditions for the
pathogen. Areas of banana plantations devastated
by wilt (Panama disease) used to be disinfected by
flooding, which is the most practicable method if
a suitable topography and accessibility of water
supplies are given. Subsequent pathogen levels
will remain reasonably low for up to 6years
(Manners 1993).
Storage temperature and duration of seeds
influence the viability of Fusarium-affected seeds.
For example, low temperature and low moisture
over several months suppress fungal metabolism
due to decreased activity of F graminearum in
grain bulks (Gilbert et al. 1997). Temperatures
below 10°C suppress growth and mycotoxin accu-
mulation in F graminearum, F moniliforme and F
proliferatum (Ryu and Bullermann 1999; Ryu et
al. 1999). Besides storage temperature, food
preservatives, such as sorbic acid and its potassium
salt, acetic acid, formic acid and propionic acid or
other propionate formulations, limit mycelial
growth, spore germination and mycotoxin forma-
tion by F proliferatum (Marin et al. 1999) and F
oxysporum (Tzatzarakis et al. 2000). Inhibition of
growth was generally best when additives were
applied in acidic form. Legislative regulations
control mycotoxin contaminations in food and
feed. The European Union has been working for
several years on the harmonization of standards
for mycotoxins derived from Fusarium spp. in
food (de Koe 1999). These standards follow the "as
low as reasonably achievable" principle and limit
mycotoxin exposition via food consumption.
B. Chemical Methods
The use of seed treatments and in-crop fungicides
can successfully complement disease control with
cultural measures. Many reports have studied the
effect of seed treatments on seed vi ability, seed
germination, seed emergence and seedling vigor
including root weight (Gilbert and Tekauz 1995;
Gilbert et al. 1997). Treatment of the propagative
stock with benomyl or application of beno-
myl sprays on the plants has helped to reduce
Fusarium rots. Benomyl as well as the other
227
benzimidazoles (carbendazim, thiabendazole,
thiophanate) are highly effective, function sys-
temically and suppress infection by fusaria. Benz-
imidazoles are known to control F avenaceum,
F culmorum, F equiseti and F solani, but not the
potato tuber affecting F sambucinum, which
exhibits high levels of resistance to benzimidazole
derivatives (Kawchuk et al. 1994). As measured
by visual and molecular tools, the demethylase-
inhibiting fungicides prochloraz and tebuconazole
significantly decrease F culmorum and F poae ear
blight on wheat (Doohan et al. 1999a). Also,
postharvest applications help to control diseases
of agricultural crops in storage. Dry rot of potato
tubers, for example, is a ubiquitous and destructive
disease which requires an injury for entry into the
host. It can be inhibited in its development by
postharvest treatment with thiabendazole (Secor
and Gudmestad 1999). Most benzimidazoles are
converted at the plant surface to methyl benzimi-
dazole carbamate (MBC, carbendazim), which is
fungistatic and is itself used as a systemic fungi-
cide (Manners 1993). This compound interferes
with nuclear division of sensitive fungi and inhibits
mitosis. Organomercury fungicides are protec-
tants, as well as eradicants, used as cereal seed
dressing in Fusarium disease control (Häni 1981;
Manners 1993). In China, organic mercury and
formalin seed treatments controlled the bakanae
disease of rice (Cook 1981b). However, the
phytotoxic effects of formalin/mercury-based
fungicides lead to critical dosage optima.
Fungicides used to partially sterilize soil in order
to control Fusarium diseases can be nonspecific.
The volatile soil fumigant methyl bromide is
applied to the soil before the crop is sown,
penetrates the soil pores thoroughly, disperses
rapidly and does not leave phytotoxic residues
(BenYephet et al. 1994). It is often applied
together with the volatile insecticide chloropicrin
(Manners 1993). The mixture is especially
employed in glasshouses. Organic fungicides
used for soil treatments include metalaxyl, dia-
zoben, pentachloronitrobenzene (PCNB), ethazol,
captan and chloroneb. They are more expensive
per unit area of crop treated than copper- or
sulfur-based fungicides, but cause less damage to
the plant, and have come into widespread use on
horticultural crops of high value. The protectant
captan inhibits thiol-containing enzymes by react-
ing with sulfhydryl groups (Manners 1993). For
broader spectrum applications these fungicides
are often sold as mixtures of different compounds.
228 K. Voigt

Progress in development of soil fungi cides is
limited by the ability of soil microorganisms to
metabolize many chemical moieties quickly into
nonfungicidal degradation products, or by binding
irreversibly to soil particles, especially those high
in organic matter or clay (Morton 1994). Many
isolates of Fusarium become resistant to fungista-
tic chemicals, which results in reduced control and
also increased incidence and severity of Fusarium
diseases (Secor and Gudmestad 1999). Conse-
quently, we must rely on additional management
strategies, including the use of clean seed, seed
treatment, appropriate cultivation measures, etc.
The following methods will provide additional
tools to control disease spread and manifestation.
C. Biological Control Strategies
Numerous reports on biological control agents
and mycorrhizal fungi describe the direct interac-
tion with phytopathogenic Fusarium spp. (Table
12.2).
Fusaria causing root or stern rot diseases are
non obligate parasites that live, grow and multiply
as soil inhabitants, usually in association with dead
organic matter. These fungi are favored by high
soil moisture and high relative humidity in the air.
The fungi can overwinter as mycelium (thickened
hyphae) in infected plant tissues or debris or as
spores. These fungal stages also serve as inoculum
for new infections (Benson 1994). Considerable
progress has been made in biological control of
root and stern rot Fusarium spp. by treating the
seed with antagonistic fungi and bacteria. Co m-
mercially, biological control agents have already
been applied as seed coatings or as water/mineral
amendment in soil-Iess greenhouse cultures
(Alabouvette et al. 1998).
Incorporating organic materials such as barley
straw and chitin in the soil favors an increase in
microorganisms antagonistic to Fusarium. Thus,
accumulation of nonpathogenic microorganisms
diminishes the pathogen population by creation of
suppressive soils that naturally limit the incidence
of fusarioses. The association between suppressive

Table 12.2. Micraorganisms evaluated as potential biological contral agents to contral phytopathogenic Fusarium spp.
in the rhizosphere

Biological control agent Pathogen Mechanism of biocontral Ref

Bacteria
Pseudomonas aeruginosa F. oxysporum Gupta et al. (1999)
Pseudomonas fluorescens F. oxysporum Alabouvette et al. (1998)
Pseudomonas fiuorescens F. oxysporum f. sp. raphani Induced systemic resistance Leeman et al. (1995)
Pseudomonas putida F. oxysporum Alabouvette et al. (1998)
Pseudomonas sp. F. oxysporum f. sp. Sneh et al. (1984)
cucumerinum
Pseudomonas sp. F. oxysporum f. sp. dianthi Rattink (1992)
Actinomycetes F. oxysporum f. sp. Chitinolytic activity Sneh et al. (1984)
cucumerinum
Alcaligenes spp. F. oxysporum f. sp. dianthi Chitinolytic activity Parry (1990)
Arthrobacter sp. F. oxysporum f. sp. Chitinolytic activity Sneh et al. (1984)
cucumerinum
Arthrobacter sp. F. oxysporum f. sp. dianthi Chitinolytic activity Sneh (1981)
Bacillus subtilis F. oxysporum f. sp. dianthi Chitinolytic activity Parry (1990)
Bacillus brevis F. oxysporum f. sp. udum Chitinolytic activity Bapat and Shah (2000)
Bacillus sp. F. oxysporum f. sp. Chitinolytic activity Sneh et al. (1984)
cucumerinum
Enterobacter cloacae F. oxysporum f. sp. Chitinolytic activity Sneh et al. (1984
cucumerinum
Enterobacter herbicola F. oxysporum f. sp. Chitinolytic activity Sneh et al. (1984)
cucumerinum
Hafnia sp. F. oxysporum f. sp. Chitinolytic activity Sneh et al. (1984)
cucumerinum
Hafnia alvei F. oxysporum f. sp. dianthi Chitinolytic activity Sneh et al. (1985)
Serratia liquefaciens F. oxysporum f. sp. Chitinolytic activity Sneh et al. (1984)
cucumerinum
Serratia liquefaciens F. oxysporum f. sp. dianthi Chitinolytic activity Sneh (1981); Sneh et al.
(1985)
 Management of Fusarium Diseases 229

Table 12.2. Continued

Biological contral agent Pathogen Mechanism of biocontral Ref

Fungi
N onpathogenic F. oxysporum Alabouvette et al. (1998)
F. oxysporum
Nonpathogenic F. oxysporum f.s p. batatas Parry (1990
F. oxysporum
Nonpathogenic F. oxysporum f. sp. basilici Induced systemic resistance Larkin and Fravel (1999)
F. oxysporum F. oxysporum f. sp. lycopersici
F. oxysporum f. sp. niveum
Nonpathogenic F. oxysporum f. sp. basilici Competition for nutrients Larkin and Fravel (1999)
F. oxysporum F. oxysporum f. sp. lycopersici
F. oxysporum f. sp. niveum
Nonpathogenic F. oxysporum f. sp. dianthi Rattink (1992)
F. oxysporum
Nonpathogenic F. oxysporum Alabouvette et al. (1998)
F. solani
N onpathogenic F. oxysporum f. sp. basilici Induced systemic resistance Larkin and Fravel (1999)
F. solani F. oxysporum f. sp. lycopersici
F. oxysporum f. sp. niveum
Chaetomium globosum F. graminearum Parry (1990)
Penicillium oxaUcum F. oxysporum f. sp. lycopersici Induced systemic resistance De Cal et al. (2000)
Penicillium purpurogenum F. oxysporum f. sp. lycoperisici Mycoparasitism Larena and Melgarejo
(1996)
Trichoderma viride F. oxysporum Mycoparasitism Tronsmo and Hjeljord
(1998)
Trichoderma viride F. moniliforme Mycoparasitism Yates et al. (1999)
Trichoderma harzianum F. oxysporum f. sp. raphani Mycoparasitism Ogawa et al. (2000)
Trichoderma harzianum Induced systemic resistance Yedidia et al. (1999)
in cucumber
( Cucumis sativus)
Zygorhynchus moelleri F. oxyporum f. sp. Uni ß-1,4-Glucanase and Brawn (1987)
proteolytic activity
(mycoparasitism)
Glomus fasciculatum F. moniliforme Enhancement of plant Vilich and Sikora (1998)
health through
endomycorrhiza
formation
Bacterium-bacterium
combinations
Pseudomonas spp. F. oxysporum f. sp. raphani de Boer et al. (1999)
Pseudomonas spp. F. oxysporum Sneh et al. (1984)
f. sp. cucumerinum
Pseudomonas sp. F. oxysporum Sneh et al. (1984)
+Arthrobacter sp. f. sp. cucumerinum
Pseudomonas sp. F. oxysporum Sneh et al. (1984)
+Enterobacter cloacae f. sp. cucumerinum
Pseudomonas sp. F. oxysporum Sneh et al. (1984)
+Serratia Uquefaciens f. sp. cucumerinum
Bacterium-fungus
combinations
F. oxysporum+ F. oxysporum f. sp. Uni Duijff et al. (1999)
Pseudomonas putida
F. oxysporum+ F. oxysporum f. sp. dianthi Rattink (1992)
Pseudomonas sp.
230 K. Voigt
soils and a sufficient proportion of montmoril-
Ionite clay has been ascribed to the high base-
exchange capacity of the clays (Manners 1993).
Biological factors (e.g., saprophytic microflora)
are involved in the complex mechanisms of soil
suppressiveness (Alabouvette et al. 1998). Clay
prevents the development of metabolic acidity
during bacterial growth and so increases their
growth rate. Montmorillonite clays are favorable
for bacterial activity. This suppresses infection on
banana rootlets by F. oxysporum f. sp. cubense.
1. Fusarium Against Fusarium
The involvement of a saprophytic microflora led
to the idea of pro vi ding soil suppressiveness by
manipulating the microbial balance. Soils sup-
pressive to Fusarium wilts support large popula-
tions of nonpathogenic Fusarium spp. (Louvet et
al. 1981; Sneh et al. 1987; Alabouvette et al. 1998).
Sometimes, the antagonistic organisms may
consist of avirulent strains of the same pathogen.
Nonpathogenic (avirulent) and low virulent
(hypovirulent) strains are capable of colonizing
infection site niches on the plants' surface and pro-
tecting susceptible plants against their respective
pathogens. They are good candidates for the
development of biocontrol preparations (Sneh
1998). Fusarium wilts of several crops (celery,
cucumber, sweet potato) caused by the respective
formae speciales of F. oxysporum were successfully
controlled by inoculating transplants or cuttings
with nonpathogenic strains of the same fungus
(Fravel and Engelkes 1994). These strains
compete with pathogenic strains and also enhance
the resistance of the host towards the pathogens.
Also, the capability of certain fusaria to form anti-
fungal compounds, e.g., alpha-pyrones from F.
semitectum, has biotechnological applications in
plant protection (Evidente et al. 1999).
2. More Biocontrol Agents
Biological antagonisms, although subject to
numerous ecological limitations, are expected to
become an important part of integrated plant pro-
tection against Fusarium diseases. Root-coloniz-
ing mycorrhizal symbionts have been shown to
provide considerable protection to Douglas fire
seedlings from F. oxysporum and to soybean from
F. solani. Ecto- and arbuscular endomycorrhizae
inhibit development of F. oxysporum, F. solani, F.
culmorum and F. graminearum on their hosts by
supporting plant health (Schönbeck et al. 1994).
Cardamom plants inoculated with the endomyc-
orrhizal fungus Glomus fasciculatum to control
damping-off supported more microorganisms in
the rhizosphere with properties antagonistic to
Fusarium than noninoculated plants (Vilich and
Sikora 1998). These indirect mechanisms associ-
ated with biological control agents and mycor-
rhizal fungi are still poorly understood.
Soilborne bacteria like Bacillus cereus provide
effective biocontrol of Fusarium rot and damping-
off diseases. Fluorescent pseudomonads, mainly
P fluorescens and P putida, are among the most
abundant bacteria in the rhizosphere (Alabou-
vette et al. 1998). The mechanisms of effective bio-
control are diverse (Lemanceau and Alabouvette
1993). Pseudomonads exhibit antifungal activity
against F. oxysporum due to antifungal antibiotics,
like N-butylbenzenesulfonamide (Kim et al.
2000). P putida can elicit antifungal phenolics sys-
temically in cucumber. This in duces an overall
defence response varying in chemical complexity
and organ specificity (Ongena et al. 2000).
Siderophores produced by pseudomonads are
related to soil suppressiveness to Fusarium wilts
(Alabouvette et al. 1998). The competition for
iron has been attributed to the suppression of
Fusarium wilts. Pseudomonas aeruginosa produce
hydroxamate-type siderophores and promote
plant growth due to induction of hydrocyanic acid
and indole acetic acid in potato plants (Gupta et
al. 1999). Growth inhibition of F. oxysporum
reaches 70.5%. Rhizobacterial siderophores, rhi-
zobacterial lipopolysaccharides and salicylic acid
are important determinants of induced systemic
resistance (ISR), which require jasmonic acid and
ethylene perception by the plant to develop (van
Loon et al. 1998). ISR, however, is involved in bio-
control via fluorescent pseudomonads, which
induce systemic resistance to Fusarium wilt dis-
eases by lipopolysaccharides of the pseudomon-
adous outer membrane (Leeman et al. 1995).
Salicylic acid is an essential key signal molecule
for the expression of multiple modes of plant resis-
tance (Mauch-Mani and Metraux 1998). A defect
in salicylic acid accumulation leads to enhanced
susceptibility to viral, bacterial and fungal
pathogens extending the normal susceptibility
pattern of the plants (Delaney et al. 1994). Rhi-
zobacteria stimulating ISR promote plant growth
by the production of indole-3-acetic acid and
cytokinin-like compounds as well as by lowering
ethylene levels in plants (Buchenauer 1998).
 Management of Fusarium Diseases
Simultaneous activation of ISR and SAR (sys-
temic acquired resistance) results in an additive
effect on the level of induced plant protection (van
Wees et al. 2000). Other mechanisms, which do not
affect the resistance status of the plant, lead to bio-
logical control too. The genus Trichoderma, for
example, inhibits growth of fusaria via mycopara-
sitism and has thus been exploited as a biological
control agent for Fusarium (Ogawa et al. 2000).
Species of Trichoderma are weIl known for their
chitinolytic activity exhibiting potential for
biocontrol of fungal pathogens. Chitinases, poly
[1 ,4-(N-acetyl-ß-D-glucosaminide)] glycanohydro-
lases (EC 3.2.1.14), hydrolyze chitin, a major com-
ponent of fungal cell walls (Manocha and
Govindsamy 1998). Recently, chitinases have
taken central stage in investigating biological
control of fungal pathogens. Trichoderma viride
decreased radial extension of F moniliforme by
46% after 6days and by 90% after 14days, sug-
gcsting that F moniliforme mycelia were under-
going lysis (Yates et al. 1999). Also, the production
of fumonisin BI by F moniliforme decreased by
85% in coinoculation experiments on corn
kerneis. Disease severity in tomato plants inocu-
lated with the wilt pathogen F. oxysporum f. sp.
lycopersici was decreased by 30% during biologi-
cal treatment with Penicillium purpurogenum
(Larena and Melgarejo 1996). The ß-l,3-glucanase
and chitin ase activities of P purpurogenum led to
lysis of hyphae and spores of the invading Fusar-
ium fungus. Synergistic interactions of chitinases
of fungal antagonists and biocontrol bacteria have
been reported to inhibit spore germination and
germ tube elongation of F solani. The production
of improved strains of Trichoderma utilizes
somatic fusions via the naturally occurring para-
sexual eyde as an alternative to sexual reeombi-
nation. To overcome vegetative ineompatibility,
protoplast fusions can also be applied for strain
improvement (reviews by Harman and Stasz 1991;
Wöstemeyer and Wöstemeyer 1998). Ogawa et
al. (2000) obtained an improved Trichoderma
harzianum strain by protoplast fusion of a wild
type with a benomyl-resistant mutant strain. The
diploid fusion derivative grew rapidlyon minimal
medium containing 100ppm benomyl and
inhibited the growth of F oxysporum f. sp. raphani
on paired plate and soil cultures. Compared
with the parental strains, the fusion strain also
exhibited improved activitics of ß-1.3-glucanases
and chitinases. Breeding of fungicide-resistant
Trichoderma strains antagonistic to Fusarium will
231
have a major impact on integrated management
protocols.
Disease suppression by compatible combina-
tions of biocontrol agents is often significantly
better compared with single strains (Sneh et al.
1984; de Boer et al. 1999; Duijff et al. 1999; Table
12.2). However, the utilization of concatenated
biocontrol agents is limited. As demonstrated by
the inter action between Trichoderma hamatum
and T. pseudokoningii, Trichoderma biocontrol
agents are prone to mutual mycoparasitism with
endogenous Trichoderma spp., which may reduce
their successful application for biological control
of fusaria (Vajna 1985). Thus, compatible combi-
nations of biocontrol microorganisms need to be
defined which do not interfere with each other and
rather synergistically enhance their activity
against the pathogen.
Antagonistic organisms mayaiso consist of
trap plants, which attract pathogens and release
into the soil substances taxic to the pathogen
(Agrios 1997).
The close relationship between beneficial
root-associated microorganisms and their specific
host plants can be used to manipulate rhizos-
phere-specific microbial communities for biologi-
cal control. For example, continuous cropping
wi th resistant varieties over 9-10 years revealed
induced suppressiveness du ring development of a
balanced rhizosphere microflora and enrichment
of nonpathogenic Fusarium spp. (Sneh et al.
1987). Utilization of genetically diverse plant co m-
munities stimulates heterogenetic niches in the
rhizosphere environment (Vilich and Sikora
1998). Due to shifts in microbial plant inter-
action by heterogenetic plantings of crops, a
diversity of new ecological niches can be created.
In mixed crop stands of barley and wheat the
spectrum of Fusarium ssp. shifted toward weak
or nonpathogenic species, whereas the amount of
pathogenic species declined (Vilich and Sikora
1998).
D. Breeding for Resistance
Rost resistanee is the most economical and poten-
tially the most effective tool for the management
of Fusarium diseases. The selection of resistant
cultivars requires no specialized equipment,
rc duces or eliminates the need for synthetic chem-
icals and may shorten rotation times (Shew and
Shew 1994). Several pioneering studies at the
232
beginning of the nineteenth century initiated the
development of resistant vanehes against
Fusarium wilt diseases of cotton, watermelon,
cowpea, flax, tomato and cabbage. Breeding
methods included hybridization and selection of
surviving plants in highly infested soil (review by
Shew and Shew 1994). All varieties of tomato
grown in greenhouses for fresh-fruit production
are resistant to the most common races of F. oxys-
porum f. sp. lycopersici (Alabouvette et al. 1998).
However, breeding for resistance is difficult when
no dominant genes conferring resistance can be
identified, or when the plant species is dioecious
(Alabouvette et al. 1998). Since currently no reg-
istered fungicide is available to control epidemics
of head blight of barley, only high levels of resis-
tance may be effective (Tekauz et al. 2000).
Recently developed barley cultivars possess
enhanced Fusarium resistance (two row malt
barley: AC Metcalfe, AC Oxbow; 6 row hulless
feed: CDC Silky). The success of breeding strate-
gies depends highlyon the lines used as the source
of resistance. Therefore, domesticated wheat lines
and wild wheat relatives have been screened in
hybridization programs as new sources of resis-
tance against Fusarium head blight (wheat scab)
with good results (Gilbert and Tekauz 2000). Five
mechanisms of resistance to Fusarium head blight
have been proposed: type I is the resistance to
initial infection - the form of resistance mani-
fes ted in the cultivar Frontana; type II prevents
spread within the head following infection - the
form of resistance expressed by the Chinese resis-
tant varieties; type III is resistance to kernel infec-
tion; type IV is tolerance whereby yields are
maintained despite the disease; and type V is the
ability of hosts to degrade the toxins secreted by
the fungus (Mesterhazy 1995). The highest
numbers of Fusarium propagules and the highest
concentrations of mycotoxins were found in the
hulls ofbarley (Clear et al. 1997). Breeding hulless
cultivars of small grains promises management of
mycotoxin problems associated with Fusarium
head blight. Clear et al. (1997) detected a twofold
decrease in deoxynivalenol trichothecenes in
barley as a consequence of laboratory dehulling,
even if disease severity and mycotoxin concentra-
tion can increase in barley at any time between
the heading stage and maturity (Prom et al. 1999).
To keep pace with rapid pathogen adaptation it
has been proposed to include phytotoxins, due to
their role in disease development, as tools in
breeding and selection of disease-resistant plants
K. Voigt
for supplementing conventional plant-breeding
programs (Buiatti and Ingram 1991). Other in
vitro selection strategies have been described to
broaden the genetic crop variation by somaclonal
variation induced by cell and tissue cultivation
(van den Bulk 1991).
Natnral selection, which occurred in regions
where the Bayoud disease of date palms had
resided for long periods of time, has given rise to
resistant palms (Louvet and Toutain 1981). These
were propagated vegetatively using mother blocks
of offshots to regenerate groves. The resistance
status of the date palm cultivars against F. oxys-
porum f. sp. albedinis appears to depend on cell-
wall-bound phenolic compounds and lignin (EI
Modafar et al. 2000). Basic pre-infection levels of
the four cell-wall-bound phenolic acids p-hydrox-
ybenzoic, p-coumaric, ferulic and sinapic acid, are
high er in resistance than in susceptible cultivars.
In response to fungal infection the maximum
increase of these compounds reached 12.3-fold for
sinapic acid. In the susceptible cultivar, the phe-
nolic acid content also increased after inoculation
but did not re ach the pre-infection level of the
resistant cultivar. Lignin contents increased in
both cultivars with a maximum accumulation on
the 15th day (EI Modafar et al. 2000). However,
the lignin level in the resistant cultivar was 1.5
times higher than in the susceptible one. Speed
and intensity of phenolic acid accumulations
strongly correlate with the resistance level of
palm cultivars against the wilt pathogen of date
palms.
E. Forecasting Fusarium Diseases
Survey results for the spread of Fusarium spp.
over long periods of time and large crop districts
(e.g., Clear and Patrick 2000) help to develop
strategies for forecasting epidemics. Metapopula-
tion models, as demonstrated by Gandon et al.
(1996), can have a high impact on the develop-
ment of forecasting schemes.
In combination, all the previously outlined
measures would greatly reduce fusarioses in agri-
cultural crops.
IV. Targets for Plant Protection
Plant-fungus interactions are extremely variable.
The inter action between a pathogen and a host
 Management of Fusarium Diseases 233

plant depends on the environment and on the germ tubes, swelling, vacuolization of the cellular
genotype of both. This relationship is often content and finally by lysis of their cell walls
expressed by the diseases tri angle of host, (Ciopraga et al. 1999). Pretreatment of the fungal
pathogen and environment (Bos and Parlevliet suspension with wheat germ agglutinin prevents
1995). Altering one of them influences the physi- fungal infection.
ology of the disease. For example, environmental
factors may change fungal growth rate, a factor
affecting population density which can lead to 2. Fungitoxic Exudates
pathogen adaptation as shown in theoretical and Phenolic Compounds
models (Gandon et al. 1996). Such changes can be
Plants can detoxify a variety of Fusarium toxins
used to define targets for plant protection.
(Karlovsky 1999). Enzymatic inactivation of
mycotoxins is an attractive strategy for the decon-
tamination of agricultural commodities and for
A. Defence Mechanisms of the Host Plant the protection of crops from phytotoxic effects of
fungal metabolites. Genes responsible for some of
1. Plant Chitinases the detoxification activities, some of which origi-
nate from toxin-producing fungi that are able to
Plants induce a variety of defence-response genes.
transform their own products, have been doned
The timing of defence-response gene induction is
and expressed in heterologous hosts (Karlovsky
correlated with the developmental stage of the
1999).
pathogen. In wheat the temporal expression
Plants produce a wide variety of low-
pattern of genes for peroxidases, thaumatin-like
molecular-weight secondary metabolites, many of
pro teins, ß-1.3-glucanases and chitinases ranged
which - phytoalexins - have antifungal activity
from 6 to 12h after F graminearum infection and
and provide a chemical barrier against phytopath-
peaked at 36 to 48h after inoculation (Pritsch et
ogenic fungi. Rapidity and extent of phytoalexin
al. 2000). Plant chitinases represent a family of
accumulation is associated with resistance against
enzymes differing in chitin affinity and optimum
fungal and bacterial diseases, although the genetic
pH values. Out of four chitin ase isozymes, three,
potential for phytoalexin synthesis is found in
chitinases E, Fand H1, had high lytic activity to F
susceptible and in resistant plants. The effective
oxysporum, whereas chitin ase G did not (Arakane
contribution of phytoalexins to the complex mcch-
et al. 2000). The genes for plant chitinases are
anisms for disease resistance resulted an the eval-
known to undergo rapid evolution to provide new
uation of strategies for their biosynthesis in
molecular targets against pathogen adaptation
disease control. Reviews of these and other
(Bishop et al. 2000). At the transcriptional level,
antimicrobial compounds as components of plant
the chitinase genes are differentially expressed
defense against fungal attack are available (Kuc
dependent on plant-microbe interaction. Salzer et
1995; Osbourn 1996). The best-investigated
al. (2000) detected an increased level of transcripts
phytoalexins in plant-Fusarium relationships are
of dass I, II and IV chitinase genes in compatible
saponins (e.g., tomatine; Roldan-Arjona et al.
interactions between Medicago truncatula and its
1999) and the isoflavonoid phytoalexins pisatin
root rot pathogen F solani f. sp. phaseoli. Bean
(Matthews and VanEtten 1983; Ciuffetti and
roots produce dass IV chitin ase when infected
VanEtten 1996), kievitone (Kuhn and Smith 1979),
with F solani f. sp. phaseoli, but proteolytic pro-
medicarpin and maackiain (Lucy et al. 1988;
cessing of the enzyme that is blocked at the N-
Stevenson et al. 1997).
terminus occurred only in compatible interactions
and was not detected in incompatible interactions
with a nonhost strain of F solani (Lange et al.
3. Resistance Genes
1996).
Apart from plant chitinases, other plant pro- Following the molecular pathways of plant-fungus
teins also exhibit high affinity to N-acetylglu- systems sharing gene-for-gene relationships (Flor
cosamine, the major component of chitin. Wheat 1946; De Wit 1992), plants prevent fungi from
germ agglutinin binds to the chitin-containing causing disease by plant-resistant (R) genes
fungal cell wall and inhibits growth of F gramin- matching fungal avirulence genes (Lauge and
earum and F oxysporum by modification of their DeWit 1998). Screening for novel R genes is
234 K. Voigt
required in plant breeding in order to keep pace
with adaptation of fungal parasites to resistant cul-
tivars. Adaptation mechanisms have been studied
by metapopulation models (Gandon et al. 1996).
These models include the distribution of R genes
among host populations in relation to virulence
genes of the parasite population and help to define
sources of improved resistance to pathogens in
plants. In a multiplicative inter action experiment
multiple plant genotypes were inoculated with
several Fusarium spp. at different locations across
Europe to assess nonspecificity of resistance in
wheat to Fusarium head blight (Vaneeuwijk et al.
1995). These experiments generate the basic pre-
requisite for an efficient resistance breeding
pro gram by the identification of genes controlling
the resistance character and resistance level. As
reviewed by Gilbert and Tekauz (2000), genes for
resistance against Fusarium head blight in wheat
(wheat scab) are located on four different chro-
mosomes. In maize a genetic linkage map has been
generated to localize loci involved in the resis-
tance to Fusarium head blight (Pe et al. 1993).
Sensitive and resistant parental inbreds were
selected for crossing to obtain Fl and F2 popula-
tions. F3 families were created by selfing the F2
plants. The segregation of 95 RFLP and 10 RAPD
markers and resistance was analyzed to obtain a
linkage map of maize, in which four to five
genomic regions are shown to carry factors
involved in the resistance to G. zeae (Pe et al.
1993). This multilocus system controls resistance
to G. zeae infection and is quantitatively inherited.
The resistance to wheat scab is horizontal and
nonspecies specific (Vaneeuwijk et al. 1995).
Hybrids can be obtained in breeding pro grams
that retain the desired resistance and can be used
in backcrosses to produce genetically stable vari-
eties. Additive inheritance effects offer the possi-
bility to accumulate different R genes to enhance
resistance to wheat scab (Bai et al. 2000). Breed-
ing strategies to improve Fusarium resistance in
wheat and rye have been reviewed by Miedaner
(1997) and Miedaner et al. (1997).
It is not only the existence of R genes that
decides between resistance and susceptibility. The
speed and quantity of transcript accumulation also
correlates with plant resistance. In resistant culti-
vars of wheat, transcript accumulation of defence-
response genes encoding thaumatin-like proteins
was greater and occurred earlier than in suscepti-
ble wheat cultivars during F graminearum infec-
tion (Pritsch et al. 2000).
B. Chemical Weapons of Fusaria Function
as Pathogenicity Factors
1. Plant-Cell-Wall-Degrading Enzymes
If pathogens prefer to enter the host by penetrat-
ing the cuticle rather than attacking via stomata or
wounds, they need to degrade the cuticle. A plant
cuticle is a complex structure composed of cutin
(up to 70%), waxes, cellulose and pectic com-
pounds (Manners 1993). The fungus penetrates
the cutin layer mechanically or due to enzymatic
degradation. Cutin, a polymer of esterified fatty
acids linked by peroxide and other link ag es, can
be degraded enzymatically by cutinases, which are
glycoproteins that hydrolyze the primary alcohol
esters of the cutin molecule. Cutinases and pecti-
nases have been found in F solani f. sp. pisi
(teleomorph N ectria haematococca MP VI; Rogers
et al. 1994; Stahl et al. 1994; Rogers et al. 2000).
The involvement of cutinases, if it occurs, in plant
infection has long been the subject of controver-
sial debates (Rogers et al. 1994; Stahl et al. 1994).
Cutinase-defective mutants, constructed by gene
disruption, tend to retain pathogenicity, an obser-
vation that does not support the model. Neither F
solani f. sp. pisi on pea (Stahl et al. 1994) nor F
solani f. sp. cucurbitae on squash (Crowhurst et al.
1997) requires cutinase activitity in infection of
fruit and hypocotyl tissue. Cutinases represent a
complex of different izozymes that are differen-
tially expressed during saprophytic and parasitic
stages of the fungus (Koller et al. 1995). It appears
that disruption of all functionally redundant genes
is required to demonstrate the role of host barrier-
degrading enzymes in pathogenesis. Single disrup-
tion of either of the two pectate lyase genes in
N haematococca alone causes no detectable
decrease in virulence on peas, whereas virulence
of double disruptants is drastically reduced. Viru-
lence can be restored by adding purified pectinase
to infection droplets (Rogers et al. 2000). Degra-
dation of the carbohydrate cell-wall components
pectin, xylan, and cellulose in F culmorum
infected wheat spikes was shown by enzyme-gold
and immuno-gold labeling in ultrastructural and
cytochemical studies (Kang and Buchenauer
2000).
Pectinase secretion occurs earlier than secre-
ti on of cellulases and xylanases. In addition, the
Fusarium wilt fungi were shown to produce
various pectinolytic enzymes including polygalac-
turonases (Garcfa-Maceira et al. 2000). Anatomi-
 Management of Fusarium Diseases
cal studies of affected tissues revealed that the cell
walls are disorganized. The presence of such
enzymes in diseased vascular tissues has been
demonstrated. Inactivation of single polygalactur-
onase genes by gene replacement does not affect
virulence of F. oxysporum f. sp. lycopersici on
tomato (Garcfa-Maceira et al. 2000). However,
substances inhibiting the overall activity of pecti-
nolytic enzymes re du ce the incidence of wilting
(Manners 1993). Mutants lacking the ability to
form certain pectinases still cause wilting. This
observation suggests the existence of a complex
set of redundant phytopathogenicity factors,
where the elimination of single factors is tolerated.
There is evidence that fusaria produce enzymes
that degrade hemicellulose, cellulose and lignin
(Manners 1993). Cellulose, poly-ß-l,4-D-glucopy-
ranoside, is the main component of cell walls in
plants and can be degraded by cellulases and ß-
glucosidases from phytopathogenic fusaria, such
as F. oxysporum f. sp. melonis, causing wilt in the
vascular system of muskmelon (Alconada and
Martfnez 1996).
2. Suppressors of Plant
Defence Response Reactions
F. solani f. sp. pisi (teleomorph N haematococca
MP VI) virulent towards pea tolerates the
isoflavonoid phytoalexin pisatin, and detoxifies it
by demethylation (pisatin demethylase ). The reac-
tion is catalyzed by a cytochrome P-450 monooxy-
genase (Matthews and VanEtten 1983). Avirulent
strains are pisa tin-sensitive and do not demethy-
late. Naturally occurring isolates of N haemato-
cocca that lack the ability to demethylate pisatin
[Pda(-)] normally lack the genes (pda) encoding
cytochrome P-450 and are not pathogenic on pea.
Transformation of Pda(-) isolates with the gene
for pisa tin demethylase increases virulence due to
increased pisatin tolerance, but for high virulence
on pea additional genes are needed (Ciuffetti and
VanEtten 1996). The same fungus also detoxifies
the phytoalexins medicarpin and maackiain by
one of three alternative oxygenations of the
isoflavonoid ring system (Lucy et al. 1988). Both
medicarpin and maackiain are fundamental com-
ponents of the resistance mechanism to wilt
(Stevens on et al. 1997). The bean isoflavonoid
phytoalexin kievitone, in contrast, is detoxified by
virulent strains of F. solani f. sp. phaseoli via hydra-
tion of the isopentenyl side chain by the extra-
cellular enzyme kievitone hydratase (Kuhn and
235
Smith 1979). The ability for maackiain detoxifica-
tion correlates with high virulence on chickpea.
One of the known maackiain-detoxifying genes,
makI encoding a flavin-containing monooxyge-
nase, is located on a meiotically unstable, 1.6 Mb
dispensable B chromosome (Covert et al. 1996;
Enkerli et al. 1998). Obviously, losing or acquiring
this chromosome by sexual or parasexual pro-
cesses will affect the behavior of the pathogen
on pisatin-producing host plants (Wöstemeyer
1997). Fortunately, the Mak( +) phenotype is spec-
ified by several independent mak genes (makI-
mak4; Covert et al. 1996) which, with the
exception of makI, map on regular chromosomes
(Enkerli et al. 1998). Natural variation in sensitiv-
ity to phytoalexins contributes to an isolate's
disease potential, as shown for the tolerance of F.
oxysporum f. sp. lycopersici to the tomato phy-
toalexins tomatine and rishitin (Suleman et al.
1996). The saponine tomatine is a plant glycoside
that is part of a preformed chemical barrier
against phytopathogenic fungi. F. oxysporm f. sp.
lycopersici pro duces an extracellular enzyme
known as tomatinase, which deglycosylate alpha-
tomatine to yield the less toxic derivatives, toma-
tidine and lycotetraose. The enzyme is encoded by
a single gene whose expression is induced by
alpha-tomatine and fully repressed by glucose
(Roldan-Arjona et al. 1999). The tomatinase gene
is expressed in planta in roots and sterns through-
out the entire disease cyde of F. oxysporum f. sp.
lycopersici, suggesting tomatinase as an important
factor for pathogenicity on tomato.
3. Mycotoxins
Fusarium spp. produce a wide range of mycotox-
ins. More than 100 toxigenic secondary metabo-
lites have been described (DeNijs et al. 1996).
Trichothecenes - a large group of Fusarium myco-
toxins - act as potent inhibitors of eukaryotic
pro tein synthesis, and thus affect human and
animal health (Desjardins et al. 1993). Phytotoxic
effects of several toxic Fusarium metabolites are
published elsewhere (McLean 1996). The specific
function, if any, for the survival of the fungi that
produce them is not obvious (Desjardins et al.
1993), although single gene disruption ex-
periments revealed reduced virulence of
trichothecene-nonproducing mutants in F.
graminearum (Proctor et al. 1995; Desjardins
et al. 1996). Similarly, virulence of enniatin-
nonproducing knock-out mutants of F. avenaceum
236 K. Voigt
was reduced (Herrmann et al. 1996). Grains
affected by fusarioses usually contain detectable
levels of toxic compounds. Most attention has
focused on toxicants affecting animal and human
health, such as trichothecenes (A and B type),
zearalenone and its derivatives, fumonisins, monil-
iformin, fusarochromanones, fusaric acid, fusarins,
cyclic peptides, and amino esters of the beau-
vericin type (D'Mello et al. 1997). However, not
all Fusarium spp. form mycotoxins in comparably
high amounts. For example, fumonisins have been
detected in two out of 32 diverse Fusarium spp., F
moniliforme and F proliferatum (Norred et al.
1999). Abramson et al. (1993) obtained different
trichothecene types, profiles and quantities among
eight Fusarium spp. Desjardins et al. (2000b)
reported significantly differing toxin profiles
among 11 species of Fusarium recovered trom
Nepalese rice seeds. The Nepalese rice itself
showed no detectable contamination with these
mycotoxins, which indicates effective traditional
practices for grain drying and storage to prevent
mycotoxin formation. Only relatively few fungal
contaminants are able to produce mycotoxins, an
encouraging observation with respect to develop-
ing efficient measures for their specific control.
Many efforts have been made to reduce mycotoxic
contamination by optimizing fungicide formula-
tions and application techniques, by combining
mycotoxin biosynthesis inhibitors and by improv-
ing predictions of infection probabilities (Matthies
and Buchenauer 2000). However, gene expression
in mycotoxin biosynthetic pathways tends to in-
crease following fungicidal treatment accompa-
nied by a decrease in fungal biomass (Doohan et
al. 1999b; for review, see D'Mello et al. 1998).
Additional criteria are required to develop
evaluation protocols for candidate fungicidal
compounds.
Excellent reviews on mycotoxin production
and toxicity including their fungal producers are
available (D'Mello et al. 1997,1998,1999; Placinta
et al. 1999).
4. Growth Regulators
Phytopathogenic fungi of the genus Fusarium are
able to form and secrete plant growth regulators,
phytohormones of the auxin and gibberellin type.
The types of all phytohormones synthesized by
fungi in relation to disease development were
reviewed in an earlier volume in this se ries
(Tudzynski 1997). The role of fungal growth hor-
mones in pathogenesis is still unclear. Auxin and
gibberellin hormones may act synergistically and
seem to activate the corresponding plant genes. F
oxysporum f. sp. cubense, causative agent of
banana wilt disease (Panama disease), induces
increased levels of indole-3-acetic acid (IAA) in
its host and is itself also capable of producing
IAA. F oxysporum f. sp. lycopersici forms ethyl-
ene, which leads to epinastical growth of leaves
during wilt of tomatoes (Manners 1993). F
fujikuroi and its ascomycetous stage G. fujikuroi,
the etiological agent of bakanae (foolish seedling)
disease of rice, produces gibberellic acid (Sun and
Snyder 1981; Tudzynski 1997). Although in the
past decade gibberellin formation has been
demonstrated in numerous plant pathogens,
there is little information on its pathological
significance.
v. Conclusions
Due to the wide distribution and large populations
of pathogenic species complexes of Fusarium, the
control of fusarioses on agricultural crops is diffi-
cult. Our present knowledge on the management
of Fusarium diseases is based mainly on cultural
measures and breeding of resistant plants. Culti-
vation methods reduce disease symptoms but, if
applied exclusively, do not suppress outbreaks of
Fusarium epidemics. Conventional plant breeding
for resistance to pathogens, although successful,
suffers from (1) a lack of sources for resistance
genes, (2) the lack of genetic variability in culti-
vated species, and (3) time-consuming procedures
for hybrid screening and selection. Traditional
plant breeding for resistance is to slow to keep
pace with pathogen adaptation, which is favored
by the globalization of agriculture and by mono-
cultures on a large scale. Recently introduced
resistant plant cultivars trigger the fast develop-
ment of novel fungal races. The establishment of
alternative breeding strategies and screening pro-
tocols will be of increasing importance. Screening
against single or complex pathogenicity factors of
the invading fungus requires the definition of
pathogenicity factors and the evaluation of their
role during pathogenesis. In order to und erstand
the molecular basis for resistance and virulence
factors, plant-fungus interactions need to be inves-
tigated with respect to gene-for-gene relation-
ships. More information on the organization and
 Management of Fusarium Diseases
the genetic traits of Fusarium genomes will be
mandatory in the future.
Acknowledgements. I thank Johannes Wöste-
meyer (Jena) for discussing with me many obser-
vations in fungus-plant interactions and critically
reading the manuscript. I would like to express my
gratitude to all those colleagues who provided lit-
erature of their research work. I need to apologize
to all those colleagues whose publications are not
cited in this paper. There are so many relevant
reports on Fusarium and its diseases that to
mention all of them would go beyond the scope of
these pages.
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13 Disease Management of Rusts and Powdery Mildews
HOLGER B. DEISING, SVEN REIMANN, ANDREAS PEIL, and W. EBERHARD WEBER
CONTENTS
I. Introduction .........................
11. Significance of Rust and Mildew Spore
Production and Dispersal .. . . . . . . . . . . . . .
III. Disease Control by Resistance Genes .....
IV. Disease Control by Fungicides . . . . . . . . . . .
V. Establishment of Fungicide Resistance
or Fungieide Insensitivity ...............
VI. Novel Approaches ....................
A. Induced Resistance . . . . . . . . . . . . . . . . . . . .
B. Resistance Mediated by Introduction
of Foreign Genes .....................
VII. Efficient Disease Management
by Combination of Different Approaches ..
VIII. Conclusion and Perspectives . . . . . . . . . . . . .
References ..........................
I. Introduction
Development of human civilization has been
closely linked to cultivation of cereals, and dis-
eases of these crops have been a concern to
mankind probably since they were cultivated
more than 10,000 years aga (McIntosh et al. 1995;
Griese et al. 1997). Biblical accounts, at ab out 1870
B.c., and records of the early Greek and Roman
literature dating from approximately 500B.c., indi-
cate that severe epidemics threatened crops and,
as a consequence, people of that time. Neither the
cause of the diseases was known, nor were cereal
diseases differentiated, but samples taken from an
excavation in Israel contained urediniospores of
the stern rust fungus Puccinia graminis, suggesting
that cereal rusts have been a problem since about
1300B.e. on (Kislev 1982). In the Roman world,
ceremonial appeasement of Robigus, the Corn
God, appeared to offer the only hope of prevent-
ing crop failure.
Martin-Luther-University Halle-Wittenberg, Faculty of
Agriculture, Department of Plant Breeding and Plant
Protection, Ludwig-Wucherer-Str. 2,06099 Halle (Saale),
Germany
The first person to recognize that rust was
243 caused by a parasitic fungus was Fontana in 1767,
but it was not until the nineteenth century that de
244 Candolle distinguished the leaf rost pathogen
245 Uredo rubigo-vera from Persoon's P graminis
248
(McIntosh et al. 1995). In the nineteenth century,
251 severe epidemics of powdery mildews and rusts
256 were recorded around the world. The first major
256
outbreaks of these diseases included powdery
259 mildew epidemics of hops in England in 1840 and
of grapes in England in 1845 and in France in 1848.
261 This disease affected almost all of Europe by 1851.
262
263
Likewise, severe coffee rost epidemics in 1869
destroyed coffee plantations in Sri Lanka and
made the English a tea-drinking nation. Serious
epidemics of cereal rusts, especially of the het-
eroecious stern rust, P graminis, were recorded in
Austria and Prussia at the end of the nineteenth
century and in the United States of America and
Canada at the beginning of the twentieth century.
The repeated occurrence of rust and powdery
mildew epidemics stimulated research on these
fungi and De Bary proved heteroecism of P
graminis on ce re als and barberry in 1866. In 1894,
Erikson defined formae speciales to describe
forms of rusts specializing on different host
species (McIntosh et al. 1995).
The first notable efforts to control rosts were
based on early observations of the proximity of
rusted cereals to barberry. In France, rem oval
of barberries from the neighborhood of cereals
was required by law already by 1660, and barberry
eradication programs reached their climax in
England and America at the beginning of the
twentieth century (Roelfs 1982). At the end of the
nineteenth and the beginning of the twentieth
century, chemical plant protection schemes were
devised, using copper and sulfur as antifungal
agents to control downy and powdery mildews.
These early treatments led to the establishment of
a multibillion dollar industry that has developed
efficient modern fungicides belonging to various
chemical classes, differing in mode of action and
The Mycota Xl
Agricultural Applications
Kempken (Ed.)
© Springer-Verlag Berlin Heidelberg 2002
244 H.B. Deising et a1.
characteristics of uptake and distribution within
the plant.
Side by side, modern fungicides and breeding
programs have allowed control of rusts and
powdery mildews in recent years. However, muta-
tions occurring in these fungi, together with their
adaptive ability, allowed them to defeat resistance
genes of their host plants and to acquire resistance
to fungicides. These challenges require the devel-
opment of efficient disease management strate-
gies. Reasonable agronomical practice must be
combined with continued breeding and provision
of plants with broad-spectrum disease resistance,
and development of efficient fungieides with novel
modes of action. Importantly, successful powdery
mildewand rust disease management will require
practiees that avoid development of fungicide
resistance or insensitivities and that minimize the
risk of breaking of genetic resistance of the host
plants.
In this chapter we will introduce the reader
to the pathogens, resistance breeding strategies
and mechanisms of resistance. We will further
describe the modes of action of modern fnngicides
used to control powdery mildews and rusts, and
we will review the current status of research
on development of fnngicide insensitivity and
resistance. Finally, we will outline concepts of
modern rost and mildew disease management
and describe novel approaches like chemical
induction of disease resistance and production of
transgenie crop plants harboring genes confering
resistance against fungal pathogens. As this article
covers a variety of subjects, a list of other reviews
covering this area is given at the beginning of each
subsection.
11. Significance of Rust and Mildew
Spore Production and DispersaI
The biology of rusts and powdery mildews has
been described comprehensively by several
authors (Gäumann 1959; Spencer 1978; J~rgensen
1988; Staples 2000), including excellent reviews by
Mendgen (1997) and Griese et al. (1997) in
Volume V/B of this Series. We will therefore only
briefly discuss features that are important for
propagation and spread of the pathogens, as
these features are critical to the success of the
pathogens (Hau and de Vallavieille-Pope 1998; de
Vallavieille-Pope et al. 2000).
As typical "r-strategists" rusts and powdery
mildews rely on the formation of large spore
numbers and anemochoric short- and long-
distance distribution to achieve high colonization
rates. A single aecium of the wheat stern rust
fungus can produce more than 10,000 aeciospores
and an average of approximately 608 uredin-
iospores per uredinium are produced per day,
representing a total of 18,240 urediniospores per
flag leaf at an infection rating of 1 % (approx. 30
pustules per flag leaf; Eversmeyer and Kramer
2000). Even moderate infection with P graminis
f. sp. tritici would thus produce 4 x 1012 uredin-
iospores/day/ha, whereas Puccinia recondita and
P striiformis would produce 3.2 x 1013 and 0.6 to
2.0 x 1013 urediniospores/day/ha (Rowell and
Roelfs 1971; Nagarajan and Singh 1990). For the
powdery mildew of cereals, Blumeria graminis,
Aust (1981) determined a spore production rate of
15x103 conidia/lesion/day, a rate very similar to
that reported for cereal rusts (Hau and de
Vallavieille-Pope 1998). These tremendous spore
numbers can be transported over long distances
and, as reported for rusts, cause new infections
hundreds or even thousands of kilometers away
from the original site of infection (Nagarajan and
Singh 1990; Eversmeyer and Kramer 2000).
Nagarajan and Singh (1990) put together meteo-
rological data clearly showing efficient long-
distance transport of fungal spores. Wind flow at
high altitudes was estimated by following a
balloon through the Global Horizontal Sounding
Technique program. In 102 days, the balloon
circled around the southern hemisphere of the
earth 10 times, at a level of approx. 12,000m, and
it took only 3--4 days to travel from the tip of
Africa to Perth, Australia. Urediniospores show
significant loss of viability only after 5 days.
Similarly, weather data support the possibility of
transatlantic spread once urediniospores have
reached an altitude of 1500 to 2000m (Nagarajan
and Singh 1990, and references therein). Spores of
the coffee rust fungus Hemileia vastatrix, pro-
duced during an outbreak of a rust epidemie in
Angola in 1966, were carried across the Atlantic
by wind and deposited 5-7 days later over the
coffee estates of Bahia, Brazil (Bowden et al.
1971). Annual long-distance transport of P
graminis occurs across the Great Plains in North
America, from northern Africa to Europe and,
frequently, from Australia to New Zealand
(Nagarajan and Singh 1990; Eversmeyer and
Kramer 2000). Furthermore, long-distance trans-
 Disease Management of Rusts and Powdery Mildews
port can be sustained and within 11 years peanut
rust was reported to have migrated from tropical
America and China to southern Asia and Oceania
and to Africa (Ivory Coast and Senegal). Similarly,
the sugar cane rust, endemie to scattered locations
in Africa and Asia, occurred in the Dominican
Republic in 1978 and subsequently spread to
alm ost all sugarcane-growing areas in America
(Nagarajan and Singh 1990; Mendgen 1997;
Staples 2000, and references therein). These cases
represent so me examples of long-distance spread
of fungal spores and efficient spread may be
regarded as one of the major reasons of the
success of these pathogens.
Short-distance migration mayaiso be signifi-
cant in epidemiology of wind-dispersed diseases.
One of the most relevant aspects is the initial
inoculum density at the beginning of the growth
season (Hau and de Vallavieille-Pope 1998; de
Vallavieille-Pope et al. 2000). Early development
of powdery mildews benefits from large spore
numbers carried over on winter cereals, obviating
the need of overwintering fruiting bodies and effi-
ciently bridging the gap between summer and
winter cereals. Growing summer and winter crops
in dose proximity may thus strongly promote
development of powdery mildew populations
(Limpert et al. 1999).
111. Disease Control
by Resistance Genes
Survival of organisms depends on genetic diver-
sity, and this is particularly evident in responses to
pathogen challenge in wild populations. Within
plant genera and wild populations of cultivated
species, pathogen-resistance occurs, offering rich
sources of resistance (R) genes. As a matter of
fact, plant breeders have efficiently exploited such
sources to provide rust and powdery mildew resis-
tance for cereals since the late nineteenth century.
Reviewing the research on rust fungi during
the twentieth century, Staples (2000) regards plant
breeding, based on increasingly sophisticated
genetic and molecular genetic tools, as the best
long-term solution to control rusts. This is cer-
tainly also true of powdery mildew disease
control.
Flor (1971) used the flax-flax rust (Melamp-
sora lini) system to demonstrate that resistance is
governed by pairs of corresponding genes, the R
245
genes of the plant and the avirulence (Avr) genes
of the pathogen. Today, several resistance genes,
induding the flax rust resistance gene L6, have
been doned, and molecular genetic studies with
different pathosystems have verified Flor's gene-
for-gene model (De Wit 1995; Rohe et al. 1995;
Knogge 1996; Ellis et al. 1997; Wubben et al. 1997).
Importantly, as defense reactions are elicited by
the interaction of R gene and Avr gene products,
virulence of the pathogen is recessive, and
simple loss-of-function mutations of an Avr gene
may confer virulence to the formerly avirulent
pathogen. In fact, as early as 1958, Flor demon-
strated widening of virulence of F1 urediniospores
of flax rust race 22 x race 1 after X-ray-induced
mutagenesis. While a loss-of-function mutation is
sufficient for the success of the pathogen, plants
must evolve new resistance functions (genes) to
newly occurring biotypes of a pathogen (Richter
and Ronald 2000). Virulence of the pathogen and
resistance of the plant are therefore subject to dif-
ferent evolutionary forces.
Resistance breeding is a competition between
incorporation of R genes and occurrence of new
races with virulence for these genes or, in other
words, rates of mutation occurring in Avr genes
corresponding to R genes. The easiest way to
introduce resistance is to use R genes already
existing in other varieties of the same species. As
this is done by many breeders, new R genes are
widely distributed in a short time, increasing the
danger of resistance breaking down rapidly.
Another important source of R genes are
pathogen-resistant wild forms of cultivated plant
species. Such plants can be found in gene banks,
growing wild in the species' center of origin or in
other geographie regions like the Vavilov centers
in northwest Asia where they have naturalized
or perpetuated as "land-races". Fischbeek and co-
workers described the introduction of resistanee
against powdery mildew from Israeli Hordeum
spontaneum populations into barley (Jahoor and
Fischbeck 1991), and Münnich et al. (2000) against
stripe rust from Ethiopian barley aecessions.
Resistance against powdery mildew of wheat was
successfully introduced from wild diploid
(Blüthner et al. 1992) and tetraploid wheat species
(Oertel et al. 1997). The aceession SV740-69 of
Aegilops markgrafii earried resistance genes
against wheat leaf rust and powdery mildew
(Schubert et al. 1993; Peil et al. 1997). Numerous
other examples exist and, for comprehensive lists
of current rust and powdery mildew R genes in
246 H.B. Deising et al.
cereals, the reader is referred to publications by
Mclntosh et al. (1995,1998) and Lutz et al. (1994).
When a cultivar with a new R gene is released
to farmers, it is often grown in dose vicinity to
susceptible and heavily infected fields. Such fields
could give rise to tremendous numbers of spores
(see Sect. II). Even if the frequency of mutation is
in the range of 1 x 10-6-1 X 10-5 , and although for
rust the dikaryotic and heterozygous uredinial
stage rather than the haploid monokaryon is
affected, significant numbers of mutated spores
would occur (Mclntosh and Brown 1997). Conse-
quently, monoculture of resistant cultivars and the
rapid and efficient wind dispersal of rusts and
mildews would select mutations in Avr genes and
promote the evolution of new fungal races capable
of defeating the newly introduced R gene. As a
matter of fact, Limpert et al. (1999) showed that
in dispersing powdery mildew populations viru-
lence complexity increases by one additional vir-
ulence every 1000 km. Walther (2000) reported
that only 2 years after the release of a summer
barley cultivar with the Rph12 gene conferring
resistance to Puccinia hordei, 100% of the rust
population over the area of the former GDR
carried virulence to that gene. Comparably, leaf
rust (P. recondita) populations with virulence to
the wheat Lrl gene increased from 0.8% in 1996
to 62.1 % in 1999 (Walther 2000).
However, examples of stable resistance medi-
ated by single dominant genes exist. The wheat leaf
rust R gene Lr13 has provided stable resistance in
Australia for 20 years, despite the fact that this gene
had been defeated in South Africa, Western
Europe, Mexico and South America (Mclntosh
and Brown 1997). Interestingly, durability of a
plant R gene can be predicted by analyzing the
loss of pathogen fitness (aggressiveness and per-
sistence) associated with the loss of avirulence
function, as demonstrated for different single bac-
terial blight R genes of rice (Vera Cruz et al. 2000).
As race-specific resistance mediated by single
R genes is easily broken, combinations of several
R genes are introduced into cultivars (Wilson et
al. 2001). One may argue that, as breaking several
R genes would require multiple simultaneous
mutations in the corresponding Avr genes, multi-
gene cultivars promise stable resistance. However,
as different R genes are usually introduced
sequentially rather than simultaneously, pathogen
races can co-evolve with the release of the new
cultivars. In fact, Hulbert and coworkers (1991)
identified races of the common maize rust Puc-
cinia sorghi that were virulent on maize lines
carrying all known Rp genes, and this and similar
reports raised concerns of super races of
pathogens occurring that cannot be controlled by
R genes (Wolfe and Finckh 1997).
Among the most interesting reports was that
on three lesion mimicry phenotypes obtained after
mutagenesis of the maize rust R gene Rpl (Hu
et al. 1996). The mutants responded with necrotic
reactions to inoculation with any race of P. sorghi
and, remarkably, any other rust species tested. If
non-race-specific resistance is mediated by single
recessively acting R genes, this type of resistance
should be durable and, from a practical point of
view, would be very attractive for breeding pro-
grams (Hulbert 1997).
The first major accomplishment in powdery
mildew resistance breeding was the use of a barley
spring cultivar carrying the MIg gene. Until 1948,
less than 10% of Germany was sown to this culti-
var, and resistance was fully effective. Although
some MIg virulent mildew isolates occurred, the
gene was considered effective in 1953. However,
after this, the area growing MIg cultivars increased
until, by 1960, they occupied some 70% of the
barley acreage. A drastic increase in Mig virulence
accompanied this increasing acreage so that by
1960 dose to 100% of the population carried the
virulence. Another example is the rapid spread of
powdery mildew isolates virulent on Mla13 plants
in former Czechoslovakia (Wolfe and McDermott
1994).
In the system barley-B. graminis different
race-specific R gene loci have been investigated in
great detail on the molecular level, as reviewed by
Schulze-Lefert and Vogel (2000). In order to fully
understand the mechanisms governed by mildew-
resistance genes, genetic analyses have been com-
plemented by detailed microscopic investigations.
Kogel and co-workers have compared mlo resis-
tance with resistance governed by the dominant R
genes Mla12 and Mig (Hückelhoven et al. 1999;
Fig. 13.1). In mlo-resistant barley lines, the mildew
fungus is arrested by effective papillae, leaving
the attacked cell alive. In plants exhibiting Mla12-
governed resistance, many, but not all of the
attacked epidermal cells undergo a hypersensitive
response. Thirty hours after inoculation, approxi-
mately 30% of the attacked cells develop a
compatible single-cell interaction and the fungus
is able to differentiate a haustorium and branched
secondary hyphae on the leaf surface. Subse-
quently, fungal growth is effectively arrested by
 Diseasc Management of Rusts and Powdery Mildews
10 hai
/
16 hai
,
~
.. .
18-30 hai
,
~ . ..~,,: .t,..,.. .
• '.
48 hai
Mla12
Fig. 13.1. Scheme of predominant interaction phenotypes
in barley mediated by the powdery mildew resistance genes
Mlal2, Mlg, and mlo5, or by the resistance-inducing
compound Bion after inoculation with Blumeria graminis
f. sp. hordei A6. Beginning at 16h after infection (a.i.),
differences in fungal development are evident on the
four near-isogenic lines. Although the fungus penetrated
the epidermal cells of susceptible and Mla12-resistant
barley, effective papillae did impede penetration in the
Mlg- and mlo5-mediated response. Mla12 mediated hyper-
sensitive response (HR) in penetrated epidermal cells by
24 to 40h a.i. If the fungus was able to cstablish a diffcr-
death of mesophyll cells subjacent to the invaded
epidermal cells, beginning 36 h after inoculation.
In Mlg-specified resistance, both formation of
effective papillae and hypersensitive response of
the epidermal cell were observed. Importantly,
H 2ü Z accumulation is closely associated with the
major defense responses of barIey against
powdery mildew, i.e., hypersensitive response and
formation of effective papillae, and can thus be
regarded as a key event in expression of resistance
(Thordal-Christensen et a1. 1997; Hückelhoven et
a1. 1999; Fig. 13.1). However, as Vanacker et a1.
(2000) pointed out from studies of Mlal-mediated
powdery mildew resistance in barley, the oxidative
processes that lead to H2Ü2 accumulation in papil-
lae and in cells that die as a result of attack may
be independent. Thus, effective papilla formation,
associated with oxidative activity, may prevent
signaling for engagement of oxidative activity
that leads to cell death. Cell death may only
result where papilla defenses fail, representing a
situation where successful infection could have
occurred in the absence of the R gene.
247
C!J
,
,
~.. .-
~, ,
.. . "
~
~
,..
~.... ,.
." . ~ :.,.
, ~
C!!J ,
~
M/g m/oS susceptible
BION
entiated haustorium and branched elongated secondary
hyphae, fungal development was arrested by spreading
HR of mesophyll cells subjacent to the attacked epidermal
cell beginning at 36 h a.i. The Mlg gene governed formation
of effective papillae and HR of the attacked but non-
invaded epidermal cells by 18-24h a.i. The same phenotype
is seen in barley treated with Bion. The recessive mlo5
gene media ted formation of effective papillae, and attacked
cells stayed alive. In susceptible plants, cell wall pen-
etration was followed by formation of a haustorium
and elongated secondary hyphae. (Hückelhoven et al.
1999)
While single major R genes like Mla12 or Mlg
have been overcome, durable mlo-mediated
broad-spectrum mildew resistance has remained
effective for more than 20 years (Schulze-Lefert
and Vogel 2000), although powdery mildew iso-
lates capable of infecting barley mlo cultivars exist
and occasional outbreaks of powdery mildew in
field-grown mlo cultivars have been reported
(Lyngkjrer et a1. 1995). Such isolates could induce
accessibility and thus modify mlo resistance to B.
graminis (Lyngkjrer and Carver 1999).
In mlo plants resistance is governed by a
single recessive gene. Isolation of the Mlo gene
confirmed that resistance is caused by loss-of-
function mutations of the wild-type gene and
revealed that the 60-kDa Mlo protein is anchored
to the plasma membrane by seven transmembrane
helices, typical of most abundant metazoan G-
protein-coupled receptors (Büschges et a1. 1997;
Devoto et a1. 1999). Expression of Mlo in single
cells of mildew-resistant mlo genotypes was suffi-
cient to confer single-cell susceptibility (Shirasu et
a1. 1999). As mutations in two genes, designated
248 H.B. Deising et al.
Rorl and Ror2 (required for mlQ resistance), par-
tially compromised broad-spectrum mlo resis-
tance, Mlo is thought to act as a repressor of
Rorl- and Ror2-mediated defense responses,
rather than a compatibility factor providing a
signal to the mildew fungus to initiate haustorium
formation (Freialdenhoven et al. 1996; Schulze-
Lefert and Vogel 2000). Mutations in genes of
other plant species, e.g., lsdl (lesion ~imulating
disease resistance response) and edr1 (~nhanced
disease resistance) of Arabidopsis thaliana or si of
rice, have also been shown to confer enhanced
disease resistance to different pathogens (Dietrich
et al. 1994; Arase et al. 1997; Frye and Innes 1998).
Importantly, recessive resistance has been shown
to be non-specific and appears to confer more
stable resistance than race-speeific resistance
mediated by dominant R genes.
As weIl as major resistance genes, combina-
tions of minor genes, conferring a type of resis-
tance called field, partial or quantitative resistance
(Vanderplank 1978), contribute to disease control.
Partial resistance is characterized by quantitative
traits. On partially resistant plants, latency periods
are usually longer and fewer generations of the
pathogen occur. In addition, colony sizes and rates
of sporulation are often reduced (Parlevliet 1979).
A significant dis advantage is the fact that the
degree of resistance is strongly inftuenced by
environmental factors so that stable yields can-
not be guaranteed. Only a few papers describing
the mechanisms by which minor genes confer
field resistance exist (Kmecl et al. 1996). Interes-
tingly, quantitative trait loci (QTLs) for disease
resistance have repeatedly been detected in chro-
mosomal regions in which major race-specific
resistance genes are located (Jahoor et al. 2000).
For example, the highly polymorphic Mla cluster
for race-specific mildew resistance and a QTL
both locate to the same region of barley chro-
mosome 1H. Likewise, the race-specific leaf rust
resistance gene Rph16, the LR2 locus and a
QTL map to the same confidence interval of
chromosome 2H in the cross between the Turkish
cv. "Hor 1063" and the German cv. "Krona". These
data suggest that the genetic base for quantita-
tive resistance may correlate with "defeated"
resistance genes or alleles for race-speeific defense
(Jahoor et al. 2000). Compiling QTLs for disease
resistance may help to avoid breaking of resis-
tance of newly released cultivars by single point
mutations in the pathogen. A cultivar with signi-
ficant QTLs conferring field resistance could
provide increased resistance (Brown et al. 2001),
even if fungal races with mutated Avr genes
should occur.
IV. Disease Control by Fungicides
In the Roman empire, the occurrence of epidemics
was attributed to the action of the god Robigus.
As this god was not to be trusted, chemical disease
control practices were introduced. The therapeu-
tic effect of sulfur was passed down from the
ancient Greeks (Hewitt 1998) and still at the end
of the nineteenth and the beginning of the twen-
tieth century, chemical plant protection almost
exclusively relied on sulfur to control powdery
mildews. Even today, 15 mildew fungicides of
the German list of authorized plant protection
products, as published by the German Federal
Biological Research Center for Agriculture and
Forestry in January 2001, are sulfur-based fungi-
eides (Table 13.1).
N owadays, fungieides belonging to several
different chemical classes exist, differing in their
mode of action and characteristics of uptake and
distribution within the plant. In 1967, the intro-
duction of benomyl as the first systemic fungicide
was regarded as alandmark in modern fungicide
development. In the late 1990s, sales figures of
fungieides to control powdery mildews and rusts
in cereals, and mildews in vines, top fruit and
protected crops in Western Europe was valued
at approx. US $750 million (Hewitt 1998). The
most extensively used fungieides in mildewand
rost control interfere with sterol biosynthesis or
mitochondrial electron transport (Table 13.1). A
total of 20 solo-sterol biosynthesis inhibitors
(SBIs) including C-14 demethylation inhibitors
(D MIs) and morpholines inhibiting fj,8 -tl.? iso-
merase/fj,14 reductase (Kerkenaar 1995; Kuck et al.
1995; Pommer 1995), one solo-strobilurin fungi-
eide, and various fungicide mixtures containing
two or more SBIs or strobilurin components are
commercially available in Germany to control
cereal powdery mildews (Table 13.1). With the
exception of sulfur, fungieides used for powdery
mildew control are almost identical to those used
to control rusts.
SBI fungi eides represent an economically and
functionally outstandingly successful class of
fungicides (Köller 1988; Kuck et al. 1995). DMIs
include azoles, pyridines, and pyrimidines, the only
 Disease Management of Rusts and Powdery Mildews 249

Table 13.1. Registered fungicides for the control of powdery mildew, brown and yellow rust in cereals in the FRG
(pesticide list of the BBA of 01.01.2001)

Tradename Active substance Powdery mildew Brown rust Yellow rust

Strobilurine
Ami star Azoxystrobin x x x
SBIs
Alto 100SL Cyproconazole x x x
Apache Propiconazole x x
Bayleton Spritzpulver Triadimefon x x x
Bumper Propiconazole x x
Calixin Tridemorph x
Capitan Flusilazole x x
Caramba Metconazole x x x
Corbel Fenpropimorph x x x
Desmel Propiconazole x x x
DesmelWG Propiconazole x
Falimorph 750 Tridemorph x
Flamenco Fluquinconazole x x x
Folicur Tebuconazole x x x
Granit Bromuconazole x x x
HORA Propiconazol Propiconazole x x x
Impulse Spiroxamine x x x
Indar 5 EC Fenbuconazole x x
Mirage 45 EC Prochloraz x
Opus Epoxyconazole x x x
Prosper Spiroxamine x x x
Others
Asulfa WG Sulfur x
Compo Mehltau-frei Sulfur x
KumulusWG
Cosan 80 Netzschwefel Sulfur x
Fortress Quinoxyfen x
Bora Thiovit Sulfur x
KumulusWG Sulfur x
Netzschwefel WG Sulfur x
Netzschwefel 80 WP Sulfur x
Netzschwefel "Schacht" Sulfur x
Netzschwefel Stulln Sulfur x
Netz-Schwefel Sulfur x
Netz-Schwefel WG Sulfur x
Stefes Instant Sulfur x
Sufran WG Sulfur x
Supersix Sulfur x
Thiovit Sulfur x
Unix Cyprodinil x
Zenit M Fenpropidine x
Combination products
Agent Fenpropidine + propiconazole x x x
AltoD Dithianone + cyproconazole x x x
Bayfidan Cyproconazole + dithianone x x x
Baytan universal Flüssigbeize Imazalile + triadimenol + x
fuberidazole
Brio Kresoxim-methyl + x
fenpropimorph
Bumper Star Propiconazole + prochloraz x x
Colt Triadimenol + tridemorph x x x
Cortil Fenpropimorph + propiconazole x x x
Flamenco FS Fluquinconazole + prochloraz x x
Fortress Top Fenpropimorph + quinoxyfen x
Gladio Fenpropidine + propiconazole + x x x
tebuconazole
Gralan Iprodione + propiconazole x x x
Granit Plus Brumoconazole + iprodione x x
Harvesan Carbendazim + flusilazole x x
250 H.B. Deising et al.

Table 13.1. Continued

Tradename Active substance Powdcry mildew Brown rust Yellow rust

Ilbex Propiconazole + tridemorph x x x

Juwel Kresoxim-methyl + epoxyconazole x x x
Juwel Top Kresoxim-methyl + epoxyconazole x x x
+ fenpropimorph
Matador Tebuconazole + triadimenol x x x
Matador 300 Tebuconazole + triadimenol x x x
Matador 300 Stähler Tebuconazole + triadimenol x x x
Opus Top Epoxyconazole + fenpropimorph x x x
Prisma Cyproconazole + prochloraz x x x
Pronto Fenpropidine + tebuconazole x x x
Pronto Plus Spiroxamine + tebuconazole x x x
RPA 10371 F Brumoconazole + prochloraz x x x
Sambarin Chlorothalonile + propiconazole x x x
Sambarin WO Chlorothalonile + propiconazole x x x
Simbo Fenpropimorph + propiconazole x x x
Sportak Delta Cyproconazole + prochloraz x x x
Sportak Plus Fluquinconazole + prochloraz x x x
Stefes Matador Tebuconazole + triadimenol x x x
Stratego Trifioxystrobine + propiconazole x x x
Taspa Difenoconazole + propiconazole x x x
Tiptor Cyproconazole + prochloraz x x x
Tiptor S Cyproconazole + prochloraz x x x

common structural feature being a heteroaromate
containing at least one nitrogen atom with a free
electron pair, a structural feature essential for
inhibitory activity (Köller 1988). Ragsdale and
Sisler (1972) were the first to present evidence
that triarimol, a pyrimidine, interfered with sterol
demethylation in the com pathogen Ustilago
maydis. This finding greatly stimulated research
on fungal sterol biosynthesis.
Like all Eumycota, rusts and powdery
mildews synthesize sterols, which are essential to
membrane functions (Weete and Gandhi 1996).
Detailed knowledge of fungal sterol biosynthesis
mainly sterns from biochemical studies of fungi
capable of saprophytic growth, e.g., the yeast Sac-
charomyces cerevisiae, the dimorphie fungus and
com pathogen U. maydis, and the filamentous
ascomycete Aspergillus nidulans. Data on obligate
biotrophs like rusts and powdery mildeware
scarce because culturing these fungi in the absence
of their host plants (i.e., axenically) is as yet either
impossible or requires significant effort (Hotson
and Cutter 1951; Maclean 1982; Fasters et al.
1993).
A detailed overview of sterol biochemistry
and molecular biology in fungi is given by Weete
and Gandhi (1996, Vol. III, this Series) and this
topic is therefore only briefly discussed here.
Lanosterol, the first fungal sterol, is synthesized
from mevalonate via the isoprenoid pathway
(Mercer 1984; Köller 1988; Kerkenaar 1995). It is
modified by methylation in the C-24 position
and subsequent C-14 demethylation, leading to
4,4-dimethylergosta-8,14,24(28)-trien-3ß-ol. The
double bond between C-14 and C-15 of this co m-
pound is eliminated by the action of a ,::l14 re duc-
tase, and two subsequent oxidative demethylation
reactions remove two methyl groups from the C-
4 position, leading to fecosterol (Mercer 1984;
Köller 1988; Kerkenaar 1995). Later steps in sterol
biosynthesis involve ,::l8_,::l7 isomerase, generating
episterol, and further re arrangements of double
bonds lead to ergosterol, which is the end product
of this pathway in most fungi. Powdery mildews
and rosts, however, show exceptional sterol pat-
terns. The main sterol identified in powdery
mildew of barley, cucurbit and apple was ergosta-
5,24(28)-dienol (Loeffler et al. 1984), and stig-
mast-7-enol was the dominating sterol in
urediniospores of wheat rust and flax rust
(Jackson and Frear 1968; Nowak et al. 1972).
Importantly, the C-14 demethylation target site for
DMIs is not altered in powdery mildews and rusts
(Köller 1988).
The exact target sites of SBI fungieides have
been identified by analyzing sterols accumulating
in se ver al fungal systems after fungicide treat-
ment. Sterol precursors containing a methyl group
in the C-14 position, i.e., lanosterol or, more fre-
quently, 24-methylenedihydrolanosterol, accumu-
 Disease Management of Rusts and Powdery Mildews
lated after DMI treatment characterizing these
fungicides as C-14 demethylation inhibitors
(Köller 1988; Baldwin and Corran 1995; Hewitt
1998). After treatment of S. eerevisiae (Baloch et
al. 1984; Baloch and Mercer 1987) and U. maydis
(Kerkenaar et al. 1981) with the morpholines fen-
propimorph, tridemorph, and fenpropidin, sterols
containing /18 and /114 double bonds accumulated,
indicating that these fungicides inhibit /18_/17 iso-
me rase as weIl as /114 reductase activity, the latter
being considered more harmful to fungal growth
(Kerkenaar et al. 1981). Fenpropimorph, in addi-
tion to the inhibitory effects mentioned above,
also interferes with /18,14 sterol reductase activity
(Kerkenaar et al. 1984).
The effect of fungicides on powdery mildews
and rusts is shown in Fig. 13.2. On the cuticle of
cereal leaves conidia of B. graminis germinate,
form a short appressorial germ tube and differen-
tiate an appressorium (Fig. 13.2A). After estab-
lishment of a haustorium, branched secondary
hyphae and extensive spore formation occur (Fig.
13.2B). The leaf rust fungus P reeondita recog-
nizes the surface features of its host and forms an
appressorium precisely over the stomatal pore
(Fig. 13.2G). After colonization of the intercellu-
lar space by rust hyphae, the plant cuticle is dis-
rupted by urediniospores that form in large
numbers (Fig. 13.2H). Hyphae and conidia of a
colony of the mildew fungus treated with the DMI
fungicide epoxyconazole collapse soon after treat-
ment (Fig. 13.2E). Rapid cell collapse is also
observed after treatment of rust germ tubes devel-
oping on a leaf surface (Fig. 13.2K). Importantly,
if spores germinate on a newly formed leaf of a
fungicide-treated plant, with no fungicides on its
surfaee, growth inhibition oeeurs after penetration
into the leaf, due to the systemic nature of SBI
fungicides. As a matter of relevance, it has been
shown that systemic DMIs are transported by an
as-yet unknown mechanism to the infection site,
increasing the fungicide concentration directly at
the site of fungal ingress. Humphreys et al. (1992)
report on translocation of 14C-Iabeled flutriafol to
wheat leave sections infected by B. graminis f. sp.
tritiei or by Septoria nodorum. Similar results
were recently also obtained with fluquinconazole
(Metcalfe et al. 2000).
Strobilurins were introduced onto the market
in the mid-1990s and are based on the methoxy-
acrylate structure identified in related naturally
occurring inhibitors of the mitochondrial electron
transport, i.e., myxothiazol, oudemansin, and stro-
bilurin A. Strobilurin A has been isolated from the
251
two Basidiomycetes Strobilurus tenaeellus and
Myeena galopoda (Anke et al. 1977; Kraiczy et al.
1996), but UV-light instability of the molecule
required modifications to yield a marketable
product. Thus far, only a few strobilurin fungicides
are commercially available (Table 13.1).
Strobilurins bind to the cytochrome bCI
complex (complex III), a component of the mito-
chondrial respiratory chain, blocking ATP synthe-
sis. In contrast to the systemic SBIs, strobilurins
are local systemic or translaminar fungicides. If
conidia contact with these compounds after
landing on the plant cuticle they fail to germinate
(Fig. 13.2C). Hyphae formed on the cuticle before
fungicide treatment collapse and die soon after
spraying (Fig. 13.2D, I). However, as these fungi-
eides are not systemic, they cannot be regarded as
efficient curative compounds.
A particularly interesting mode of action is
exhibited by the powdery mildew fungicide
quinoxyfen, belonging to the new class of
qninolines. This protectant fungicide has been
developed to control mildews of crop plants,
including wheat, barley and grapes. It inhibits pre-
infectional development, i.e., germination and
appressorium differentiation (Wheeler et al.
2000). Quinoxyfen is thought to interfere with a
Ras-type GTPase-activating pro tein and thus to
prevent transduction of plant surface signals.
Germ tube elongation occurs on the leaf surface,
but, as the signal for appressorium formation is not
transduced, these specialized infection cells are not
differentiated (cf. Fig. 13.2A, F; Wheeler et al.
2000). Heterotrimeric G proteins have been shown
to be involved in transduction of surface signals
and induction of infection-related morphogenesis
in several plant pathogenie fungi (Dean 1997;
Bölker 1998; Deising et al. 2000). Like quinoxyfen-
treated mildews, magB and egal mutants of
Magnaporthe grisea and Coehliobolus heterostro-
phus deficient in Ga subunits of heterotrimeric
G proteins do not recognize an inductive surface
and thus fail to differentiate appressoria (Liu and
Dean 1997; Horwitz et al. 1999).
v. Establishment of Fungicide
Resistance or Fungicide Insensitivity
A requirement for modern fungicides is that they
show a specific mode of action. They usually
inhibit speeifie (single) metabolie processes and
are therefore referred to as single-site inhibitors.
 Disease Management of Rusts and Powdery Mildews 253

Almost all single-site fungieides are considered as
high-risk fungicides, as single mutations in their
target moleeule may prevent binding of the
chemical and fungicidal effect (Hewitt 1998). The
extended use of modern single-site inhibitors
could lead to occurrence of either fungieide resis-
tance or insensitivity. Reduced fungicide efficacy
can be based on different mechanisms. Mutational
alterations of genes encoding fungieide targets
can lead to reduced accessibility to the drug.
Mutation-based fungicide resistance is thus inher-
itable, and the progeny is able to develop in the
presence of the particular fungicide, even if no
prior fungicide treatment has occurred. A resis-
tant population can shift to increased sensitivity
only if resistant individuals exhibit reduced fitness
as compared with sensitive wild-type individuals.
In addition to mutations in fungieide targets,
fungicide insensitivity can result from adaptation,
i.e., induction of systems that keep the intracellu-
lar fungieide concentration below the inhibitory
threshold. This could be media ted by enzymes
capable of decomposing fungicides or by mem-
brane efflux transporters. In this case, individuals
may shift to increased sensitivity on ce synthesis
of fungicide-degrading enzymes or efflux trans-
porters is not progressing. This could be the case
after some time without fungieide application.
Utilization of alternative metabolie pathways
could also be involved in reduced fungieide effi-
cacy. Only understanding the mechanisms govern-
ing these phenomena and a competent fungieide
management will allow to prevent or delay loss of
fungieide efficacy (Veverka 1996; Reimann and
Deising 2000).
The following case studies illustrate reduction
of fungieide efficiencies only a few years after their
introduction and indicate the urgent need for
rational fungieide management.
The single-site inhibitor benomyl was intro-
duced onto the market as the first systemic fungi-
eide in 1967. The benzimidazole derivative binds
to ß-tubulin and therefore prevents cell division.
Specific amino acids of ß-tubulins are involved in
fungieide binding, and in several plant pathogenic
fungi like Venturia, Botrytis, Monilia and Penicil-
lium species, specific amino acid exchanges
resulted in reduced binding of and resistance to
carbendazim, which is the fungicidal compound
intracellularly derived from benomyl (Davidse
and Ishii 1995, and literature therein). As the
amino acids involved in fungieide binding are
highly conserved in most fungi, including powdery
mildews (Sherwood and Somerville 1990; Davidse
and Ishii 1995), mutation-based benomyl resis-
tance involves the same amino acid exchanges in
fungi belonging to different taxa. Two years after
the introduction of benomyl, powdery mildew of
cucurbits in New York State was the first to be
reported as resistant (Schroeder and Provvidenti
1969), and since then many other powdery mildew
fungi have developed resistance against this fungi-
eide dass (Delp 1987). To reduce the resistance
risk associated with these fungieides, the benzimi-
dazole carbendazim has been combined with the
DMI flusilazole to yield the commercial product
Harvesan, which still has a license for control of
powdery mildewand of leaf rust in wheat, barley
and rye in Germany (Table 13.1).
Since their introduction onto the market in
1996, strobilurins have played a major role in
powdery mildewand rust contro!. The success of
this fungieide dass has been attributed to the
broad spectrum of pathogens that can be con-
trolled and to the long half-life after application. In
addition, strobilurins affect the metabolism
of the plants by reducing the CO 2 compensation
point and by influencing the phytohormone
balance, mediating a phenomenon known as the
"greening-effect" (Grossmann and Retzlaff 1997).
EarlY studies demonstrated that single amino
acid exchanges in the cytochrome b target of the

Fig. 13.2. Effect of mildewand rust fungieides on fungal development. Scanning electron microscopy showed that on
an untreated leaf, conidia of the powdery mildew fungus Blumeria graminis germinate to form an appressorium
(arrow) (A). In a compatible interaction the fungus shows extensive development of secondary hyphae and conidiophores
with chains of conidia (H). Conidia treated with the strobilurin kresoxim-methyl fail to germinate (C) and hyphae that had
formed before fungieide treatment collapse after fungieide application (D). Hyphal collapse can also be observed after
treatment with the DMI epoxyconazole (E). Conidia treated with the mildew fungieide quinoxyfen apparently do not
recognize the plant cuticle, fail to form an appressorium and continue to grow until the nutrients of the spore are exhausted
(F). On untreated leaves urediniospores of the rye leaf rust Puccinia recondita germinate and position an appressorium
precisely over astomatal pore (arrow) (G). After fungal establishment in the intercellular system of the leaf rust pustules
breach the epidermis to produce tremendous numbers of urediniospores (H). Germ tubes collapse after contacting the
strobilurin kresoxim-methyl (I) or the DMI epoxyconazole (K). All figures except F were provided by BASF AG; original
scanning electron micrographs were taken by Richard Guggenheim, REM Laboratory, University of Basel. F was provided
by Dow AgroSciences
254 H.B. Deising et al.
yeast S. cerevisiae result in strobilurin resistance
(DiRago et al. 1989). In 1998, only 2 years after the
introduction of strobilurins, strobilurin-resistant
isolates ofpowdery mildew of wheat were detected
in northern Germany, where strobilurin fungieides
had been used intensively (Kröcher 1998; Erichsen
1999; Reschke 1999; Barteis 2000). Recent reports
suggest that two to four strobilurin applications
may be suffieient for resistant isolates of
powdery mildew to occur (Bartels 1999; Barteis
2000). Interestingly, a resistant barley powdery
mildew isolate has also been detected recently
in northern Germany (FRAC, Fungicide Resis-
tance Action Committee; http://www.gcpf.org).
Like mildews, rusts and the economicaHy impor-
tant wheat pathogen Pyrenophora (Drechslera)
tritici-repentis (DTR) are also expected to develop
resistance to strobilurin fungicides (Reschke
1999).
In addition to mutational modification of the
cytochrome b target, alternative respiration may
by-pass fungicide-mediated block of ATP synthe-
sis and represent a further mechanism of reduced
fungieide efficiency, as has been demonstrated
with the wheat pathogen Septoria tritici. Under
field conditions, however, this does not seem to
represent a significant problem (Ziogas et al.
1997).
SBI fungicides account for about half of the
systemic fungicide market worldwide, i.e., more
than US $1.5 billion in 1993, and represent the
leading group of chemicals used to control patho-
genic fungi on crop plants. Importantly, the major
usage of SBI fungieides is in European cereals,
with rusts and mildews as the major target organ-
isms (Baldwin and Corran 1995). These fungicides
were introduced onto the market in 1972 (Baldwin
and Corran 1995), and DMIs have been used inten-
sively since 1976. The first publications on devel-
oping DMI resistance described the situation in
northern England and Scotland and in Schleswig-
Holstein in northern Germany. In powdery
mildews, the level and frequency of insensitivity
against one of the prominent DMI fungicides, tri-
adimenol, correlated with the frequency of fungi-
eide applications (Limpert 1987). In the period
1960-1990,42% of the wheat yellow rust (P. stri-
iformis) isolates tested in the United Kingdom
were less sensitive to the DMI epoxiconazole than
amid-range reference isolate, and only 2% were
less sensitive than a reference isolate classified as
insensitive. In 1997 and 1998, severe yellow rust
epidemics occurred, and DMI fungieides were
again used to control the disease. The repeated
exposure of the P. striiformis population coincided
with declining fungicide effectiveness, as indicated
by 54 % of the isolates tested being less sensitive
than the insensitive reference (Bayles et al. 2000).
Several publications indicate that DMI insensitiv-
ity developed so on after introducing these fungi-
eides (Buchenauer 1984; Heaney 1988; Koller et al.
1992; Pons and Hau 1992), indicating the enormous
flexibility of fungal pathogens. However, reports of
decreasing DMI sensitivities in the field do not
give any information on the mechanism underly-
ing this phenomenon. While benzimidazol and
strobilurin resistance is often due to single-point
mutations (DiRago et al. 1989; Davidse and
Ishii 1995), Butters et al. (1986) showed by recom-
bination experiments with DMI-sensitive and
-insensitive isolates of powdery mildew of barley
that DMI sensitivity is under the control of multi-
ple genes. As a consequence, gradual sensitivity
shifts in fungal populations, rather than a transition
from sensitive to resistant as in the case ofbenomyl
and strobilurin, have been observed that correlate
weH with the number of DMI treatments (De
Waard et al. 1986; Kunz et al. 1997).
Fungicides are regulatory compounds in
modern agricultural ecosystems, and fungi have
evolved qualitative and quantitative mechanisms
to cope with these chemicals. Such mechanisms
are induced by exposure to chemical challenge
and usually operate to keep the intracellular tox-
icant concentration low. While reports on enzy-
matic inactivation of fungieides are scarce (labs et
al. 2001), fungal membrane efflux pumps have fre-
quently been reported to reduce the net uptake of
the fungicide (De Waard and van Nistelrooy 1987,
1988; Dekker 1995). Such efflux systems may
have evolved to protect fungi against naturally
occurring toxic substances, e.g., phytoalexins
formed by plants as a response to pathogen
attack (Nicholson et al. 1987; Nicholson and
Hammerschmidt 1992), and also confer fungieide
insensitivity and multidrug resistance (De Waard
and van Nistelrooy 1980; De Waard 1997; DeI
Sorbo et al. 2000). Two families of plasma-
membrane transporters are known to be involved
in secretion of toxicants, i.e., ATP-.Ilinding
.c.assette (ABC) transporters and the Major
,Eacilitator S,uperfamily (MFS) transporters
(Fig. 13.3). ABC transporters utilize the energy
of hydrolysis of nucleotide triphosphates, mainly
of ATp, to transport different substrates against a
concentration gradient across the plasma mem-
 Disease Management of Rusts and Powdery Mildews 255

(---\ V (
~

I Cytopla m I
Cell wall I ~ JS<0 •

~
Epoxyconazol

~~
~2N

o ö NAcH]
Ethidium bromide
}(resoxiIn Dßethyl

Fig.13.3. Representation of an ABC transporter consisting pumps can transport structurally diverse molecules such as
of two repeats of a nucleotide binding domain (NBF) and strobilurin and DMI fungicides as well as the fiuorescent
six transmembrane domains and an MSF transporter with dye ethidium bromide. (After Dei Sorbo et al. 2000 with
12 transmembrane domains. Importantly, these membrane modifications)

brane. MFS transporters, in contrast, are unable to
hydrolyze ATP. These membrane pumps use the
proton motive force to facilitate the transport of
toxicants, sugars, peptides and ions. More than 350
uni-, sym- and antiporters are known to belong to
this superfamily (Pao et al. 1996).
ABC transporters represent a rapidly growing
efßux pump superfamily (Saurin et al. 1999). They
possess a highly conserved ABC module, display-
ing the WalkerA and WalkerB motifs (Walker et al.
1982), which are found in all pro teins requiring
ATP. In addition to the Walker motifs, ABC
modules show a short, highly conserved LSGGQ
consensus sequence, known as the linker peptide
or ATP signature. The major difference between
the various ABC systems is related to the polarity
of transport. Transporters relevant to fungicide
insensitivity are exclusively members of the export
pumps, classified as ABC-A transporters (Saurin
et al. 1999).
As ABC transporters export substrates
belonging to different chemical c1asses rather non-
specifically, they are thought to be of particular
importance in adaptation of pathogenic fungi to
various fungicides (DeI Sorbo et al. 2000). The
role of ATP-dependent transporters has been ana-
Iyzed by transformation-mediated gene inactiva-
tion. Hamer and co-workers have c10ned the ABC
transporter gene ABCl of the rice blast fungus M.
grisea (Urban et al. 1999). The transporter is
strongly induced by the rice phytoalexin saku-
ranetin and by azole fungicides. An ABCl-
deficient mutant penetrates the rice epidermis
normally but dies soon thereafter. Since the muta-
tion does not lead either to increased sensitivity to
sakuranetin or various fungicides, the exact role of
this transporter needs to be defined. The ABC
transporter gene of Botrytis fuckeliana, BeatrB, is
induced by the phenylpyrrole fungieides fen-
pic10nil and ftudioxonil and by the grapevine
phytoalexin resveratroI; mutants with disrupted
BcatrB display increased sensitivities to these
compounds and show reduced virulence on
grapevine leaves (Schoonbeek et al. 2001). Five
ABC transporter genes, designated MgAtrl to
MgAtr5, have been c10ned from the wheat
pathogen Mycosphaerella graminicola. MgAtrl
and MgAtr2 are differentially up-regulated by the
azole fungicides cyproconazole and imazalil, and
different expression patterns between yeast-like
256 H.B. Deising et al.
cells and the mycelium were found (Zwiers and
DeWaard 2000). The ABC transporter LMABC2
of the crucifer pathogen Leptosphaeria maculans
is thought to be involved in multidrug resistance,
as it restores cycloheximide and 4-nitroquinoline
N-oxide sensitivity after complementation of
yeast PDR5- and Candida SNQ2-transporter null
mutants (Taylor and Condie 2000). Azole fungi-
cides strongly induce the ABC transporter
gene PMRl in the post-harvest fruit pathogen
Penicillium digitatum, and disruption of this gene
causes high fungicide sensitivity (Nakaune et al.
1998). Interestingly, experiments with S. cerevisiae
indicated that ABC transporters confer insensi-
tivity not only against azole fungicides, but also
against strobilurins (Kolaczkowski et al. 1998).
This is of particular relevance because quantita-
tive shifting of sensitivity, which may be indicative
of efflux transport activity in field populations, has
recently been observed in apple scab populations
treated with these fungicides (Schnabel and Jones
2001).
Yeast genome analysis revealed the presence
of nine families of MFS transporters (Pao et al.
1996). The pro teins exhibit 12-14 putative trans-
membrane domains. The only motifs with con-
served amino acids in all MFS transporters locate
to the N-terminus [N(E/D)(X)z-4GR] and to the
loop between transmembrane domains 5 and 6
[W(R/S)W] (DeI Sorbo et al. 2000). However,
members of specific MFS clusters show some
high er degrees of homology (Goffeau et al. 1997;
DeI Sorbo et al. 2000). Fungal MFS transporters
are thought predominantly to playa role in secre-
tion of toxins. The TaXA gene of the maize
pathogen Cochliobolus carbonum encodes an
MFS transporter required for secretion of HC
toxin (Pitkin et al. 1996). As toxA mutants cannot
be obtained after gene disruption experiments, the
TOXA MFS-efflux pump probably functions to
protect C. carbonum from its own toxin (Pitkin et
al. 1996). Likewise, disruption of the MFS trans-
porter gene of the soybean pathogen Cercospora
kikuchii and of the maize pathogen Fusarium
sporotrichioides, respectively, resulted in reduced
virulence and increased toxin sensitivity
(Alexander et al. 1999; Callahan et al. 1999). Some
MFS transporters, however, also confer fungicide
insensitivity. For example, benomyl insensitivity in
the human pathogen Candida albicans is not only
mediated by mutation al modifications in the ß-
tubulin gene (Yarden and Katan 1993), but also by
the action of a MFS efflux pump (Fling et al.
1991), and the Atr1 transporter mediates amino-
triazole resistance in S. cerevisiae (Gömpel-Klein
and Brendel 1990).
ABC or MFS transporters have not yet been
shown to be involved in reduced fungicide efficacy
in rust or powdery mildew populations and the
above-mentioned cases do not allow any conclu-
si on on the basic mechanism of fungicide insensi-
tivity. It would be of interest to clone genes
encoding ABC or MFS transporters and to
compare transcript levels in fungicide-sensitive
and -insensitive populations. As techniques to
stably transform powdery mildew (Chaure et al.
2000) are available, gene inactivation experiments
to analyze the role of transporter gene(s) in this
obligate biotroph can be envisaged. It would also
be of interest to see whether microscopical tech-
niques that have been developed to visualize
efflux pump activity in the wheat pathogen
Pyrenophora trztlCl-repentis (Reimann and
Deising, unpublished data) could be used to test
whether or not transporters are induced by
routine fungicide applications in the field.
VI. Novel Approaches
A. Induced Resistance
In the early 1930s, Chester (1933) observed devel-
opment of 'acquired immunity' in plants that had
been inoculated with a pathogen, and Ross (1961)
coined the terms local and systemic acquired
resistance (LAR and SAR) to describe resistance
induced exclusively in the inoculated leaf or in the
entire plant after treatment of one leaf. Resistance
can also be induced by non-pathogenic rhizos-
phere bacteria (van Loon et al. 1998) and by
mycorrhizal fungi (Cordier et al. 1998). Charac-
teristically, the mechanisms induced in plants are
active against a broad spectrum of pathogens,
including viruses, bacteria and fungi. Resistance
has been induced in a large number of plants,
including mono- and dicots, and can last for days,
weeks or even months (Kessmann et al. 1994;
Schneider et al. 1996; Steiner and Schönbeck 1997;
Sticher et al. 1997). Interestingly, an increase in
salicylic acid has been observed at the onset of
systemic acquired resistance in cucumber
(M6traux et al. 1991a), and experiments with
transgenic plants expressing the nahG gene from
Pseudomonas putida encoding salicylate hydroxy-
 Disease Management of Rusts and Powdery Mildews
lase show that this metabolite plays a central role
in disease resistance (Gaffney et al. 1993; Delaney
et al. 1994). In an elegant experiment, Ryals and
co-workers (1996) expressed the nahG gene in the
Arabidopsis thaliana cim3 (~onstitutive immunity)
mutant, which shows elevated levels of free and
conjugated salicylic acid, elevated levels of tran-
scripts of the SAR markers PR-I, PR-2 and PR-5,
and exhibits constitutive immunity against viru-
lent pathogens. In the case of nahG expression,
both elevated levels of salicylic acid and constitu-
tive immunity were lost. Shulaev and co-workers
(1997) showed that salicylic acid can be converted
into methyl salicylate after pathogen attack. This
metabolite, in comparison to salicylate, exhibits
increased vapor pressure and is thought to func-
tion as an airborne signal between plants. Signal
pathways involving salicylic acid and leading to
expression of defense-related genes have been
described in detail and the reader is referred to
other recent publications (Dong 1998; Scheel
1999; Feys and Parker 2000; Yang et al. 2001, and
references therein).
In accordance with the role of salicylic acid
in resistance, exogenous application of salicylic
acid or of structurally related chemieals like ace-
tylsalicylic acid (aspirin), 2,6-dichloroisonicotinic
acid (DCINA) or benzo(1,2,3)thiadiazole-7-
thiocarbonic acid S-methyl ester (BTH), the latter
being the active ingredient of the plant strength-
ener Bion, efficiently induce resistance in
plants (Metraux et al. 1991b; Kessmann et al. 1994;
Görlach et al. 1996). As they are not active against
microorganisms and therefore do not exert a
selection pressure, development of resistance or
insensitivity against these compounds is of no
concern. Chemical inducers have thus been
expected to widen the spectrum of disease control
agents and they were supposed to reduce the risk
for growers (Uknes et al. 1996).
An important question relates to the mecha-
nisms activated by chemical induction of resis-
tance. Kogel et al. (1994) demonstrated that
2,6-dichloroisonicotinic acid-induced resistance is
a phenocopy of a genetically based mechanism
governing race-specific resistance against powdery
mildew in barley, that requires the functionally
active Mlg gene. This also holds true for Bion (Fig.
13.1). In response to infection and prior to forma-
tion of haustoria, short epidermal cells und ergo
hypersensitive cell collapse, along with accumula-
ti on of fiuorescent material in papillae at the time
of fungal arrest. Furthermore, the onset of
257
acquired resistance correlates with the accumula-
tion of PR-I, chitinase and peroxidase transcripts.
Peroxidases play a role in hypersensitive cell
death, papilla formation and oxidative cross-
linking of hydroxyproline-, glycine- or threonine-
rich glycoproteins, which may result in physical
strengthening of the cell wall (Brisson et al. 1994;
Boudet et al. 1995; Scott-Craig et al. 1995;
Thordal-Christensen et al. 1997). Interestingly,
electron microscopy revealed that in interactions
between wheat and stern rust (F graminis f. sp.
tritici) and leaf rust (F recondita) , and between
barley and the powdery mildew fungus (E. gra-
minis f. sp. hordei), threonine-hydroxyproline-rich
glycoproteins accumulated specifically over the
extrahaustorial matrix. In the latter interac-
tion, highly localized accumulation was also seen
over the center of penetration sites, suggesting
that the host establishes a physical barrier
between itself and the pathogen (Hippe-Sanwald
et al. 1994). When BTH is sprayed onto wheat
plants at an early developmental stage, resistance
against the biotrophic rust F recondita and the
powdery mildew B. graminis f. sp. tritici was
induced (Görlach et al. 1996). As in barley, chem-
ical induction of resistance resulted in restrietion
of fungal development by papillae and hypersen-
sitive response. Using differential screening and
differential display techniques, Görlach et al.
(1996) identified five WCI (~heat ~hemically
induced) genes that did not reveal homology with
previously described SAR genes (Ward et al.
1991). Chemically induced genes of wheat and
barley were not activated in response to powdery
mildew infection, suggesting at least partially
distinct signal transduction pathways for R-
gene-specified and chemically induced resistance
(Schaffrath et al. 1997; BeBer et al. 2000).
Although sequence homology between certain
chemically induced genes and defense-related
genes with known activity suggests their involve-
ment in restriction of fungal development, it is
necessary to assess their function by tedious and
time-consuming generation of transgenic plants
expressing anti-sense constructs. To functionally
test chemically induced genes, Schweizer et al.
(1999) established a transient assay system
employing bombardment of wheat leaves with
tungsten particles coated with plasmids carrying
the ß-glucuronidase (GUS) reporter gene and
different genes to be tested. Subsequent challenge
inoculations indicated that WCI5 (a protein of
unknown function), a chitinase, a glucose oxidase,
258 H.B. Deising et al.

and a putative peroxidase significantly reduced

penetration by B. graminis f. sp. tritici. As an
alternative to high-level expression of genes,
gene silencing by the recently established dsRNA
technique could be utilized (Schweizer et al.
2000).
In chemically induced plants, a complex
defense system consisting of different defense
responses is thought to contribute to protection
against the attacking pathogen (Ward et al. 1991;
Schneider et al. 1996; Sticher et al. 1997).
However, in certain systems, the induction of
single factors appears to be sufficient to confer
resistance. Rauscher et al. (1999) used salicylic
acid and 2,6-dichloroisonicotinic acid to chemi-
cally induce resistance in broad bean (Vicia faba)
to the broad bean rust fungus, Uromyces fabae.
Light microscopy studies revealed that infection
structure differentiation was inhibited in the inter-
cellular space, immediately after the fungus had
penetrated through the stomatal pore. Low tem-
perature scanning electron microscopy studies of
freeze fractures showed that intercellular hyphae
were formed abundantly in water-treated control
plants, as compared to scarce hyphae in salicylate-
induced plants. Those hyphae of U. fabae that
grew in the intercellular space of salicylate-
induced plants showed severe distortion, i.e.,
development of protrusions at the hyphal tips
(Fig. 13.4). Interestingly, Western blot analyses of
washing fluids clearly showed that several anti-
fungal proteins, including ß-1,3-glucanases and
chitinases, are constitutively synthesized in broad
bean and secreted into the extracellular space. The
only extracellular PR protein that was truly
induced by salicylic acid, 2,6-dichloroisonicotinic
acid and the necrotizing chemical AgN0 3 was a
PR-1 homologue of tobacco. Importantly, purified
PR-1 did not interfere with germination or germ
tube growth and appressorium formation of Fig.13.4. Freeze fractures of broad bean leaves treated
Uromyces fabae, but all subsequently synthesized with A water or B,C 0.5 mM salicylic acid and inoculated
structures, which naturally occur in the intercellu- with urediniospores of the broad bean rust fungus
lar space and thus make contact with this protein, Uromyces fabae 24h later. Leaves were excised 1 day after
inoculation. ih lnfection hypha; protrusions are marked
were inhibited. These data indicate that PR-1 of with arrows. (Rauscher et al. 1999)
broad bean is a stage-specific inhibitor of rust
infection structure differentiation (Rauscher et al.
1999).
Although induced resistauce was strongly after Bion treatment, but yields were not reliably
advocated as a new strategy supporting plant increased as compared with non-treated and fully
protection (Görlach et al. 1996; Uknes et al. 1996), infected fields (Andersch 1998; Stadnik and
practical experiences have cast some doubt on the Buchenauer 1999). As the multiple defense
yield re li ability of these chemicals. Often B. responses stimulated by chemical inducers, e.g.,
graminis disease severity was significantly reduced formation of cell wall appositions and synthesis of
 Disease Management of Rusts and Powdery Mildews
phenols and PR proteins, demand significant
energy (Smedegaard-Petersen and Stolen 1981;
Smedegaard-Petersen and Toistrup 1985), the
bonus of reduced disease severity may be accom-
plished at the expense of yield reduction (Stadnik
and Buchenauer 1999). It is thus questionable
whether or not chemical induction of resistance
can be regarded as an alternative in disease man-
agement under practical conditions (Lyon and
Newton 1997; Andersch 1998). Responding to
these difficulties, the product Bion has recently
been withdrawn from the market.
Interestingly, Thieron et al. (1998) reported on
a fungicide with two different modes of action.
Carpropamid, the active ingredient of the rice
fungicide WIN, inhibits melanin biosynthesis in
appressoria of the rice blast fungus M. grisea and
induces resistance by stimulating lignification
and phytoalexin synthesis in the plant. Combining
fungicidal and resistance inducing activity may
increase the efficacy of the product and be more
attractive for farmers than chemical resistance
inducers alone.
B. Resistanee Mediated by Introduetion
of Foreign Genes
When disease resistance of plants is overcome by
a pathogen, breeders need to produce new resis-
tant varieties of a crop plant. The question of
whether genes conferring resistance can be uti-
lized largely depends on the breeding strategy or
the technique of introgression of new genes. Three
strategies can be pursued to introduce new genes
into a given genetic background.
Traditional breeding, i.e., crossing of plants, is
the oldest technique to produce varieties harbor-
ing new genes, and introduction of TUst and
powdery mildew resistances into cultivars via
crosses from wild species has been accomplished
with many different species, e.g., a powdery
mildew resistance major gene from wild emmer to
bread wheat (Reader and Miller 1991) and leaf
TUst resistance from an amphiploid of Aegilops
speltoides x Triticum monococcum to hexaploid
wheat (Kerber and Dyck 1990). Today, the time-
consuming breeding process can be accelerated by
using resistance-linked markers. A typical way is
the use of bulked segregant analyses (Michelmore
et al. 1991) to identify RAPD (random amplified
polymorphie DNA) or AFLP (amplified fragment
length polymorphism) markers and to subse-
259
quently convert them into a SCAR (sequenee
eharaeterized amplified region) marker. This tech-
nique allows direct PCR-based detection of a
resistance gene in a test tube. A SCAR marker has
been developed for Lr24 in wheat (Dedryver et al.
1996). The source of resistance genes that can be
used in breeding approaches is restricted by sexual
incompatibilities. A clear dis advantage of conven-
tional crossing is that not only the desired trait,
e.g., an unbroken resistance gene, but rather a set
of chromosomes is transferred from a wild species
into a crop plant, demanding a long recurrent
selection to eliminate undesired traits. Due to veg-
etation cycles, this process usually takes several
years.
Fusion of protoplasts has been utilized to
transfer new genes, with the advantage that sexual
incompatibilities and crossing barriers can be
overcome if the different genomes are compatible.
However, as in conventional breeding, the intro-
gression of a single trait requires displacement
crossing of all other traits and the breeding time
is not reduced.
DNA-mediated transformation of plants
made it possible to introduce single genes, irre-
spective of their source, and to maintain other
characteristics of a variety that is well established
on the market. The advantage of genetically engi-
neered transgenic plants is, therefore, that only the
desired trait is conferred and time-consuming
displacement crossing is no longer required. Some
methodological difficulties in stable introgression,
reliable hereditability and avoidance of gene
silencing to allow high-level expression must be
taken care of in the laboratory before transgenic
plant can be tested in the field. It should be
emphasized that, for many reasons, genetic engi-
neering in food production is controversially dis-
cussed. A major concern is the allergenicity of
food derived from transgenic plants, as has been
reported for a Brazil nut pro tein expressed in
soybean (Nordlee et al. 1996; Franck-Oberaspach
and Keller 1997).
In various experiments, individual genes or
combinations of genes encoding antifungal pro-
teins or enzymes forming fungitoxic compounds
have been used. The greatest emphasis has been
placed on trans genes encoding PR-proteins,
ribosome-inactivating proteins (RIPs) or enzymes
synthesizing phytoalexins (Swords et al. 1997).
PR proteins such as PR-1, or the hydrolytic
enzymes chitinases and ß-1,3-glucanases, have
been shown to inerease plant resistance against
260 H.B. Deising et al.

various pathogenes, including rusts and powdery Phytoalexins are low molecular weight
mildews (Bliffeld et al. 1999; Yamamoto et al. antimicrobial compounds formed in plants in
2000). While antifungal activity of chitinases has response to pathogen attack (Hammerschmidt
clearly been demonstrated in vitro (Mauch et al. 1999). They are synthesized locally at the site of
1988), the significance of these enzymes in vivo is pathogen ingress, and their concentration some-
unclear, as rusts, and this is possibly also true of times exceeds that required to affect pathogen
several other fungi, convert the chitin exposed at growth several thousand-fold (Nicholson et al.
the hyphal surface into chitosan (Deising and 1987; Snyder and Nicholson 1990).
Siegrist 1995). This polymer may not readily be While phytoalexin synthesis usually requires
degradable by plant chitinases (Ride and Barber complex biosynthetic pathways involving several
1990). However, in some cases, chitinases have enzymes, synthesis of the grapevine stilbene phy-
been demonstrated to be efficient against powdery toalexin trans-resveratrol is formed in a single
mildew. Toyoda et al. (1991) showed that, after enzymatic step, using p-coumaryl-CoA and
microinjection of chitinase into barley coleoptile malonyl-CoA as substrates. As these substrates
epidermal cells, development of the powdery are commonly present in higher plants (Rupprich
mildew pathogen was suppressed. Two homozy- and Kindl 1978), synthesis of resveratrol may
gous transgenic wheat lines (T3 generation) con- occur in transgenie plants expressing a stilbene
stitutively expressing an antifungal barley-seed synthase capable of resveratrol synthesis. Stilbene
dass II chitinase under the control of a ubiquitin synthase genes from Vitis vinifera (Vst) have been
promoter showed increased resistance to infection transferred into several economically important
with B. graminis, as indicated by significant plants induding tobacco (Hain et al. 1993), rice
reduction of colony development by up to 54% (Stark-Lorenzen et al. 1997), and barley and
(Bliffeld et al. 1999). The level of reduction of wheat (Leckband and Lörz 1998). The efficiency
symptom development in both lines correlated of resveratrol has been demonstrated by increased
with the level of immunodetectable chitinase. In a resistance of transgenic plants to fungal pathogens
comparable approach, Yamamoto et al. (2000) like Boytritis cinerea (Hain et al. 1993) and M.
expressed the rice dass I chitinase gene RCC2 grisea (Stark-Lorenzen et al. 1997). In barley and
under the control of the 35S promoter in trans- wheat, the Vstl promoter was induced by infection
genic grapevine (Vitis vinifera) and observed with B. graminis. Vstl-mRNA was specifically
enhanced resistance against the powdery mildew found at infection sites but not in non-infected
fungus Uncinula necator. Scanning electron leaves of the same plant (Leckband and Lörz
microscopy revealed that both mycelial growth 1998). Whether or not resveratrol can enhance
and formation of conidia were suppressed on the resistance against powdery mildew needs to be
leaf surface of the transformants. thoroughly analyzed.
Plant RIPs are enzymes that inhibit protein Not only the level of resistance, but also the
biosynthesis (see review by Barbieri et al. 1993). specificity of recognition may be altered in trans-
RIPs inactivate ribozymes irreversibly by the genic plants. Ellis et al. (1999) constructed intra-
release of adenine from a highly conserved rRNA genic exchanges at the flax rust resistance locus
loop sequence that is present in all ribozymes and L involving the L2, L6, and LlO alleles. Transfor-
crucial for the interaction with elongation factor mation experiments with some alleles of the rust
2 in eukaryotic ribosomes (GuteIl and Fox 1988; avirulence gene confirmed that rust resistance is
Brigotti et al. 1989; Girbes et al. 1996). Bieri et al. specifically mediated by certain regions of the 13
(2000) tested barley seed RIP for its efficiency alleles of the L locus that determine gene-for-gene
against powdery mildew in transgenic wheat. specificity. For example, the flax line 'Forge', which
Expression of the RIP30 gene of barley was con- is susceptible to rust strain Sp-y, was transformed
trolled by an enhanced CaMV 35S promoter, and with the L2 allele under the control of the 35S
a leader peptide directed the RIP protein to the promoter. The strain Sp-y carries an avirulence
apoplastic space. Plants exhibiting high-level gene matching the L2 allele. Two transgenic lines
expression and synthesis of RIP30 were protected resistant to rust strain Sp-y were obtained, and
moderately against infection by powdery mildew. the resistance phenotype of all transgenic plants
Also, in combination with other antifungal pro- was unambiguous. Small hypersensitive flecks
teins, RIPs have been shown to enhance resistance appeared on infected leaves, and no urediniospore
(Logemann et al. 1992; Wang et al. 1998). formation occurred. The Toll/interleukin receptor
 Disease Management of Rusts and Powdery Mildews
and the leucine-rich repeat regions of this R gene
thus determine specificity and, by taking compa-
rable approaches, it may be possible to produce
transgenic plants with desired race specificity.
While resistance media ted by introduction of
transgenes as described above usually relies on
constitutive expression of genes giving rise to anti-
fungal compounds, pathogen-inducible expression
of such genes may be advantageous. Stuiver (1999)
fused the avr9 gene of Cladosporium fulvum to a
promoter that is inducible by fungi, virus es, bacte-
ria and nematodes, and transformed the construct
into tomato plants containing the Cf9 plant re-
ceptor. Attacking pathogens trigger expression of
the avr9 gene and thus initiate the hypersensitive
response, leading to cell death specifically in the
area of ingress (De Wit 1992; Jabs and Slusarenko
2000). Resulting transgenic plants displayed
enhanced resistance not only against fungi, but,
remarkably, also against tomato spotted wilt virus.
These examples demonstrate that, although a
significant amount of research is still needed,
genetic engineering has the potential to contribute
to producing pathogen-resistant crop plants. Such
plants mayaiso help to reduce the amount of
fungicides applied in disease control.
VII. Efficient Disease Management
by Combination
of Different Approaches
To meet the ecological and economieal demands of
modern agriculture, "Integrated Plant Protection"
or" Integrated Disease and Pest Management" has
been the strongly promoted by plant pathologists.
Recently, the European Union has included a defi-
nition of"Integrated Control" in Council Directive
91/414 EEC, closely resembling that of the German
Plant Protection Act of 1986: "Integrated control
implies the rational application of biological,
biotechnical, chemical, cultural or plant breeding
measures, whereby the use of chemical plant pro-
tection products is limited to the strict minimum
necessary to maintain the pest population at levels
below those causing economically unacceptable
damage or loss". Integrated disease control thus
requires reduced fungicide usage and strengthen-
ing of other agronomie measures, e.g., breeding and
growing resistant cultivars, performing crop rota-
tion and reasonable mineral nutrition, and using
plant strengtheners and biocontrol agents, if avail-
261
able (Mendgen 1981; Avis et al. 2001). As a pre-
assumption for integrated disease control, modern
and realistic modeling and fore casting, i.e., deci-
si on support systems to estimate disease progres-
sion, need to be developed and optimized (de
Vallavieille-Pope et al. 2000; Verreet and Klink
2000, and references therein). Also, and this may
become increasingly important in the future, con-
struction of genetically modified organisms, either
plants expressing defense genes against certain
pathogens or microorganisms with enhanced abili-
ties to control pathogens, could be used, depending
on the legal provision. A long-standing issue that
mayaiso contribute to the concept of integrated
disease control is the use of cultivar mixtures to
reduce disease severity.
Plant breeding has provided farmers with cul-
tivars with improved resistance against important
pathogens such as powdery mildews and rusts. In
wheat, significant decreases in susceptibility
against powdery mildew, stripe rust and leaf rust
have been recorded from 1986 to 1994, accompa-
nied by dramatic yield increases (Hartleb et al.
1997). On a scale from 1 to 10 (1 being resistant,
10 being susceptible), average mildew susceptibil-
ity shifted from 4.2 in 1986 to 2.2 in 1994. Like-
wise, susceptibility for stripe rust and leaf rust
shifted from 3.9 to 2.9 and from 5.4 to 2.4, respec-
tively. Thus, growing resistant cultivars may help
to reduce the amount of fungicides and, impor-
tantly, to reduce the ecological risk. In semi-arid
regions of Sachsen-Anhalt, Germany, P recondita
on wheat and rye, and P hordei on barley, caused
yield losses of approximately 20% in susceptible
cultivars without fungicide treatment in 1993 and
1994; the use of resistant wheat and spring barley
cultivars, however, allowed growers to reduce
fungicide applications significantly without losses
in net return (Hartleb et al. 1995). During the
twentieth century large-scale repeated monocul-
ture of varieties with one or more resistance genes
has been performed, and this applied significant
selection pressure for pathogen genotypes that are
able to overcome resistance. Resistance mono-
culture thus led to massive epidemics of wheat
stern rust (McIntosh et al. 1995), especially in
times when efficient fungicides were not available
(Kislev 1982), and Harlan (1972) coined the term
"genetics of disaster" to indicate the severe
mistakes associated with this strategy of disease
management.
Previously, local bulk-selected land races of
crops variable in genetic structure of disease resis-
262 H.B. Deising et al.
tance were grown. In fact, mixed cropping has
many advantages in reducing the severity of dis-
eases (Wolfe and Finckh 1997). For example,
mixtures of malting quality barley cultivars were
introduced into the former GDR in 1984 and, by
1988/89, the entire spring barley crop of approxi-
mately 300,000 ha was grown as mixtures. This
strategy kept powdery mildew at a low level and
allowed considerable reduction of fungieide appli-
cation. There was also a restriction of leaf rust,
although this was not a target disease in the choice
of the components of the mixture. High crop
yields and grain quality were obtained, in spite of
the large field size of up to 100 ha, and in spite of
the fact that only three resistance genes were com-
bined. However, in order to prevent the selection
of races able to overcome all of the resistance
genes present in the mixture, the components of
the mixture should be changed as often as pos-
sible (Wolfe and Finckh 1997, and references
therein). Even today, crop heterogeneity may
support control of fungal diseases of crop plants.
In Yunnan Province, China, genetically diversified
rice crops were planted in all fields of five town-
ships in 1998, and of ten townships in 1999.
Disease-susceptible rice varieties grown in mix-
tures with resistant varieties had 89% greater
yield and rice blast (M. grisea) severity was
reduced by 94%. By the end of the 2-year
program, fungicide sprays were nolonger applied,
indicating that intraspecific crop diversification
provides a highly effective, ecologically based con-
tribution to sustainable crop protection over a
large area (Zhu et al. 2000).
Chronological as well as spatial distribution of
R genes is of critical importance. For efficient
control of powdery mildew the use of identical
major R genes in summer and winter cereals
should strictly be avoided, as this would apply
uniform selection pressure throughout the entire
growing season. Chronological and spatial alter-
ation of R genes would help to prevent or delay
occurrence of the corresponding virulences and
support R gene management in these crops.
VIII. Conclusion and Perspectives
In this chapter we have described historical devel-
opments and re cent trends in powdery mildewand
rust disease management. An important conclu-
sion to be drawn from the data presented is that,
due to the f1exibility of the fungal genome,
complex rather than single plant proteetion strate-
gies should be recommended. Failure of plant
protection relying on single aspects has become
evident especially at times of major epidemics
occurring after the plants' resistance genes were
defeated by Avr gene mutations. Likewise, con-
tinued use of fungieides with identical or similar
modes of action has resulted in rapidly decreasing
fungicide efficacy, often due to mutation al
changes of the fungieide target and reduced
binding of the drug. Even variable fungicide man-
agement pro grams may not guarantee durability
of the compounds used, and the possibility of
induction of ABC- or MFS-transporter-based
insensitivity urgently requires a continued search
for new antifungal strategies. In fact, we are con-
cerned that such transporters may become a
major challenge during the next decades in
modern agro-industrial societies. Simultaneous
loss of fungieides with different modes of action
mediated by transporters may have dramatic
consequences.
This scenario clearly requires high-level
research and development of novel disease man-
agement expertise. In addition to continued clas-
sical plant breeding and plant protection, the
application of molecular genetics will help to
define novel fungieide targets, e.g., by saturating
restrietion enzyme-mediated DNA integration
mutagenesis. Precise knowledge of potential novel
fungieide targets could be combined with com-
puter-assisted fungicide design to discover new
drugs. However, irrespective of the mode of
search for and design of fungieides, new plant
protection chemieals can only be successful and
durably established on the market if they are sup-
ported by well-coordinated modern agronomie
measures (reasonable agricultural practice). This
also holds true for new pathogen-resistant crop
varieties, either produced by classical breeding
programs or by transgene technology. These
complex requirements will involve many different
research disciplines and can only be met if scien-
tists in universities and industry, plant proteetion
advisors and farmers share intensive cooperation.
Acknowledgements. The authors are indebted to
Tim L.W. Carver, Institute of Grassland and Envi-
ronmental Research,Aberystwyth, UK; Karl-Heinz
Kogel, Institute of Phytopathology and Applied
Zoology, University of Gißnen; and Horst Hartleb,
Landesanstalt für Landwirtschaft und Gartenbau,
 Disease Management of Rusts and Powdery Mildews
Magdeburg, for critical discussions and helpful
comments on the manuscript. The financial support
of fungicide resistance projects in H.D.'s laboratory
by Ministerium für Landwirtschaft und Forsten
(grant No. 3205AI0020H), Kultusministerium des
Landes Sachsen-Anhalt (grant No. 3282AI0080R)
and Deutsche-Fosschunqs-Gemeinschaft is grate-
fully acknowledged. The donation of scanning elec-
tron micrographs by Richard Guggenheim, REM
Laboratory, University of Basel, BASF AG and
Dow AgroSciences is also acknowledged.
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Genet BioI30:115-125
Update on Host-Parasite Interactions
14 Signal Transduction Pathways in Phytopathogenic Fungi
MICHAEL BÖLKER
CONTENTS
I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . .
11. Signal Transduction Pathways . . . . . . . . . . . . .
A. Receptors ............................
B. Heterotrimeric G Proteins ...............
C. cAMP Signaling . . . . . . . . . . . . . . . . . . . . . . . .
D. MAP Kinase Cascades ..................
E. Ca2+ICalmodulin Signaling . . . . . . . . . . . . . . . .
III. Model Organisms ......................
A. Magnaporthe grisea . . . . . . . . . . . . . . . . . . . . .
B. Ustilago maydis ........................
IV Other Phytopathogenic Fungi . . . . . . . . . . . . .
A. Cryphonectria parasitica .................
B. Erysiphe graminis ......................
C. Botrytis cinerea ........................
D. Colletotrichum spp. .....................
V Conclusions...........................
References . . . . . . . . . . . . . . . . . . . . . . . . . . . .
I. Introduction
Fungal plant pathogens have been the focus of sci-
entific research for many years since they cause
some of the most devastating plant diseases. Inter-
estingly, of the many known fungal species, only
a few succeed to overcome the barrier of nonhost
resistance and to proliferate within the plant
tissue. It is apparent that fungal pathogens co-
evolved for a long time with their specific hosts.
Evolution results in an arms race of disease and
resistance leading to highly specific adaptations
on both sides (Dawkins and Krebs 1979). This is
evident at the molecular level in gene-for-gene
relationships between fungal avirulence (avr)
genes and plant-resistance genes, the products of
which directly or indirectly interact with the prod-
ucts coded for by the avr genes (for review, see
Stahl and Bishop 2000; Chap.15, this Vol.). During
their long history, fungal pathogens developed
Universität Marburg, Fachbereich Biologie, Karl-von-
Frisch-Strasse 8,35032 Marburg, Germany
elaborate mechanisms to recognize their specific
273 hosts, to penetrate the plant cell wall and to avoid
276 host resistance reactions. Therefore, it is obvious
276 that it is not a single virulence trait that makes a
276
277 fungus pathogenic, but that many genes contribute
277 to fungal virulence.
278 In recent years, the study of signal transduc-
278 tion cascades in fungal pathogens has revealed the
279
280 importance of signaling pathways during the infec-
283 ti on process. Parasites have to recognize their
283 specific hosts and react to certain environmental
284
284
conditions. In addition, morphogenetic develop-
284 ment and differentiation is often triggered by
285 ambient signals. This review will focus on an
285 update of current knowledge on signaling path-
ways in phytopathogenic fungi. Due to the limited
genetic and biochemical tools that can be applied
to most of these organisms our knowledge is far
from complete. However, an increasing number of
components have been identified in re cent years,
many of which play pivotal roles for morphogen-
esis and pathogenic development (see Table 14.1).
Comparison of different species with regard to
their signaling networks reveals not only common
themes, but also highly sophisticated adaptations
for specific needs. Research into fungal signal
transduction processes profi ted in many aspects by
the great scientific progress reached in Saccha-
romyces cerevisiae. In particular, molecular analy-
sis of the pheromone response pathway and
pseudohyphal differentiation in yeast revealed
major insights into the complex network of fungal
signal transduction. The modular structure of sig-
naling cascades in yeast and their interconnections
with each other often serve as useful paradigms
for the study of related signaling processes even in
higher organisms (Herskowitz 1995). Surprisingly,
some components of signaling pathways are used
in different signaling cascades (Liu et al. 1993),
which raised the problem as to how the specificity
of the response is guaranteed.
The first part of this chapter will give an
overview of the different elements that are used
The Mycota XI
Agricultural Applications
Kempken (Ed.)
© Springer-Verlag Berlin Heidelberg 2002
Table 14.1. Genes involved in signal transduction

Organism Gene Function Phenotype of mutants Accession no. Reference( s)

li
Magnaporthe MPGl Hydrophobin Required for recognition of L20685 Talbot et al. (1996)
grisea hydrophobie surfaces;
reduced pathogenicity
MACI Adenylyl cyclase defect in appressoria AF012921 Choi and Dean (1997)
MAGA G protein a subunit development
No effect on vegetative growth, AF011340 Liu and Dean (1997)
conidiation, or appressorium
formation
MAGB G proteiu a subunit Reduced appressorium formation, AF011341 Liu and Dean (1997)
virulence, and vegetative growth
MAGC G protein a subunit Reduced conidiation AF011342 Liu and Dean (1997)
CPKA (PTH4) Catalytic subunit of Reduced pathogenicity U12335 Mitchell and Dean (1995);
cAMP-dependent Xu et al. (1997);
protein kinase Sweigard et al. (1998)
PMKI MAP kinase Loss of pathogenicity; defect in U70134 Xu and Hamer (1996)
appressoria development
OSMI MAP kinase, similar to Sensitivivity to osmotic stress, AF184980 Dixon et al. (1999)
yeast Hog1, regulating morphological defects under
response to hyperosmotic hyperosmotic conditions ~
tx:I
stress 0:
MPS1 MAP kinase, similar to yeast Female sterility; defect in AF020316 Xu et al. (1998) ~
(1)
>;
Slt2, involved in maintaining appressoria development
cell wall integrity
PTHI Similar to yeast Grr1 involved Loss of pathogenicity Sweigard et al. (1998)
in regulating glucose repression
PTHll Integral membrane protein, Loss of pathogenicity AF119670 DeZwaan et al. (1999)
effector of appressorium
differentiation
Usilago maydis bW/bE Pair of homeodomain transcription Sterile, nonpathogenic M84179,M58553 Schulz et al. (1990);
factors, multiallelic mating Kronstad and Leong (1990;
type determinants Gillissen et al. (1992)
mfallmfa2 Secreted mating-type specific Sterile M84159/U37796 Bölker et al. (1992)
lipopeptide pheromones
pral/pra2 Pheromone receptors Sterile M84157/U37796 Bölker et al. (1992)
ruml Similar to retinoblastoma Induction of b-dependent genes AF286030 Quadbeck-Seeger et al. (2000)
binding protein, interaction in the absence of a bW/bE
with histone deacetylases heterodimer
prfl Transcription factor Sterile, nonpathogenic U40753 Hartmann et al. (1996)
ncpl Regulator of Prfl expression No effect on mating and AF082296 Hartmann et al. (1999)
pathogenicity
 gpa3 G protein a subunit Sterile, nonpathogenic U85777 Regenfelder et al. (1997)
uac1 Adenylyl cyclase Filamentous growth L33918 Gold et al. (1994)
ubc1 Regulatory subunit of Multiple budding L33917 Gold et al. (1994)
cAMP-dependent
protein kin ase
adrl Catalytic subunit of Filamentous growth, U23730 Orth et al. (1995);
cAMP-dependent nonpathogenic Dürrenberger et al. (1998)
protein kinase
ukal Catalytic subunit of No effect on morphology AF025290 Dürrenberger et al. (1998)
cAMP-dependent and pathogenicity
protein kin ase
kpp2/ubc3 MAP kin ase (MAPK) Attenuation of mating AF193614 Mayorga and Gold (1999);
and pathogenicity Müller et al. (1999) f/)

fuz7/ubc5 MAP kinase kinase (MAPKK) Attenuation of mating U07801 Banuett and Herskowitz äö·
:=
and pathogenicity (1994); Andrews et al. (2000) e:..
ubc4 MAP kin ase kinase Attenuation of mating AF197562 Andrews et al. (2000) ;:;l
kinase (MAPKKK) and pathogenicity :=
'"
Cryphonectria CPG-l G pro tein a subunit Attenuation of virulence, L32176 Choi et al. (1995) '"P-
t::
()
parasitica pigmentation g.
CPG-2 G pro tein a subunit Reduction in growth rate L32177 Gao and Nuss (1996) :=
CPGB-I G protein ß subunit Attenuation of virulence, U95139 Kasahara and Nuss (1997) '"1:l
pigmentation '::;:fT"
BDM-l Phosducin related protein Attenuation of virulence, AF140555 Kasahara et al. (2000)
pigmentation '<'"
Erysiphe graminis cPKA Catalytic subunit of Not determined AJ243654 Hall et al. (1999) '"
5·
cAMP-dependent '"1:l
::r-
protein kinase '<
BKRI Regulatory subunit of Not determined AJ304829 GcnBank cntry Ö
'0
cAMP-dependcnt '"fT0
protcin kin ase (JQ
Botrytis cinerea BMPl MAP kin ase Loss of pathogenicity AF205375 Zhcng ct al. (2000) (1)
:=
Colletotrichum CLKl Serinc/threonine Loss of pathogenicity AFOO0309 Dufresne et al. (1998) Ci·
lindemuthianum protein kin ase 'Ti
t::
Colletotrichum trifolii TB3 Protein kinase similar Mutants not available U14989 Buhr et al. (1996) :=
'-(9.
to N.crassa Cot!
Colletotrichum CMK Calmodulin-dependent Mutants not available AF034963 Kim et al. (1998)
gloeosporioides protein kin ase

N
~
276 M. Bölker
for signal transduetion purposes in plant patho-
genie fungi. In the seeond part seleeted fungal
systems will be deseribed in more detail focusing
on those species in which the study of signaling
mechanisms is most advanced. Several reviews
that cover similar aspects or address certain
aspeets of pathogen-host communication have
appeared recently (Kronstad 1997; Kronstad et al.
1998; Kahmann et al. 1999; Borges-Walmsley and
Walmsley 2000; Lengeler et al. 2000).
11. Signal Transduction Pathways
Signals are physical or chemical stimuli that
induce a specific response by the cello Recognition
can occur both at the surface and inside the cello
The latter case applies not only for physical para-
meters such as temperature, but also for
hydrophobic chemical substances that can pass
the membranes, e.g., steroidal hormones. Whereas
temperature can influence the virulence of some
phytopathogenic bacteria by inducing tempera-
ture-responsive gene loci (Ullrich et al. 2000),
little is known about such regulation with respect
to fungal plant pathogens. This is in contrast to
human fungal pathogens such as Candida albicans
in which the dimorphic switch is controlled by
many factors among which temperature plays
an important role (Manning and Mitchell 1980).
Intracellular recognition of hydrophobie eom-
pounds that can pass the membrane has not yet
been addressed in fungi. This may be an interest-
ing field for future research in phytopathogenic
fungi because plants secrete a number of
flavonoids in response to wounding or to attract
symbionts. In root-nodulating bacteria, these sub-
stances bind directly to intracellular transcription
factors and induce the expression of nodulation
genes (for review, see Schultze and Kondorosi
1998).
A. Receptors
Most studies on fungal signaling pathways have
concentrated on receptors and cytoplasmic com-
ponents that transmit specific signals from the
membrane to the nucleus. Receptors are mem-
brane-bound proteins that are able to bind extra-
cellular ligands and to transmit this information
using signaling molecules such as G pro teins,
protein kinases, ion channels, etc. Binding of
ligands to their cognate receptors can induce not
only general responses like the induction of gene
expression, but also specific temporal and spatial
re arrangements of cytoskeletal elements that help
cells to respond to the direction of the incoming
signal, e.g., in orienting their growth along a con-
centration gradient. In general, receptors belong
to different families such as receptor tyrosine
kinases, G-protein-coupled receptors, membrane
channels or other membrane-bound proteins that
can recognize certain stimuli. It is worth mention-
ing that a mechanosensitive channel helps the
germlings of Uromyces appendiculatus, a rust
fungus, to find bean leaf stomata by recognizing
topographical information (Zhou et al. 1991). In
most phytopathogenic fungi only receptors that
are coupled to heterotrimeric G proteins have
been studied in any detail. These receptors exhibit
a highly conserved topological structure and
contain seven membrane-spanning domains with
the N-terminus of the protein placed outside of
the cell and the C-terminal end inside the cyto-
plasm (for review, see Strader et al. 1994). Upon
ligand bin ding these receptors activate het-
erotrimeric G proteins, which contact the cyto-
plasmic loops and the C-terminal end of the
protein. Conservation at the amino acid sequence
level is rather low within the large family of G-
protein-coupled receptors; only members of
subfamilies with related ligand specificity show
extensive sequence similarity.
B. Heterotrimeric G Proteins
G proteins consist of three subunits, the
GDP/GTP binding a subunit, and the ßly het-
erodimer. Upon receptor stimulation, exchange
of GDP to GTP is induced in the a subunit. This
leads to a conformational change resulting in dis-
sociation of the GTP-bound Ga subunit from the
ßlyheterodimer (Hamm and Gilchrist 1996). Sig-
naling by heterotrimerie G proteins in fungi has
been the subject of several reviews, one of which
appeared in this series (Borkovich 1996; Bölker
1998). The GTP-bound a subunit can stimulate
other components such as adenylyl cyclase or
ion channels. The a subunit has a high intrinsie
GTPase activity, which leads to rapid inaetivation.
This ensures that signaling is switched off imme-
diately when the receptor is no longer activated.
The catalytic activity of this intrinsic GTPase can
 Signal Transduction Pathways in Phytopathogenic Fungi
still be enhanced by RGS proteins (regulator of G
protein ~ignaling), which thus antagonize G
protein signaling (Koelle 1997). In Aspergillus
nidulans, the RGS protein FlbA acts as a negative
regulator of the FadA Ga subunit to block
mycelial proliferation and activate asexual sporu-
lation (Yu et al. 1996).
Several G protein a subunits have been iden-
tified in a variety of fungal species (Borkovich
1996; Bölker 1998). By sequence comparison and
susceptibility to modification by pertussis toxin,
the a subunits can be placed into different sub-
families, which correlate with different functions
such as stimulation or inhibition of adenylyl
cyclase activity. Although up to four different Ga
subunits have been isolated in a single species,
only very few ß and ysubunits have been charac-
terized so far. It is yet unknown whether in fungi
combinatorial interactions of different a, ß and y
generate a high diversity of heterotrimeric G pro-
teins similar to mammalian systems (Sirnon et al.
1991).
C. cAMP Signaling
Regulation via the intracellular level of cyclic
AMP (cAMP) is a common motif of cell signaling
in phytopathogenic fungi (Kronstad 1997; Borges-
Walmsley and Walmsley 2000). The secondary
messenger cAMP is produced from ATP by the
activity of adenylyl cyclase (see Fig.14.1). Adeny-
lyl cyclase can be positively and negatively regu-
lated through inter action with G protein a
subunits or Ras protein. Depending on the activ-
ity of both adenylyl cyclase and the cAMP-
degrading enzyme phosphodiesterase, increasing
or decreasing levels of cAMP serve as a trigger
for cellular processes like morphogenesis, sexual
development and differentiation. The cAMP
signal is transmitted by the cAMP-dependent
protein kinase A (PKA). PKA is a heteromeric
protein that consists of catalytic and regulatory
subunits. The binding of cAMP to the regulatory
subunits results in dissociation from the complex
and activation of the catalytic subunits (Fig.14.1).
Active PKA phosphorylates downstream targets,
e.g., transcription factors. In many fungi, nutri-
tional sensing has been found to occur via cAMP
signaling. In yeast, the phenotype of ras mutants
and extragenie suppressors implicated Ras pro-
teins with some function in regulating adenylyl
cyc1ase in response to nutrient limitation (Tatchell
277
~GUi d1 Adenylyl
cyclase
~
cAMP ATP
t]
J
n
cAMP . depen den t -- l:t~M!) ./ Regulatory
subunit
protem kmase A
Q)
(inactive)
~ Catalytic
subumt
(active)
LI\
~ c§V
Fig. 14.1. cAMP signaling. The membrane-associated
adenylyl cyclase is positively and negatively regulated by
stimulatory (Ga.) and inhibitory (Ga;) G protein a sub-
units. Adenylyl cyclase produces cyclic AMP (cAMP)
which serves as a secondary messenger. cAMP binds to the
regulatory subunits of the tetrameric cAMP-dependent
protein kinase A. Upon binding of cAMP the negatively
acting regulatory subunits dissociate and release the active
catalytic subunits. The cAMP signal is transmitted via
target proteins, which are activated by phosphorylation
et al. 1985). Recent experiments demonstrated
that glucose sensing involves a seven transmem-
brane receptor, Gpr1, which is coupled to the Ga
subunit Gpa2 (Yun et al. 1997; Xue et al. 1998;
Lorenz et al. 2000). Gpa2 is also involved in the
regulation of pseudohyphal growth and together
with Ras2 stimulates the activity of adenylyl
cyclase (Kübler et al. 1997; Lorenz and Heitman
1997). The study of pseudohyphal development in
yeast revealed a clear interconnection between
cAMP signaling and the MAP kinase pathway
(Mösch et al. 1999), which may be a common
theme of regulation in many fungal species (for
review, see Kronstad et al. 1998).
D. MAP Kinase Cascades
Mitogen-activated protein kinase (MAP kin ase )
has been detected in fungi as part of the
pheromone response pathway in S. cerevisiae (for
arecent review, see Elion 2000). Recognition of
the specific pheromone signal by the membrane-
bound receptor results in dissociation of the G
pro tein a subunit. The free ß/yheterodimer acti-
vates a MAP kinase cascade, which consists of
three pro tein kinases. MAP kinase phosphorylates
278 M. Bölker
dawnstream targets, same af which act as tran-
scriptian factors. The MAP kinase itself is acti-
vated by phosphorylation through MAPK kinase
(MAPKK), a dual specificity serine/threonine
tyrosine kinase. Phosphorylation of MAP kinase
takes place in the cytoplasm but the phosphory-
lated MAP kinase is shuttled to the nucleus
(Fig.14.2). At the top of this kinase cascade a
MAPKK kinase (MAPKKK) is placed, which
phosphorylates MAPKK. Several MAPK signal-
ing modules exist in yeast and are involved in
sensing high osmolarity, regulating cell-wall
biosynthesis in response to activation of the low-
osmolarity sensing pathway, and inducing pseudo-
hyphal growth in response to the nutrient status
(Herskowitz 1995; Banuett 1998). Interestingly,
~~2V @
~/
C
Scaffold
C
protein
r--------·
mRNA
Fig. 14.2. MAP kinase cascade. Signals can be transmitted
to the nucleus by the conserved MAPK cascade. Aseries
of kinase reactions results in phosphorylation of MAP
kinase, which is then shuttled to the nucleus where it can
activate transcription factors by phosphorylation. Speci-
ficity of parallel MAPK cascades is reached by the use of
scaffold proteins that tether the components of these trans-
duction cascades together. Known upstream effectors
include Ste20-like kinases and members of the protein
kinase C (PKC) family
subsets of these modular components are used by
different signaling pathways (Liu et al. 1993;
Roberts and Fink 1994). This poses the question,
how can the specificity of the response reactions
be maintained? As first observed in yeast, scaffold
proteins like Ste5p tether the kinases of a MAPK
module to prevent cross-talk with other signaling
modules (Fig.14.2; Printen and Sprague 1994;
Sprague 1998; Burack and Shaw 2000).
E. Ca2+/Calmodulin Signaling
Ca2+ is an important second messenger in many
systems. The regulatory functions of Ca2+ ions are
exerted mostly by the small cytoplasmic protein
calmodulin. Calmodulin has four characteristic
binding domains for calcium. On binding Ca2+,
calmodulin undergoes a major conformational
change that allows it to bind to calmodulin-
dependent enzymes, modifying their activity.
Two such enzymes are the calmodulin-
activated protein phosphatase, calcineurin, and
the calmodulin-dependent protein kinase
(CDPK). Because binding of Ca2+ to calmodulin is
co operative, small changes in cytoplasmic Ca2+
concentrations can "switch on" the active form of
calmodulin. Inhibitor studies have implied that
Ca2+ and calmodulin may funetion in dimorphie
switching and during infeetion processes of some
phytopathogenic fungi (Muthukumar and Nieker-
son 1984; Kim et al. 1998; Lee and Lee 1998). A
calmodulin kinase (CaMK) has been claned
from Colletotrichum gloeosporioides where it is
involved in germination and appressorium forma-
tion (Kim et al. 1998).
III. Model Organisms
In recent years, many phytopathogenic fungi have
been analyzed for genetic and bioehemieal pro-
cesses involved in the infcetion proeess. Many
of these studies are aimed at identifying poten-
tial targets for the development of fungicides to
control these economically important plant dis-
eases. Among the fungal plant pathogens, two
species, Magnaporthe grisea and Ustilago maydis,
have reached the status of model organisms
because both have been studied extensively and
both are very amenable to genetic analysis
(Hamer and Talbot 1998; Bölker 2001). Whole
 Signal Transduction Pathways in Phytopathogenic Fungi
genome sequencing projects are underway for
both species that will allow parallel analysis of
expression levels of thousands of genes during dif-
ferent stages of host-pathogen interactions in the
near future. This will greatly enhance our under-
standing of fungal virulence.
A. Magnaporthe grisea
The heterothallic ascomycete M. grisea causes the
economieally important rice blast disease and can
infect many other grass species. During the initial
phase of infection on the surface of the plant leaf,
asexual spores form germ tubes, whieh differen-
tiate at their tips into a specialized structure,
the appressorium. Appressoria serve as hydrome-
chanical devices to penetrate the epidermal cuticle
of the plant leaf using high pressure (for review,
see Deising et al. 2000). Within the appressorium,
osmotic pressure is built up by accumulation of
high concentrations of solutes (Howard et al.
1991). After penetration, the fungal cells prolifer-
ate within the plant tissue and cause disease
lesions. Propagation of disease occurs by asexual
sporulation at aerial conidiophores. During early
and late stages of development, signaling pro-
cesses play important roles (Hamer and Talbot
1998). The signal transduction pathways involved
in the regulation of morphogenesis and pathogen-
related development have been analyzed in great
detail.
The formation of appressoria is specifically
induced on the surface of plant leaves. Chemie al
and physical signals have been implicated to reg-
ulate infection-related development in M. grisea.
The activity of fungal cutinase results in the
release of cutin monomers, which serve as chemi-
cal signals to induce appressorium formation in M.
grisea (Gilbert et al. 1996). It is presently unclear
by which receptors this chemical cue is perceived.
The leaf surface can be partially mimicked by
other hard, hydrophobie materials like poly-
styrene (Lee and Dean 1994). Recognition of
the hydrophobie surface might involve a fungal
hydrophobin encoded by the MPGl gene (Talbot
et al. 1993; Talbot et al. 1996). Deletion of MPGl
results in decreased pathogenicity due to reduced
appressorium formation. Hydrophobins are
amphipathic moleeules which are able to self-
assemble and to cover the hydrophobie surface of
plant leaves with a hydrophilie layer of polymer-
ized protein. This function has been proposed to
279
be an important factor in conditioning appresso-
rium formation (Kershaw and Talbot 1998).
A first hint for the critical role of cAMP sig-
naling during the early development of M. grisea
came from the observation that addition of exoge-
nous cAMP stimulates appressorium formation
on noninductive surfaces (Lee and Dean 1993).
Mpg1 appears to act upstream of cAMP signal-
ing because addition of exogenous cAMP can
induce appressoria formation in mpgl mutants
(Beckerman and Ebbole 1996; Talbot et al. 1996).
To study the function of cAMP signaling, the gene
encoding adenylyl cyclase (MACl) was cloned
and deletion mutants were generated by gene
re pI ace me nt (Choi and Dean 1997). The loss of
adenylyl cyclase results in a pleiotropic pheno-
type. Mutants are unable to develop appressoria
on inductive surfaces and are nonpathogenic on
susceptible rice leaves. In addition, mac1 mutants
have a sterility phenotype and show reduced veg-
etative growth and conidiation (Choi and Dean
1997). Activation of adenylyl cyclase in response
to the recognition of surface cues is likely to occur
by a heterotrimerie G protein. In M. grisea, three
genes encoding Ga subunits, MAGA, MAGB and
MAGC, have been identified (Liu and Dean
1997). Disruption of MAGB caused pleiotropic
effects and results in reduced appressorium for-
mation, virulence, and vegetative growth, indieat-
ing the importance of G-protein signaling in
regulating cellular activities. Addition of exoge-
nous cAMP restores appressorium formation in
magB mutants implying that this Ga subunit is
critical for stimulation of adenylyl cyclase. This
was surprising because MagB contains some fea-
tures characteristie for members of the mam-
malian GlXj family that are known to inhibit
adenylyl cyclase activity (Liu and Dean 1997). To
dissect the various functions of the MagB pro tein,
dominant active and dominant negative mutants
of this G protein a subunit were generated by
site-directed mutagenesis and introduced into
M. grisea. Expression of a constitutively active G
protein a subunit allowed appressorium forma-
tion on both hydrophobie and hydrophilie sur-
faces. In addition, autolysis of aged colonies and
reduced sexual and asexual reproduction were
observed (Fang and Dean 2000). The phenotype
of a mutant version of MagB, whieh is unable to
dissociate from the ß/rheterodimer, implicated a
function for the free ß/r heterodimer during G-
protein signaling in M. grisea (Fang and Dean
2000).
280 M. Bölkcr
To identify further components of the cAMP-
signaling pathway, the CPKA gene encoding the
catalytic subunit of cAMP-dependent protein
kinase A (PKA) was c10ned (Mitchell and Dean
1995; Xu et al. 1997; Sweigard et al. 1998). cpkA
mutants exhibit a delay in appressorium formation
and are dramatically reduced in pathogenicity (Xu
et al. 1997). Interestingly, cpkA mutants are still
responsive to exogenous cAMP indicating the
existence of an additional catalytic subunit of
PKA. The isolation of suppressor mutants, which
rescue the defects of cpkA mutants, leads to the
identification of the regulatory subunit of cAMP-
dependent pro tein kinase (Adachi and Hamer
1998).
A number of MAP kinases have been identi-
fied in M. grisea by degenerate peR screens. The
MAP kin ase Pmk1 is required for pathogenicity
and appressorium formation (Xu and Hamer
1996). !J.pmkl mutants are not complemented by
exogenous cAMP but undergo early hooking
stages of appressorium development in its pres-
ence. Monitoring the movement of vesic1es during
appressorium formation revealed that Pmk1 is
required for mass transfer of storage carbohy-
drates and lipids to the appressorium. The
generation of turgor, which occurs by compart-
mentalization and rapid degradation of lipid and
glycogen reserves, appears to be under control of
the cAMP-dependent pro tein kinase A (Thines
et al. 2000). OSM1, encoding a homologue of
the yeast Hog1 MAP kinase, which regulates in
yeast the response to hyperosmotic stress, has
been identified in M. grisea (Dixon et al. 1999).
Although OSMl triggers arabitol accumulation in
the mycelium in response to hyperosmotic stress,
it does not appear to playa role during appresso-
rium formation. osml mutants do form normal
appressoria and are fully pathogenic on rice leaves
(Dixon et al. 1999). Another MAP kin ase, Mps1,
is involved in maintaining cell-wall integrity (Xu
et al. 1998). This pathway has been particularly
weIl studied in the yeast S. cerevisiae, in which >20
cell-wall maintenance genes have been identified
(for review, see de Nobel et al. 2000). M. grisea
mpsl mutants are female sterile and cannot form
functional appressoria. Although mpsl mutants
cannot penetrate the plant surface they still induce
plant-defense reactions if applied direct1y into the
leaf tissue (Xu et al. 1998).
The variety of signaling components involved
in pathogenic development in M. grisea poses the
question at which levels a cross-talk between these
signal transduction pathways occurs and how the
specificity of the response to different environ-
mental stimuli is reached? To identify further com-
ponents required for virulence, which may act
up- or downstream of the known signaling path-
ways, a screen for nonpathogenic mutants through
insertional mutagenesis by restriction enzyme
mediated integration (REMI) has been success-
fully established (Sweigard et al. 1998). Several
pth mutants that showed a reproducible patho-
genicity defect were isolated. Two of these genes
are supposed to have a regulatory function. PTHl
encodes a protein with similarity to yeast Grr1,
which is involved in glucose repression. PTH4
codes for the catalytic subunit of cAMP-depen-
dent protein kinase A (CpkA), which was also
identified by alternative approaches (see above).
A potential membrane-bound receptor protein,
Pthll, was identified and has been suggested to
function at the cell cortex as an upstream effector
of appressorium differentiation in response to
surface cues (DeZwaan et al. 1999).
With the complete genome sequence of M.
grisea being accessible in the near future, the
advent of postgenomic techniques will dramati-
cally increase our knowledge of the dynamics of
gene expression, in particular during pathogene-
sis. The parallel screening of many genes with the
help of microarray techniques will allow a precise
determination of stage-specific gene expression.
The differences in expression patterns in mutants
affected in specific signaling pathways will help to
define the regulatory function of these signaling
processes during pathogenic development.
B. Ustilago maydis
The heterobasidomycete Ustilago maydis is the
causative agent of corn smut disease and infects
maize (Zea mays ssp. mays) and its progenitor
plant teosinte (Zea mays ssp. parviglumis). U
maydis is highly amenable to genetic analysis and
thus serves as one of the model systems to study
the interaction between phytopathogenic fungi
and their hosts (Bölker 2001). Two privately
funded genome projects on U maydis have been
finished recently emphasizing the importance of
this model organism. Upon infection, large gaUs or
tumors are induced on all green parts of the host
plant. Within the plant gaUs, proliferation occurs
and, at the end of infection, masses of black
teliospores are produced (Christensen 1963).
 Signal Transduction Pathways in Phytopathogenic Fungi
Upon germination, these diploid spores undergo
meiosis. U maydis has a dimorphic lifestyle,
haploid sporidia grow vegetatively by budding and
are saprophytic. Cells of compatible mating type
can fuse to form a stable dikaryon, which grows as
a filament. Only in this stage is the fungus able to
infect maize plants (for review, see Banuett 1995).
Cell fusion and pathogenicity are controlled
by two genetically unlinked mating-type loci. The
contributions of the a and b mating-type loci to 61-
amentous growth and pathogenicity have been
dissected by molecular genetic analysis. Cell
fusion is solely triggered by the a-Iocus, whereas
pathogenesis is under the control of the b-Iocus.
Filamentous growth, however, requires different
alleles at both the a-and the b-locus. The a-Iocus
exists in two alleles, al and a2, and codes for a
pheromone-based cell recognition system. The
mating-type specific region contains the structural
genes for precursors of farnesylated lipopeptide
mating factors, mfal and mfa2, and their cognate
receptors, pral and pra2 (Bölker et al. 1992;
Spellig et al. 1994). The multiallelic b-Iocus
encodes a pair of transcription factors of the
homeodomain protein family (Kronstad and
Leong 1990; Schulz et al. 1990; Gillissen et al.
1992). After cell fusion, the bW and bE pro teins
can form heterodimers only if they are derived
from different alleles (Kämper et al. 1995). It is
assumed that this heterodimeric complex acts as a
regulator of filamentous growth and pathogenic-
ity by regulating the expression of a specific set of
genes. This has allowed the construction of a
haploid strain that carries a chimeric b-allele. This
strain can be used to study directly the effect of
mutations on the virulence of the fungus (Bölker
et aI. 1995). Recently, a direct target sequence for
the bW/bE heterodimer has been identified within
the promoter region of a gene of unknown func-
tion in the a2 allele (Romeis et al. 2000). This
binding motif may help to identify other binding
sites for the bW/bE heterodimer, which may be in
the vicinity of other genes required for pathogenic
development. In a screen for mutants that bypass
the requirement for a functional bW/bE het-
erodimer, the ruml gene has been isolated, which
encodes a protein with high similarity to a human
retinoblastoma binding protein (Quadbeck-
Seeger et al. 2000). Since its human homologue is
known to interact with histone deacetylases, it has
been proposed that alterations in the chromatin
structure may be involved during the induction of
b-dependent development.
281
Signal transduction plays an eminent role in
the mating reaction and during the pathogenic
stage of U maydis (Kahmann et al. 1999). Recog-
nition of haploid cells of opposite a mating type
occurs by specific bin ding of secreted pheromones
to their cognate receptors. The pheromone recep-
tors belong to the family of G-protein coupled
seven transmembrane receptors (Bölker et al.
1992). Upon pheromone stimulation, cells stop
budding and form mating tubes that grow towards
each other and eventually fuse at their tips
(Snetselaar 1993; Spellig et al. 1994; Snetselaar et
al. 1996). Pheromone stimulation induces the
expression of several genes which all carry char-
acteristic DNA elements upstream of their pro-
moter region. These short pheromone response
elements are necessary and sufficient to confer
pheromone induction (Urban et al. 1996). Among
the genes regulated by pheromone induction are
the pheromone and the receptor genes them-
selves. This positive feedback results in strong
expression of these genes during the mating
process. After fusion of compatible cells, continu-
ous autocrine stimulation of the pheromone
receptors is observed albeit at a lower level than
during paracrine stimulation of unfused cells
(Urban et al. 1996). The pheromone-dependent
regulation of gene expression is media ted by a
central transcription factor, Prfl. Prfl belongs to
the family of HMG box pro teins and is able to
bind to the pheromone-response elements (Hart-
mann et al. 1996). The basal level of expression of
the pheromone and receptor genes is strongly
influenced by the nutritional status of the cells.
This was supported by the observation that
expression of Prfl itself is und er complex envi-
ronmental control (Hartmann et al. 1999). Tran-
scriptional activation of prfl was observed in the
presence of carbon sources, such as glucose and
fructose, and could be attributed to a cis-acting
element in the prfl promoter that mediates these
effects. The same element provides for negative
control of prfl gene transcription at high cAMP
levels (Hartmann et al. 1999). Within the amino
acid sequence of Prfl several consensus sites for
phosphorylation by MAP kinase and cAMP-
dependent kinase have been identified which are
essential for function (Hartmann et al. 1999).
Deletion of prfl results not only in sterility, but
also in loss of pathogenicity in a virulent haploid
strain that carries a chimeric b-allele (Hartmann
et al. 1996). Pathogenic development can be
restored by constitutive expression of the b genes,
282 M. Bölker
indicating that induction of these genes is the sole
function of Prf1, which is required for patho-
genicity (Hartmann et al. 1996).
It is still unclear which heterotrimeric G
pro tein is coupled to the pheromone receptors.
Four different genes encoding Ga subunits have
been cloned from U maydis. Only disruption of
gpa3 interfered with the mating re action suggest-
ing that Gpa3 transmits the pheromone signal
from the pheromone receptor (Regenfelder et al.
1997). By its amino acid sequence, Gpa3 belongs
to the subgroup of Ga subunits which stimulate
adenylyl cyclase (Bölker 1998). Accordingly, the
phenotype of gpa3 mutant cells resembles that of
uac1 mutants, which lack adenylyl cyclase (see
below and Gold et al. 1994). These strains have
been identified in a screen for haploid cells that
show bE/bW-independent filamentous growth
(Gold et al. 1994). The morphological phenotype
of both gpa3 and uac1 mutants can be rescued by
addition of cAMP to the growth medium (Gold et
al. 1994; Krüger et al. 1998). This points to an
interesting cross-talk between the pheromone-
response pathway and cAMP signaling which is
normally involved in nutritional sensing and envi-
ronmental control. It is difficult to establish
whether Gpa3 is functioning primarily as part of a
nutritional sensing pathway regulating the expres-
sion level of the pheromone genes or whether it
couples directly to the pheromone receptor. It is
still feasible that Gpa3 may be involved in multi-
ple signaling pathways. This would imply that the
pheromone response could be the result of paral-
lel activation of cAMP signaling through the Ga
subunit Gpa3 and stimulation of a MAP kin ase
cascade, probably by the free ß/ydimer. Introduc-
tion of a constitutive active allele of gpa3, in which
a single amino acid exchange abolishes the intrin-
sic GTPase activity, induces a pleiotropic pheno-
type which is characterized by multiple budding
and reduced fungal proliferation during the in-
fection (Krüger et al. 2000). This indicates that
Gpa3 is probably involved in yet unknown signal-
ing pathways during later stages of pathogenic
development.
The importance of cAMP signaling for mor-
phogenesis and virulence was further emphasized
by the analysis of mutants that grow in a filamen-
tous manner in the haploid stage. Mutations in the
gene for adenylyl cyclase (uac1) result in consti-
tutive filamentous growth and loss of pathogenic-
ity (Barrett et al. 1993; Gold et al. 1994). Addition
of exogenous cAMP res tores anormal budding
pattern. In an extensive search for suppressors of
the uac1 phenotype, several genes have been iden-
tified whose products are involved in either cAMP
signaling or components of a MAPK module,
pointing to interconnections between these two
signaling pathways (Kronstad et al. 1998). The
suppressor gene ubc1 encodes the regulatory
subunit of the cAMP-dependent protein kinase A
(Gold et al. 1994). This subunit inhibits the kinase
activity unless it binds cAMP and dissociates from
the complex. Whereas uac1 and ubc1 double
mutants look like wild-type cells, ubc1 in the
genetic background of wild-type cells results in a
cytokinesis defect with a multiple budding pheno-
type (Gold et al. 1994).
Two catalytic subunits of PKA have been
identified in U. maydis. The adrl gene was first
isola ted as being responsible for conferring resis-
tance against the dicarboximide fungicide vinclo-
zolin (Orth et al. 1995) and ukal was cloned by
sequence similarity (Dürrenberger et al. 1998).
When mutants for these genes were tested for
their phenotype, only adrl mutants were impaired
in virulence and showed filamentous growth in
haploid cells. Interestingly, additional suppressors
of adenylyl cyclase mutants were found to encode
putative members of a MAP kinase cascade point-
ing to a strong interconnection between these two
signaling machineries (Mayorga and Gold 1998,
1999; Andrews et al. 2000). Some of these kinases
were identified independently by degenerate
peR screens as part of the pheromone-response
pathway (Banuett and Herskowitz 1994; Müller et
al. 1999). The MAP kinase kinase Fuz7 was the
first member of a MAP kinase cascade identified
in U maydis. fuz7 mutants fail to form conjuga-
tion tubes in response to mating pheromone
(Banuett and Herskowitz 1994). Although Fuz7
was presumed to transmit the pheromone signal,
induction of mfa expression in response to
pheromone is not affected (Regenfelder et al.
1997). This could be explained if the pheromone
signal is, at least partially, transmitted by a cAMP-
dependent signaling pathway. It has been sug-
gested that the MAP kin ase Kpp2/Ubc3 is directly
involved in phosphorylation of the transcription
factor Prfl, which is responsible for pheromone-
dependent gene expression. Since the protein
sequence Prf1 contains consensus sites for both
MAP kinase and PKA-specific phosphorylation,
cross-talk between these two pathways may occur
at the level of Prfl phosphorylation. It is, however,
unclear which function the MAP kinase cascade
 Signal Transduction Pathways in Phytopathogenic Fungi
has for the induction of filamentous growth
in adenylyl cyclase deficient mutants. In these
mutants, MAP kinase signaling is apparently
required for the dimorphie switch from budding to
filamentous growth, because mutations that in ac-
tivate members of the MAP kin ase pathway result
in suppression of filamentous growth (Mayorga
and Gold 1998; Mayorga and Gold 1999; Andrews
et a1. 2000). One of the challenges for the future
will be to identify the cross-talk between these two
important signaling pathways. The large number
of genes which have been already identified in U.
maydis and the vast number of genes which can
now be extracted from the genome sequence are
an invaluable resource for the molecular dis sec-
tion of signal exchange( s) during the infection
process.
IV. Other Phytopathogenic Fungi
Besides the well-studied model organisms men-
tioned above, many other phytopathogenic fungi
are the subject of intensive investigations, since
they cause economically important diseases. It is
hoped that the elucidation of signaling pathways
will support the development of potential targets
for novel fungicides. In addition, the apparent
diversity of signaling cascades and regulatory net-
works even in highly related fungi makes it nec-
essary to study as many different species.
A. Cryphonectria parasitica
The ascomycete Cryphonectria parasitica infects
chestnut trees and has destroyed the once domi-
nant chestnut tree population in eastern North
America in the last century. The interest in signal
transduction in C. parasitica has been stimulated
by observed alterations of the fungal phenotype
that result on infection by virulence-atteuuating
hypoviruses (for review, see Nuss 1996). This
double-stranded RNA virus reduces drastically
the virulence of the fungus and its high prevalence
in Europe has prevented a similar spread of chest-
nut blight in the Old World. The different suscep-
tibility of European and American strains to viral
infection may be related to their vegetative incom-
patibility systems (Milgroom and Cortesi 1999),
which allowed in the Old World a much easier
spreading of the virus through fungal anasto-
283
moses. Since the hypovirus-mediated attenuation
of virulence can be regarded as a model for natu-
rally occurring biocontrol, the understanding of its
underlying mechanism has attracted special inter-
est (Nuss 1992). Beside a marked reduction in vir-
ulence, called hypovirulence, viral infection is
associated with phenotypic alterations such as
reduced orange pigmentation and sporulation. It
has been demonstrated that expression of viral
cDNA is sufficient to induce the observed effects
(Choi and Nuss 1992a,b). It is still unknown by
which mechanism the virus causes these symptoms
but it has been shown that expression of certain
fungal mRNA species is repressed in virus con-
taining strains of C. parasitica (Powell and Van
Alfen 1987; Kazmierczak et a1. 1996).
In particular, accumulation of CPG-1, the a
subunit of a heterotrimeric G protein, is signifi-
cantly reduced in hypovirus-infected strains (Choi
et a1. 1995). To find out whether the drastic reduc-
tion of the Ga subunit is responsible for the virus-
induced hypovirulence, expression of CPG-l was
abolished by transgenic suppression (Choi et a1.
1995). Although not all of the phenotypes associ-
ated with hypovirulence could be mimicked by Ga
suppression, these results confirm the notion that
hypovirus infections fundamentally alter signal
transduction processes in the fungal host. Subse-
quently, additional genes encoding Ga subunits
were cloned and strains carrying targeted disrup-
tions of these genes were tested for virulence and
other characteristic phenotypes. Whereas dis-
ruption of cpg-l resulted in a set of phenotypic
changes similar to, but more severe than, those
induced by virus infection, disruption of cpg-2
resulted only in slight reductions in growth rate
(Gao and Nuss 1996). Using mRNA differential
display it was observed that largely similar
changes in gene expression pattern resulted from
CPG-l co suppression and virus infection. It was
also reported that both virus infection and CPG-
1 cosuppression elevate cAMP levels three- to
fivefold. These results suggest that, similar to
mammalian G~ subunits, CPG-1 functions as a
negative modulator of adenylyl cyclase (Chen et
a1. 1996). Among the genes regulated by the
Cpg-1 pathway a potential virulence gene, cbh-l,
encoding the plant cell wall degrading enzyme
cellobiohydrolase, was found (Chen et a1. 1996).
Cloning and disruption of the C. parasitica gene
CPGB-1 encoding a G protein ß subunit was
reported. The ß subunit obviously plays a positive
role in signaling since strains carrying a cpgb-l
284 M. Bölker
deletion exhibit a similar phenotype to cpg-l-
disrupted strains (Kasahara and Nuss 1997). Dis-
ruption of a gene, bdm-l, containing significant
similarity to mammalian phosducin resulted in a
phenotype indistinguishable from that previously
observed after disruption of the Gß subunit gene,
cpgb-l (Kasahara et al. 2000). Since phosducin is
implicated in binding to the G ß/y heterodimer,
these data suggest that BDM-1 is required in C.
parasitica for G ß/yfunction. Moreover, disruption
of either BDM-l or CGPB-l resulted in a signifi-
cant reduction in the accumulation of CPG-1
(Kasahara et al. 2000). These results strengthen
and extend the view that hypovirus infection
causes a significant and persistent alteration of
fungal gene expression media ted by G-protein-
regulated cAMP accumulation.
B. Erysiphe graminis
Erysiphe graminis f. sp. hordei is the causal agent
of barley powdery mildew. The obligate biotrophic
fungus produces asexual spores, conidia, which
infect barley leaves. On leaf surfaces, the conidia
germinate and form primary germ tubes. This
is followed by the emergence of an additional
appressorial germ tube from the spore. The
appressorial hypha penetrates the leaf surface.
This developmental program is triggered by
contact to the host surface (Carver and Ingerson
1987). Apparently, the fungus recognizes different
signals since neither hydrophobicity of the surface
alone nor the presence of cuticle monomers is
sufficient to induce the full response (Carver et
al. 1996). Recent experiments implied a role for
cAMP signaling dnring conidiaI differentiation
(Hall et al. 1999; Kinane et al. 2000). A cDNA en-
coding the catalytic subunit of cAMP-dependent
pro tein kin ase (PKA) has been cloned and PKA
activity was detected during conidial differentia-
tion (Hall et al. 1999). The intracellular cAMP
level increases before germ tube emergence. The
application of exogenous cAMP at a very low con-
centration (l.uM) induced germ tube formation
on moderately inductive cellulose membranes but
not on noninductive surfaces. High amounts of
cAMP resulted in inhibition of conidial differen-
tiation (Hall et al. 1999). Further experiments
demonstrated a differential role of cAMP on germ
tube initiation and appressorium formation.
Whereas cAMP and PKA appear to control germ
tube emergence, they had no effect on appressor-
ial formation (Kinane et al. 2000). The complex
pattern of cAMP-dependent signaling in this
organism may reflect the high specificity of host
recognition in this organism. The re cent establish-
ment of a stable transformation system for this
obligate biotrophic fungus (Chaure et al. 2000)
may help to elucidate the molecular organization
of cAMP signaling and its cross-talk with other
signal transduction pathways.
C. Botrytis cinerea
Botrytis cinerea is a necrotrophic plant pathogen
which exhibits an unusual broad host range. It
causes the grey mold disease on many economi-
cally important plants. The mechanisms by which
B. cinerea penetrates its host and kills the sur-
rounding cells are largely unknown. To test
whether signaling in B.cinerea is important for the
establishment on its host, the gene for a MAP
kinase has been cloned and disrupted (Zheng et
al. 2000). The amino acid sequence of the Bmp1
pro tein is highly similar to the corresponding
Pmk1 protein from M. grisea. Deletion of the
BMPl gene did not lead to any reduction in
conidia germination but affected significantly the
vegetative growth rate of the mutant (Zheng et al.
2000). Interestingly, the bmpl mutants were non-
pathogenic and induced no lesions on detached
flower petals, even after prolonged incubation.
Closer inspection of the nonpathogenic mutants
by scanning electron microscopy revealed that the
mutant conidia germinated but the germ tubes
grew on the plant surface without any penetration
(Zheng et al. 2000). Although the precise function
of the MAP kinase Bmp1 during infection is still
unknown, it is speculated that Bmp1 regulates the
formation of appressoria-like structure and/or
the production of elicitors or toxic substances
required for penetration and cell death. In B.
cinerea, genes can be very efficiently replaced by
homologous recombination (Levis et al. 1997).
This feature makes B. cinerea a very attractive
system to study the contribution of diverse sig-
naling compounds for pathogenic development of
this phytopathogenic fungus with an exceptional
broad host range.
D. Colletotrichum spp.
Several species of the fungal genus Colletotrichum
cause anthracnose disease on a wide range of eco-
nomically important plant species. Infection starts
 Signal Transduction Pathways in Phytopathogenic Fungi
with germination of spores and formation of a
melanized appressorium on the plant surfaee.
After penetration, infeetious hyphae grow and
extend inside the plant eells. During early stages
of infeetion, the fungus exhibits a hemi-biotrophie
lifestyle in whieh the host eells remain viable. This
is followed by tissue eolonization and host eell
death resulting in water-soaked lesions. Col-
letotrichum has been used to study the induetion
of appressorium formation by eontaet to hard sur-
faees (Kolattukudy et al. 1995). Chemie al sub-
stanees are also involved in triggering appressorial
development (Parberry and Blakeman 1978).
Several approaehes have been used to elucidate
the underlying moleeular meehanism of these
reeognition processes (for review, see Perfeet et al.
1999). By differential display of mRNA, three
genes were identified that are expressed early on
hard surfaees (Liu and Kolattukudy 1998). One
of these genes, CHIPI, eneodes an ubiquitin-
eonjugating enzyme (Liu and Kolattukudy 1998).
The CHIP2 and CHIP3 genes eneode proteins
with no homology to any other known proteins
and ean be disrupted without eausing a signifieant
deerease in appressorium formation or virulenee
(Kim et al. 2000). A serine/threonine protein
kinase, Clk1, whieh is required for pathogenicity
in C. lindemuthianum, was identified by inser-
tional mutagenesis (Dufresne et al. 1998). Restric-
tion enzyme-mediated integration was used to
identify mutants with unpigmented spores or
weakened eell walls (Epstein et al. 1998). In C. tri-
folii, another protein kinase, TB3, was isolated,
whieh closely resembles the Neurospora crassa
serine/threonine pro tein kinase, Cot -1, required
for hyphal elongation and branehing. TB3 is
expressed during pathogenie development and is
able to eomplement the cot-I mutant of Neu-
rospora crassa, demonstrating the funetional eon-
servation of this kinase between a pathogenic
and a saprophytic fungus. (Buhr et al. 1996). The
Colletotrichum system will be interesting for the
identification of signals that are involved in
the transition from biotrophic to necrotrophic
development during infection.
v. Conclusions
The study of signal transduction pathways in
fungal plant pathogens has opened new avenues
to understanding the intricate networks of eOffi-
munications between fungi and their hosts. The
285
diversity of life styles and infection strategies is
reftected in specific adaptations to recognize the
speeific hosts. However, common themes of signal
recognition and transduction emerge from the
comparison of different species. Genome projects
are underway for an increasing number of phy-
topathogenic fungi. The use of genomic and
postgenomic technologies will allow the parallel
analysis of thousands of genes. This may lead to
the determination of global regulatory patterns
involved in fungal development and virulence. In
addition, the identification of fungal genes and
proteins, which are erueial for essential functions
during pathogenesis, may help to develop novel
strategies to protect erop production from losses
due to fungal infections.
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15 Avirulence Determinants and Elicitors
WOLFGANG KNOGGE
CONTENTS
I. Introduction .........................
11. Considerations on the Evolution
of Pathogen Recognition ...............
III. General Elicitors . . . . . . . . . . . . . . . . . . . . . .
IV. Elicitors with Restricted Specificity .......
A. Glucan Elicitors ......................
B. The Elicitins of Oomycete spp. . . . . . . . . . . .
C. Other Elicitors from Phytophthora spp. ....
D. Products of the Species-Specific PWL
Genes of Magnaporthe grisea . . . . . . . . . . . .
V. Avirulence Gene Products ..............
A. AVRs and ECPs of Cladosporium
fulvum·. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
B. NIP1 from Rhynchosporium secalis .......
C. AVR-Pita from Magnaporthe grisea . . . . . . .
D. Novel Avirulence Genes . . . . . . . . . . . . . . . .
VI. Perception of Avirulence Signals .........
VII. Recognition-Based Enhancement
of Plant Disease Resistance .............
VIII. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . .
References ..........................
I. Introduction
Being surrounded by putatively hostile micro-
organisms, but immobile and hence unable to
escape, plants constantly need to be prepared for
defensive battle. During their coevolution with
heterotrophie parasites they have therefore
acquired efficient passive, preformed barriers that
provide protection against the majority of aggres-
sors. In addition, however, plants have an arsenal
of offensive weapons for counterattack at their
disposal once the passive bulwark has failed.
Usually, this weaponry is launched rapidly and
decisively, thus negating any further progression
of the invader in order to maintain the plant's
structural and functional integrity.
Department of Plant Science, Adelaide University, Gien
Osmond, SA 5064, Australia
In contrast to bacteria and viruses, fungi and
289 Oomycetes have the capacity to actively penetrate
plant tissues. However, of all the known fungal
290 species, those that have worked out the means
291 to break through or bypass the plant defensive
292
292 barriers and to avoid, to delay or to suppress the
293 plant counterattack or to cope with its defensive
296 weapons have remained a tiny minority. Colo-
296
nization of host tissues usually leads to plant
298 disease. While the conqueror does not necessarily
kill the entire plant, it forces it to deliver supplies
298 for its own well-being and propagation, thus
299
300
weakening the plant's viability and endangering
300 its reproductive capacity. Furthermore, if the plant
300 happens to be a crop plant, there is an additional
consequence; it can no longer fulfill its function to
302
304 yield food for man and income for the farmer.
304 The erucial proeess for the plant active
defence response is reeognition, the capacity to
detect the presence of an invader as early as pos-
sible to be able to effectively launch the counter-
attack. To this end, plants have evolved an efficient
surveillance system as well as the communica-
tion lines that transfer the information from the
reconnaissance units at the cell periphery to
the command-and-control unit, the cell nucleus.
The last decades have seen the identification and
characterization of a vast variety of plant defence
reactions that has been summarized in several
reviews (Kombrink and Somssich 1995, 1997;
Rushton and Somssich 1999). In addition, in
recent years, a number of the factors involved in
reconnaissance and signaling have been identified
and their role in plant defence discussed (Ryals et
al. 1995; Hammond-Kosack et al. 1996; Low and
Merida 1996; Durner et al. 1997; Yang et al. 1997;
Alvarez et al. 1998; Ebel and Mithöfer 1998;
Higgins et al. 1998; Hutcheson 1998; Innes
1998; Scheel 1998; Somssich and Hahlbrock
1998; Maleck and Dietrich 1999; Martin 1999;
Nürnberger 1999; Piffanelli et al. 1999; McDowell
and DangI2000). These factors include around 20
resistance genes from various plants that recog-
The Mycota XI
Agricultural Applications
Kempken (Ed.)
© Springer-Verlag Berlin Heidelberg 2002
290 W. Knogge
nize different classes of pathogens (Jones 1996;
Meyers et al. 1999; Richter and Ronald 2000). Fur-
thermore, several signal molecules of fungal origin
were identified and characterized that, upon inter-
action with the plant, elicit defence reactions
(Darvill and Albersheim 1984; Rieci et al. 1993;
Ebel and eosio 1994; Nürnberger and Nennstiel
1998). Their number has remained low, however,
and the primary recognition events are still
obscure. This chapter focuses on the structure and
possible role of different types of elicitors in deter-
mining the specificity of the interaction between
the producing microbes and their plant counter-
parts. In addition, recognition-based strategies to
engineer improved plant disease resistance are
briefly outlined.
11. Considerations on the Evolution
of Pathogen Recognition
It can be assumed that the autotrophie plants
acquired resistance very early in their coevolution
with heterotrophic parasites (Heath 1991a). As a
consequence, all extant plants exhibit parasite
nonspecific, basic resistance to the vast majority of
putative pathogens (Heath 1981). This nonhost
resistance is based on a variety of passive and
active defence mechanisms. Some of these mech-
anisms appear to be similar in all plants while
others are diverse in different plant species. In
general, however, in an individual plant, this resis-
tance is innate, i.e., inherited through the germ-
line, and not adaptive. The primary goal of this
"innate immunity" system is to detect molecules
that are unique to infectious agents and that allow
discrimination between potentially noxious
("non-self") and innocuous agents. For this
purpose the plants evolved abasie, nonspecific
recognitionßystem for microbial signatures that
differ from eukaryotie and, in particular, from self
features. Mierobial molecules recognized through
this system may be typieal cell-wall or membrane
components or secreted molecules that define
large groups of putative pathogens. A different
concept of recognition involves "self" molecules
such as fragments released from the plant cell wall
by the activity of pathogen enzymes (Doares et al.
1995; Knogge 1997). Thereby, the plant be comes
aware of a localloss of its structural integrity. This
type of recognition therefore may have some sim-
ilarity with the perception of wounding (Bowles
1998) and indeed several plant genes are activated
in response to pathogen attack as well as to
wounding (Durrant et al. 2000). It has been
pointed out, however, that these types of general
elicitor molecules may provide the basic informa-
tion of a putative threat and that plants probably
need to perceive additional signals that allow
them to discriminate between different kinds of
stress such as wounding and pathogen attack or
between pathogens and symbionts such as mycor-
rhizal fungi (Boller 1995). As a consequence,
plants are able to adjust their response according
to the nature of the incoming signals.
To successfully parasitize a plant, a fungal
pathogen needs to evolve pathogenicity factors
that enable it not only to actively penetrate plant
tissues, but also to ne gate the basic resistance
mechanisms (Heath 1981, 1991a, 1996). For this
purpose, suppressors of disease resistance may act
at different levels of the interaction, e.g., avoid-
ing recognition, interfering with the signaling
processes that lead to the activation of resistance
reactions, or inhibiting the activity of defence
factors. As the result, the fungus has established
basie susceptibility of a plant species. Or, in other
words, a plant species has become a host species
for the fungus.
Reestablishment of resistance in a host
species requires the evolution of a novel recogni-
tion capacity. Since the original general elicitors
have become inefficient or inactive, novel fungal
signals need to be utilized in the recognition
process.. These elieitors may be specific for a
wider/ar narrower group of pathogens, thus
allowing a plant to recognize an entire pathogen
family, a fungal species or even partieular fungal
isolates. In the plant, resistance is only activated
if the pathogen expresses the elicitor-encoding
gene. Interactions at or above the species level
are not amenable to genetic analysis. However,
below the species level, single genes frequently
control resistance in individual genotypes of
otherwise susceptible host species to a limited
number of fungal genotypes. In these gene-
for-gene-type interactions (Flor 1955, 1971), the
expression of a partieular resistance gene allows
a plant cultivar to recognize a fungal strain
containing the complementary gene for aviru-
lence. A number of plant resistance genes to
different types of pathogens are being studied,
as are a few avirulence genes from fungi
(Table 15.1). However, the biochemical basis of
the interaction between resistance and avirulence
genes, and in particular of signal perception,
remains only partly understood.
 Avirulence Determinants and Elicitors 291

Table 15.1. Avirulence genes from phytopathogenic fungi and pseudomycota

Organism Gene Activity Specificity Reference( s)

Cladosporium fulvum Avr9 HR in Cf-9 lines of L. esculentum, Cultivar van Kan et al. (1991;
HR on L. pimpinellifolium lines Lauge et al. (2000)
Avr4 HR in Cf-4 lines of L. esculentum Cultivar Joosten et al. (1994)
ECP1 HR in Cf-ECP1lines of Cultivar van den Ackerveken
L. pimpinellifolium et al. (1993); Lauge
et al. (2000)
ECP2 HR in Cf-ECP2 lines of Cultivar van den Ackerveken
L. esculentum, HR on et al. (1993); Lauge
L. pimpinellifolium line, et al. (2000)
HR in Nicotiana
paniculata accessions
ECP3 HR in L. esculentum line Cultivar Lauge et al. (2000)
ECP4 HR in Cf-ECP4lines of Cultivar Lauge et al. (2000)
L. pimpinellifolium
ECP5 HR in L. esculentum lines Cultivar Lauge et al. (2000)
Rhynchosporium secalis NIPI Defence reactions in RrsI Cultivar Rohe et al. (1995)
barley lines
Magnaporthe grisea AVR-Pita HR in Pi-ta rice lines Cultivar Orbach et al. (2000)
PWLl Prevents infection of Species Valent and Chumley
weeping lovegrass (1991, 1994)
PWL2 Prevents infection Species Sweigard et al. (1995)
of weeping lovegrass
Phytophthora infestans inf1 Recognition on Species Kamoun et al. (1998)
Nicotiana benthamiana

111. General Elicitors Recently, another type of membrane lipid has

General elicitors of (at least part of) the defence
response are expected to be indicative for a wide
group of pathogens. These are likely to be struc-
tural components that are present on the surface
of many if not all fungi. An example is ergosterol,
the predominant sterol of most higher fungi. In
cultured cells of tomato (Lycopersicon esculen-
turn), an extremely sensitive system has been
detected that enables the perception of ergosterol,
but not of structurally related "self" sterols
present in plants, with an extremely low threshold
for detection of -10-12 M (Felix et al. 1993;
Granado et al. 1995). Media alkalinization, a
typical response of cell cultures to elicitors, was
used as an assay system for the activity of ergos-
terol. It is not c1ear yet how this re action relates
to other typical defence reactions such as the acti-
vation of genes encoding pathogenesis-related
(PR) pro teins or phytoalexin biosynthesis. Inter-
estingly, however, some of the biotrophic fungal
pathogens such as Blumeria spp. or Uromyces spp.
that develop very intimate associations with host
cells do not contain ergosterol, suggesting an
actual function of this membrane component in
pathogen recognition.
been found to have elicitor activity. From the rice
blast fungus Magnaporthe grisea as well as from
several other plant pathogenic fungi, the struc-
turally c10sely related cerebrosides A, Band C
were isolated (Koga et al. 1998; Umemura et al.
2000). These glycosphingolipids nonspecifically
induce defence reactions in rice such as the
biosynthesis of PR proteins and phytoalexins, the
hypersensitive response (HR) and systemic
acquired resistance (SAR). In contrast, animal
glucocerebrosides failed to stimulate these reac-
tions suggesting precise structural requirements
for elicitor activity. Therefore, rice appears to
possess a specific recognition system for fungal
cerebrosides. It remains to be shown whether
other plants are also capable of responding to
these lipids. Structurally related glycosphin-
golipids have been isolated from fungi with other
host species (Sakaki et al. 2001) and future exper-
iments will show whether they are elicitors in
plants other than rice. Perception of these com-
pounds by a wide group of plant species would
suggest glycosphingolipids to represent another
type of non-self-signaling molecule. Ceramide-
related signal compounds are involved in animal
development and apoptosis. In addition, induction
of apoptosis is among the reactions to sphinga-
292 W. Knagge
nine-analogous fungal toxins. This suggests that
signaling based on these types of compounds may
represent a new biochemical link between pro-
grammed cell death and plant disease response
(for review, see Gi1christ 1997,1998).
Plants appear to have found a way to also
recognize bacteria by evolving a general sensitive
perception system for bacterial flagellin. It is
specifically targeted to the most highly conserved
domain within the N-terminus of the protein
(Felix et al. 1999). Synthetic peptides comprising
15-22 amino acids of this domain act as elicitors
of defence reactions at subnanomolar concentra-
tions in cells of tomato and several other plant
species. In contrast, however, the respective
peptides from the plant-associated bacteria
Agrobacterium tumefaciens and Rhizobium
meliloti are elicitor-inactive. In Arabidopsis
thaliana, a locus, termed FLS1, was detected that
determines flagellin sensitivity (Gomez-Gomez
et al. 1999). In addition, a second locus, FLS2,
contains a ubiquitously expressed gene encoding
a putative receptor kinase that shares structural
and functional homologies with known plant
resistance genes (Gomez-Gomez and BoIler
2000).
Chitin, a homopolymer of ß(1......,4)-linked N-
acetyl-D-glucosamine, is a major constituent of the
cell walls of most high er fungi. Fragments of
somewhat varying degrees of polymerization were
found to function as elicitors of plant defence
reactions in dicots such as pea (Akiyama et al.
1995), tomato (Felix et al. 1993) and melon (Roby
et al. 1987), as weIl as in monocots such as wheat
(Barber et al. 1989), barley (Kaku et al. 1997), oats
(Ishihara et al. 1996, 1998; Miyagawa et al. 1996),
and rice (Kuchitsu et al. 1993; Kikuyama et al.
1997). Furthermore, specific, high-affinity binding
sites for chitin fragments have been detected in
membranes from tomato (Baureithel et al. 1994)
and rice cells (Shibuya et al. 1993, 1996). In the
latter, a 75-kDa membrane protein was identified
by photoaffinity labeling that represents the func-
tional receptor for chitin fragments (Ito et al.
1997). This suggests that chitin fragments may
function as general elicitors in a wide variety of
plant species. Generation of chitin fragments
appears to be achieved through the activity of
plant chitinases that are either constitutively
expressed or, in addition, induced upon fungal
infection or elicitor treatment (Wubben et al.
1996; Münch-Garthoff et al. 1997; Kaestner et al.
1998). These enzymes are also induced by chitin
fragments (He et al. 1998; Nishizawa et al. 1999)
suggesting that at least some of the plant chiti-
nases may be involved in a mechanism to amplify
the elicitor signal rather than being part of the
plant counterattack. In this context, it may be
speculated that the function of fungal chitinases
may be to hydrolyze the released chitin fragments
to a size nonrecognizable by plants (Sahai and
Manocha 1993).
Membrane lipids such as ergosterol and prob-
ably also glycosphingolipids and cell-wall com-
ponents such as chitin occur in mutualistic,
mycorrhizal fungi and neutral endophytes as weIl
as in antagonistic pathogens (BoIler 1995). This
indicates that their role in the interaction with
plants may lie in alerting the plant to the presence
of a fungus. However, in the case of pathogens, a
combination of factors may be required to trigger
an efficient plant defence response, maybe similar
to a model recently proposed for regulating gene
expression during leaf senescence (Morris et al.
2000). Furthermore, successful pathogens may
need to inhibit or suppress the activity of the
general elicitors to be able to penetrate plants.
Recent results on a class IV chitinase induced in
fungus-infected French bean (Phaseolus vulgaris)
roots (Lange et al. 1996) may point in this direc-
tion. In the compatible inter action with Fusarium
solani f. sp. phaseoli, this secreted chitinase is post-
translationally processed by an induced protease
to yield three smaller isoforms. In contrast, such
proteolysis does not occur in interactions with the
pea pathogen F. solani f. sp. pisi or with an arbus-
cular mycorrhizal fungus, Glomus mosseae.
Although lacking the chitin-binding domain, the
isoforms retained enzyme activity, possibly
however with altered catalytic characteristics
(Iseli et al. 1993). It is not known yet whether the
chitinase-processing protease is of fungal or plant
origin. Nevertheless, the results indicate a fungal
strategy to inactivate at least part of the plant's
defence response.
IV. Elicitors with Restricted Specificity
A. Glucan Elicitors
The majority of presently known elicitors of plant
defence reactions is not active in all plants, but
rather functions in a more or less restricted group
of plants. The surface of fungal and oomycete
 Avirulence Determinants and Elicitors
pathogens of plants have attracted early research
interest as a source of elicitors. In addition to
chitin, (1 ~3,1 ~6)-branched ß-glucans represent
components of the walls of fungi and they are the
major constituents of the chitin-free cell walls of
Oomycetes. Chitin has a linear structure and, thus,
does not have the potential to carry a high level of
information specifying its origin. It can therefore
only serve in a non-self-recognition process (see
Sect. III). In contrast, the branching pattern of ß-
glucans may vary in different organisms enabling
plants to evolve a more specific recognition
system, including the possibility to enzymatically
release different oligoglucan entities that can be
utilized in the recognition process.
The first glucan elicitor that was characterized
in structural detail was isolated from cell walls of
the Oomycete Phytophthora sojae (Sharp et al.
1984). A specifically branched (1~3,1~6) hepta-
ß-glucan, but not structurally related compounds,
were shown to elieit defence reactions in the host
plant, soybean (Glycine max; Cheong et al. 1991).
The various reactions triggered by this glucan elic-
itor have been previously reviewed (Cöte and
Hahn 1994; Ebel and Cosio 1994; Ebel and Scheel
1997). High-affinity binding sites for this elicitor
were detected on plasma membranes from
soybean and other legurnes (Cosio et al. 1996) and
genes encoding homologous binding proteins
were cloned from soybean (Umemoto et al. 1997)
and French bean (Mithöfer et al. 1999). The hepta-
ß-glucan appears to be specifically recognized by
species of the legurne family, not however by an
Apiacean species, parsley (Petroselinum crispum;
Parker et al. 1988), and by a Poacean species, rice
(Oryza sativa; Yamaguchi et al. 2000).
Recently, three ß-glucan fragments have been
isolated from cell walls of M. grisea that induce
phytoalexin biosynthesis in suspension-cultured
rice cells (Yamaguchi et al. 2000). Highest activity
was found with a penta-ß-glucan of a specific ß1,6-
branched structure, whereas two structurally
related ß-glucan fragments displayed lower activ-
ities. The rice-active component did not however
function as an elicitor in soybean. This suggests
that both plant species, soybean and riee, have
the capacity to recognize ß-glucan fragments.
However, their perception systems indeed target
different structural motifs.
Similar to chitin fragments, (1 ~3,1 ~6)­
branched ß-glucans are thought to be released
from cell walls of fungal and oomycete pathogens
by plant enzymes, endo-l,3-ß-glucanases, that are
293
induced upon pathogen attack (Stintzi et al. 1993).
Recently, an endo-1,3-ß-glucanase inhibitor
protein was purified from P. sojae that inhibits
only one of two soybean endo-1,3-ß-glucanases,
but not the enzymes from tobacco (PR-2c) and
P. sojae (Harn et al. 1997). This suggests that
pathogens have evolved the me ans to very speci-
fically inhibit the generation of ß-glucan elicitors.
In addition, ß-glucanases may be secreted to inac-
tivate these elicitors.
B. The Elicitins of Oomycete spp.
Species of the oomycete genera Phytophthora and
Pythium (Pseudomycota) are among the most
destructive plant pathogens causing disease on a
diverse range of plant species. From Phytophthora
culture filtrates elieitor proteins, collectively
named elicitins, were isolated (Bonnet et al. 1985;
Ricci et al. 1989). These pro teins have two dis-
tinctive effects on tobacco (Nicotiana tabaccum) ,
the test plant most frequently used. At nanomolar
concentrations, they cause extended necrosis rem-
iniscent of the HR in leaves of tobacco as well as
SAR to infection by normally virulent pathogens
(Ricci et al. 1989; Kamoun et al. 1993b; Keller et
al. 1994, 1996; Yu 1995; Bonnet et al. 1996). In
addition, subnecrotic concentrations suffice to
stimulate defence-related plant reactions such as
the initiation of ion fluxes across the cell mem-
brane, the generation of active oxygen, protein
phosphorylation, ethylene production, phyto-
alexin and PR protein biosynthesis (Blein et al.
1991; Milat et al. 1991; Rieci et al. 1993; Keller et
al. 1994; Viard et al. 1994).
Meanwhile, elicitins and elicitin-encoding
genes have been detected in all Phytophthora
species as well as in a few species of the related
genus Pythium (Huet et al. 1995; Gayler et al.
1997; Panabieres et al. 1997). In contrast, other
oomycete families appear to lack elicitins. The
original elicitins were characterized by their size
of typically 98 amino acids, by their amino acid
composition with a high content of Ser and Thr but
lacking Trp, His and Arg, and by the occurrence of
six invariant Cys residues in three disulfide bonds.
Recently, the crystal and solution structures of
the elicitin cryptogein have been determined
(Boissy et al. 1996; Fefeu et al. 1997; Gooley et al.
1998). It is a globular pro tein revealing a novel
protein fold with five a-helices concentrated on
one face of the molecule. The other face consists
294 W. Knagge
of an antiparallel two-stranded ß-sheet and an Q-
loop with one edge of the ß-sheet and the adjacent
face of the Q-loop forming a hydrophobie cavity.
Due to their high degree of sequence conserva-
tion, the tertiary structures of the different elicitins
can be expected to be very similar.
Originally, two classes of elicitins were
defined, basic a-elicitins (pI >7.5) and acidic ß-
elicitins (pI <5). While a-elicitins are produced by
all Phytophthora species, ß-elicitins are restricted
to a subset of species. The availability of more
gene sequences encoding elicitins or elicitin-like
pro teins from additional Phytophthora species
allowed an expansion of the original two-class
system. The acidic elicitins were combined in class
I-A, the basic elicitins in class I-B (Kamoun et al.
1997a). Most of the elicitin-like sequences identi-
fied within Pythium species display new features
such as the presence of His residues, flexible
amino acid numbers (98-101) and putative N-
glycosylation sites and are therefore pooled in
subclass l' (Ponchet et al. 1999). In P. cryptogea
(Panabieres et al. 1995) and P. cinnamomi (Duclos
et al. 1998), genomic sequencing revealed open
reading frames encoding 103-104 amino acid long,
highly acidic elicitins that form class II (Kamoun
et al. 1997a). Finally, class III contains elicitin-
encoding genes from P. infestans that encode pro-
teins consisting of the conserved 98 amino acid
elicitin domain followed by an approximately 70
amino acid C-terminal domain, the latter being
rich in Ser, Thr, Ala and Pro reminiscent of 0-
glycosylation domains. It was therefore proposed
that these proteins may be surface or cell-wall-
associated. In addition, a 32-kDa glycoprotein was
isolated from P. megasperma that induced elicitin-
like plant responses (Baillieul et al. 1995). It
showed immunological similarity with P.
megasperma elicitins (Baillieul et al. 1996) indi-
cating that post-translational modification of elic-
itins or elicitin-like proteins occurs.
Despite a substantial amount of data, no clear-
cut picture has emerged to date regarding the role
of elicitins in pathogenesis or resistance (Grant
et al. 1996; Ponchet et al. 1999). A survey of
Phytophthora isolates originating from different
plant species revealed that elicitin production is
almost ubiquitous (Ricci et al. 1992; Kamoun et al.
1994). The major exception is the species P. para-
sitica from which tobacco isolates were found to
not secrete elicitins while isolates nonpathogenic
to this plant are elicitin producers (Pernollet et al.
1993; Kamoun et al. 1994). Since tobacco is sensi-
tive to all tested elicitins, it was suggested that
these pro teins might be major determinants of the
basic resistance of tobacco to Phytophthora
(Kamoun et al. 1994; Yu 1995). However, the
hypothesis that elicitins are species-specific aviru-
lence determinants was shaken by the identifica-
tion of a few elicitin-producing isolates that are
virulent on tobacco, aIthough to a lesser extent,
indicating a role in virulence (Bonnet et al. 1994;
Mouton-Perronnet et al. 1995). After cloning of
the first elicitin gene, parAl, Southern analysis
revealed the presence of muItigene families in
elicitin-producing and nonproducing isolates of
P. parasitica and other Phytophthora species
(Kamoun et al. 1993a). AIthough at least three iso-
forms of cryptogein are expressed by P. cryptogea
(Le Berre et al. 1994), it is not known whether all
elicitin genes are transcribed. The isolation of a
genomic cryptogein clone led to the characteriza-
tion of four clustered genes. Two of these genes
encode secreted proteins. In contrast, the other
two genes encode a class of highly acidic elicitins
whose products have not been detected yet (Le
Berre et al. 1994; Panabieres et al. 1995). A similar
elicitin gene cluster with tandem pairs of genes
encoding an elicitin isoform and a highly acidic
elicitin was found in P. cinnamomi (Duclos et al.
1998). This suggests a structural conservation of
the elicitin cluster throughout Phytophthora
species. The occurrence of several elicitin genes
prompts the question as to how the expression of
different members of multigene families is regu-
lated during pathogenesis. In this context, it was
recently found that an elicitin-encoding gene of
P. infestans was down-regulated during infection
of its host plant, potato (Solanum tuberosum;
Kamoun et al. 1997b). Clearly, further detailed
analyses of gene expression are required to shed
light on the role of elicitins in the interaction of
Phytophthora species with their hosts.
Although there is some debate in the litera-
ture concerning the host specificity of elicitins (cf.
Kamoun et al. 1993b; Pernollet et al. 1993), it
appears now that most plant species lack the
capacity to sense and respond to elicitins. There-
fore, the genus Nicotiana represents a special case
with 10 of 11 species tested being sensitive to elic-
itin treatment. In addition, some but not all culti-
vars of two brassicacean species, radish (Raphanus
sativus) and turnip (Brassica campestris; Kamoun
et al. 1993b), developed necrosis. From these
data, it was concluded that elicitins might be
genus-specific elicitors within the Solanaceae and
 Avirulence Determinants and Elicitors
cultivar-specific elicitors within the Brassicaceae
(Kamoun et al. 1993b).
Recent, detailed analysis of the effect of elic-
itins on two radish cultivars has led to the descrip-
tion of an additional elicitin response phenotype
(Keizer et al. 1998). Cultivar "Daikon" has been
described to respond to elicitin treatment with
developing strong leaf necrosis (type I response),
while cultivar "White Icide" did not show any
symptoms (Kamoun et al. 1993b). The latter culti-
var did, however, develop an accelerated senes-
cence phenotype (type II response) that could
also be induced in "Daikon" with elicitin treatment
at 100% relative humidity that abolished the
necrotic response. A cross between these two cul-
tivars led to an F 1 population segregating for type
land II responses in a 1: 1 or 7: 9 ratio. F 2 popula-
tions from type I F 1 plants gave three phenotypes:
the parental types land II and, in addition, a null
phenotype, in a 1 : 2: 1 ratio. When protoplasts from
the different F 2 plants were treated with several
elicitins, extracellular pH changes (H+ uptake, K+
extrusion) and a transient Ca2+ uptake were
induced only in type I -derived protoplasts. These
reactions could be suppressed by preincubation
with a pro tein phosphorylation inhibitor, stau-
rosporine, suggesting that protein phosphorylation
is required. The strict correlation between proto-
plast responsiveness and the type I phenotype sug-
gests a causal linkage between the presence of a
functional signal perception and/or transduction
system and the induction of type I necrosis. Future
genetic analysis needs to provide the tools to
unravel the mechanism by which elicitins interact
with radish.
Several studies aimed at demonstrating that
elicitins are race-specific elicitors. However, these
proteins do not appear to be recognized by host
resistance genes. In P. sojae, elicitin production of
21 isolates belonging to different races did not
correlate with the avirulence phenotype of these
races (Mao and Tyler 1996). Furthermore, infiltra-
tion experiments using cryptogein and parasiticein
failed to induce necrosis in the presence of any of
the 12 Rps genes encoding resistance of soybean
to P. sojae. These elicitins were therefore not rec-
ognized by any of the Rps genes. However, the
purified elicitins from P. sojae were not used in
these experiments. Therefore, their inter action
with a soybean Rps gene currently cannot be ruled
out entirely.
P. infestans is considered to be specialized on
the genera Solanum and Lycopersicon but does
295
not attack Nicotiana. The molecular basis of this
specificity is not understood. P. infestans was
transformed with a DNA fragment from P. cryp-
togea carrying the cryptogein-encoding cry-b gene
along with an apparently not transcribed elicitin
gene (Panabieres et al. 1995,1998). In contrast to
wild-type strains, transformants secreting the alien
elicitin in addition to the endogenous elicitin
caused necrosis on tobacco. However, the P. infes-
tans mutants did not gain the capability to invade
the plant. This suggests that the interaction of
cryptogein with the plant does not promote
disease development and that resistance in this
case is not associated with the development of
necrotic lesions.
A role of elicitins in host specialization has
been revealed however by another set of experi-
ments. The elicitin-encoding inf1 gene was
silenced by transformation of P. infestans with an
inf1 antisense construct. Transformants were
obtained that had gained the capa city to grow and
sporulate on N benthamiana, but not on N rustica
and N tabacum (Kamoun et al. 1998). This
suggests that the INF1 protein functions as an
avirulence factor conditioning resistance in N
benthamiana probably at the species level accord-
ing to a previously proposed model (Heath
1991b).
A high-affinity binding site for cryptogein
was identified in tobacco plasma membranes
(Wendehenne et al. 1995). The Kd value was found
to be in good agreement with the cryptogein con-
centration required for biological activity. While
the functional molecular mass of the binding
protein was 193kDa, cross-linking experiments
revealed two cryptogein-linked N-glycoproteins
of about 162 and 50kDa (Bourque et al. 1999).
Interestingly, similar results were obtained using
plasma membrane preparations from Arabidopsis
thaliana and Acer pseudoplatanus, two species that
are not sensitive to cryptogein. This finding sug-
gests the presence of nonfunctional elicitin-
binding sites or subsequent signaling components
in these species.
The question as to what is the intrinsic fune-
tion of elicitors secreted by pathogens remains to
be answered in most cases. Recently, however, it
has been revealed that the hydrophobic cavity of
elicitins represents a high-affinity bin ding site for
sterols. Elicitins may therefore have an extracel-
lular sterol carrier function (Mikes et al. 1997,
1998; Vauthrin et al. 1999). In addition, the for-
mation of a sterol-elicitin complex appears to be
296 W. Knagge
the prerequisite for elicitin interaction with its
receptor on plant membranes (Ponchet et al.
1999). Numerous Oomycetes are known to be
devoid of sterol biosynthesis and it was therefore
assumed that elicitins pick up sterols from plant
membranes. However, whether and to what extent
these lipids are indeed required by Phytophthora
remains an open question.
As a conclusion, the role of elicitins from
Phytophthora species in pathogenesis and deter-
mination of specificity below the species level
remains unclear. Therefore, the identification of
factors of oomycete pathogens that are specifically
recognized by host resistance genes needs to await
the cloning and characterization of avirulence
genes from Phytophthora. Furthermore, silencing
of elicitin genes provides a technique to investigate
the function of these genes (van West et al. 1999).
c. Other Elicitors from Phytophthora spp.
From culture filtrates of P. sojae a 42-kDa glyco-
protein has been purified that induces defence
reactions in cultured cells of the nonhost species,
parsley (Parker et al. 1991), but not in the host
species, soybean (Parker et al. 1988). The gene
encoding the protein moiety of the elicitor was
cloned and found to be a member of a multigene
family in P. sojae (Sacks et al. 1995). Homologous
gene families of differing sizes were found in all
Phytophthora species tested, but not in species of
other Oomycetes or in ascomycete species. A 13
amino acid peptide, Pep-13, was identified within
the primary sequence of the glycoprotein elicitor
that was shown to be necessary and sufficient for
the activity of the intact pro tein to stimulate the
defence reactions in parsley cells (H+/Ca2+
influxes, K+/Cl- effluxes, an oxidative burst, activa-
tion of defence-related genes, and phytoalexin
biosynthesis; Nürnberger et al. 1994). The elicitor
represents a Ca2+-dependent transglutaminase (T.
Nürnberger, pers. comm.) that interacts with a
100-kDa binding protein on parsley plasma mem-
branes (Nennstiel et al. 1998). Sequencing of the
elicitor-encoding gene from several Phytophthora
species and alignment of the deduced amino acid
sequences revealed two invariant regions, one of
which is almost identical to Pep-13. Introduction
of specific mutations into the pro tein resulted in a
strict correlation between enzyme inactivity and
loss of binding activity. These results indicate that
parsley may have acquired the capacity to recog-
nize an evolutionary conserved peptide sequence
in an enzyme that is essential in the genus
Phytophthora.
Another, apparently Phytophthora-specific
elicitor was detected in P. parasitica cell walls.
A 34-kDa glycoprotein was isolated that elicits
defence-related reactions such as HR-like necrosis,
the biosynthesis of PR proteins and SAR in
tobacco (Mateos et al. 1997; Sejalon-Delmas et al.
1997). This protein is restricted to the flagellum
surface of the wall-less zoospores and abundantly
synthesized upon encystment. The protein moiety
comprises two directly repeated homologous,
Cys-rich domains, connected by a Thr/Pro-rich
linker region reminiscent of a head-hinge-head
organization. The amino acid sequence did not
reveal any general homology to published sequ-
ences. However, an amino acid pattern identical
to that of the cellulose-binding domain of several
fungal glucan hydrolases was identified. The pro-
tein binds to fibrous cellulose and plant cell walls
without however exerting detectable hydrolytic
activity. In addition, hemagglutination assays
revealed that it has lectin-like activity. To date it has
not been decided whether this protein is identical
to or different from a proteinaceous elicitor from
P. parasitica that induces defence reactions in
parsley similar to but quantitatively different
from the 42-kDa protein (Fellbrich et al. 2000).
The function of this pro tein remains unknown.
However, if it were required for attachment to host
cell walls, it may represent another example for
an essential surface protein from Phytophthora
that is utilized in the plant recognition process.
D. Products of the Species-Specific PWL Genes
of Magnaporthe grisea
The aforementioned elicitors have all been
directly isolated from plant pathogens based on
their elicitor activity upon application to plants.
An alternative strategy to identify factors that
restrict the host range of a fungal pathogen is the
genetic analysis of strains that differ in their host
specificity. In addition to the interfertility of fungal
strains with different host range, this approach
requires a simple, preferably monogenie, inheri-
tance of host species specificity (Heath 1991b).
The PWL (p-athogenicity toward .n:eeping loveg-
rass ) genes of M. grisea represent an example for
 Avirulence Determinants and Elicitors
this situation. As a species, M. grisea has a wide
host range causing disease on many grasses, with
rice blast being the most prominent. Individual
isolates, however, are often restricted to only a few
species. In a cross of two isolates, one from
weeping lovegrass (Eragrostis curvula), the other
from finger millet (Eleusine coracana), a single
segregating gene, PWL1, was identified. Another
nonlinked gene, PWL2, was found in a cross
between two rice-pathogenic strains differing in
their pathogenicity on weeping lovegrass (Valent
and Chumley 1991, 1994). Both genes prevent
M. grisea from infecting weeping love grass.
The PWL2 gene was isolated by positional
cloning (Sweigard et al. 1995) and shown to
encode a hydrophilic, Gly-rich 145 amino acid
protein. The presence of a putative 21 amino acid
signal peptide suggests its extracellular location.
However, preliminary attempts to demonstrate
elicitor activity of the PWL2 gene product, as pro-
duced in E. coli, upon infiltration into E. curvula
tissue have failed suggesting that its mode of
action may not be the direct interaction with a
plant surface receptor (Sweigard et al. 1995).
Mutations to a PWL2-deficient phenotype
occurred with a high frequency. These were mostly
caused by deletion of the PWL2 gene and sur-
rounding sequences, possibly due to homologous
recombination between ftanking repetitive DNA
sequences. However, one fungal strain carried an
allele, pwI2-2, that encodes a protein with a single
amino acid alteration creating a glycosylation site
(Sweigard et al. 1995). It is currently not known
however whether glycosylation of the protein
causes the altered phenotype.
The molecular analysis of M. grisea strains
with diverse host specificities has led to the dis-
covery that PWL2 is a member of a multigene
family (Kang et al. 1995) that appears to be
present in most fungal strains. Based on their
sequence homology with PWL2, three additional
genes were cloned, PWLl and PWL3 from a
finger millet isolate and PWL4 from a weeping
love grass isolate. While PWLl and PWL2 map to
different positions in the fungal genome, PWL3
and PWL4 are allelic. Although the product of the
PWLl gene shows only 75% sequence identity
with the PWL2 protein, it also prevents infection
of weeping love grass. In contrast, PWL3 and
PWL4 are nonfunctional genes. When these genes
were expressed from the PWLl or PWL2 pro-
moter, only PWL4, but not PWL3, acted as host
297
specificity determinant. This suggests that PWL3,
being 72 % identical to PWL4, does not encode an
active protein.
It is not known whether the different
PWL genes match single species-specific plant-
resistance genes. If so, it would demonstrate that
nonhost resistance of related plant species could
be governed by the same gene-for-gene-type prin-
ciple as observed within species (Heath 1991b;
see Sect. V). This would allow similar genetic
approaches to be followed with other fungal plant
pathogens. However, many plant pathogenic fungi
lack a sexual state precluding genetic crosses. With
these fungi, mutational strategies, e.g., transforma-
tion- or transposon-based insertion al mutagenesis
(de Groot et al. 1998; Kahmann and Basse 1999;
Nakayashiki et al. 1999), may enable the identifi-
cation of mutants that have gained pathogenicity
on originally nonhost-resistant species that are
related to the host species. Isolation of the
affected genes and characterization of their prod-
ucts would allow a comparison between the mech-
anisms that control nonhost and host resistance
within a plant species. Since nonhost resistance is
usually more durable than race-specific host resis-
tance, the genes involved in the recognition
process between a nonhost plant and a fungal
plant pathogen are prime targets to be utilized in
gene technological strategies to improve plant
disease resistance (see Sect. VII).
The "artificial" , recessive resistance gene mlo
of barley may serve to illustrate this situation. A
variety of mutations in the wild-type gene, Mlo,
have been obtained that result in broad-spectrum
resistance to all known isolates of the powdery
mildew fungus, Blumeria (syn. Erysiphe) graminis
(Jorgensen 1992). The gene encodes a 60-kDa
integral membrane protein with seven transmem-
brane helices that is reminiscent of metazoan G-
protein-coupled receptors (Devoto et al. 1999).
These pro teins transduce extracellular signals
through ligand binding into amplified intracellular
responses through activation of the a subunit of
heterotrimeric G pro teins. It is as yet not known
how defects in the products of the different mlo
alleles affect the signaling function of the protein
or what fungalligand is responsible for the elici-
tation of the plant response. Nevertheless, mlo
is an example of a single gene responsible for
non-race-specific plant resistance. Interestingly,
however, several mlo mutants displayed enhanced
susceptibility to M. grisea suggesting that wild-
298 W. Knagge
type Mlo is involved in resistance to this fungus
(Jarosch et al. 1999).
v. Avirulence Gene Products
A. AVRs and ECPs of Cladosporium fulvum
Cladosporium fulvum, the causal agent of leaf
mold on tomato, interacts with its host in a gene-
for-gene manner (de Wit 1992). In apoplastic
washing fluids isolated from susceptible, infected
plants, the first fungal cultivar-specific elicitors,
AVR9 and AVR4, were purified and shown to be
the products of avirulence genes (van Kan et al.
1991; van den Ackerveken et al. 1992; Marmeisse
et al. 1993; Joosten et al. 1994). The Avr9 gene is
identical in all analyzed fungal strains that are
avirulent on tomato plants carrying the Cf9 resis-
tance gene. It is absent, however, from fungal
strains virulent on Cf9 plants. The Avr9 gene is
only transcribed in planta and encodes a prepro-
protein of 63 amino acids. In vitra, using a consti-
tutive fungal promoter to control Avr9 expression,
cleavage of the signal peptide for extracellular
targeting and additional N-terminal proteolytic
processing leads to a mixture of elicitor-active
peptides with 32, 33, and 34 amino acids (van den
Ackerveken et al. 1993b). Further N-terminal
processing by plant proteases releases the origi-
nally purified AVR9 elicitor peptide of 28 amino
acids.
IH-NMR analysis revealed that AVR9 con-
tains a small triple-stranded ß-sheet region and
two solvent-exposed loops. Its six Cys residues
form three disulfide bonds in a cystine knot
fashion (Vervoort et al. 1997; van den Hooven et
al. 1999). This structural feature is common in
small, Cys-rieh pro teins such as serine protease
inhibitors, ion channel blockers and growth factors
(Isaacs 1995). AVR9 is structurally most related to
a member of the inhibitor cystine knot protein
subgroup, carboxypeptidase inhibitors (Vervoort
et al. 1997).
To pinpoint the amino acids that are required
for AVR9 elicitor activity a mutational approach
was followed (Kooman-Gersmann et al. 1997). A
series of Avr9 constructs in which each co don was
independently changed into an Ala codon was
tested using the potato virus X (PVX) expression
system (Chapman et al. 1992; Hammond-Kosack
et al. 1995). Comparison with a wild-type
PVX::Avr9 construct revealed that the hydropho-
bic amino acid residues in the solvent-exposed
loops are essential for the necrosis-inducing
activity of AVR9. This was further supported by
substituting surface-exposed amino acid residues
by other residues and, in addition, by using
chemically synthesized AVR9 mutants (Mahe et
al. 1998).
The Avr4 gene that is complementary to
the tomato resistance gene Cf4 encodes a 135
amino acid preproprotein (Joosten et al. 1994).
Cleavage of a secretory signal peptide and
additional proteolytic processing releases an 86
amino acid protein. In contrast to Avr9, an avr4
allele is present in all fungal strains virulent on
Cf4 plants. All avr4 alleles from virulent strains
exhibit single nucleotide substitutions translating
into amino acid alterations. Although they are
strongly expressed during infection, the AVR4
isoforms were not detected in apoplastic washing
fluids indicating their instability. Using the PVX
system, Avr4 and avr4 alleles were expressed in
Cf4 plants (Joosten et al. 1997). Inoculation
with PVX::Avr4 induced the development of
spreading lesions whereas the various PVX::avr4
derivatives induced symptoms ranging from
severe necrosis to no lesions at all. This suggests
that several of the AVR4 isoforms still possess
elicitor activity. However, plant recognition of
fungal strains expressing these alleles is precluded
by the instability of the AVR4 isoforms in
planta.
Avr9 disruption mutants and wild-type avr4
strains are not affected in their growth patterns in
vitro and in planta and an intrinsic function of
these genes remains to be shown. In contrast, two
other extracellular pro teins of C. fulvum, ECP1
and ECP2, have a role in virulence expression
(van den Ackerveken et al. 1993a; Lauge et al.
1997). The Ecpl gene encodes a 96 amino acid
pro tein that is processed to yield a mature protein
of 65 amino acids. The Ecp2 gene encodes a pre-
cursor pro tein of 165 amino acids that is processed
to result in a 142 amino acid protein. Both Ecp
genes are present in all tested fungal strains and
highly expressed in planta. Single and double dis-
ruption mutants showed reduced virulence on the
host plant (Lauge et al. 1997). The Ecpl-deficient
strain was not affected in its ability to invade,
to colonize and to reemerge from the host, but
conidiophore differentiation is drastically reduced
resulting in significantly lower amounts of conidia.
In contrast, the Ecp2-deficient strain was inhibited
 Avirulence Determinants and Elicitors
during development and hardly produced conidia
at all.
All pro teins secreted by a pathogen are poten-
tial elicitors of plant defence reactions and, thus,
candidates for avirulence factors. Therefore, three
additional extracellular proteins from C. fulvum,
ECP3, ECP4, and ECP5, were recently purified
and cDNAs encoding the latter two were isolated
(Lauge et al. 2000). Subsequently, a "reverse
screening" for resistance genes was performed
either by injecting the purified ECPs into plant
leaves or by using nucleotide sequences encoding
the ECPs in the PVX expression system in planta.
Among a total of 28 tomato breeding lines, four
were identified that respond to the presence of
ECP2 with an HR (Lauge et al. 1998, 2000). The
resistance is based on a single dominant gene,
termed Cf-ECP2 for resistance to C. fulvum
through recognition of ECP2, which was intro-
gressed into the four lines from the same L.
pimpinellifolium accession. An Ecp2-disruption
strain is pathogenie, although only weakly viru-
lent, on Cf-ECP2 plants demonstrating that resis-
tance is solely based on ECP2 recognition by the
plant. All 25 C. fulvum strains from a worldwide
collection were found to produce an HR-inducing
ECP2 protein. Therefore, the Cf-ECP2 gene
appears to operate through recognition of an
important virulence factor of C. fulvum (Joosten
and de Wit 1999; see Sect. VII). In addition to the
Cf-ECP2 lines, two lines were identified that
responded to ECP3 and ECP5, respectively, while
no HR was detected upon injection with ECP1 or
ECP4.
Since most Cf resistance genes in L. esculen-
tum originate from L. pimpinellifolium, 40 acces-
sions of this wild relative were screened for the
presence of recognitional specificities for the C.
fulvum ECPs and AVRs (Lauge et al. 2000).
Among the 13 accessions showing HR-associated
resistance, six recognized AVR9 and none AVR4.
A single line each recognized ECP1, ECP2 and
ECP3, respectively, while ECP4 triggered HR in
two lines and ECP5 in three lines, one line recog-
nizing both ECP4 and ECP5. As for ECP2, inher-
itance of resistance to ECP1 and ECP4 is based
on single dominant genes, Cf-ECPl and Cf-ECP4,
respectively.
Homologues of Cf genes were detected in
Nicotiana species (Kooman-Gersmann et al.
1996). Therefore, 44 accessions covering 25 dif-
ferent nonhost species belonging to the genus
Nicotiana were screened for HR-associated recog-
299
nition of AVRs and ECPs (Lauge et al. 2000).
While no response was observed in most of these
interactions, two N paniculata accessions spe-
cifically developed an HR to inoculation with
PVX::Ecp2. These data confirm the expectation
that basically all extracellular proteins of a fungal
pathogen can function in the recognition process
and that they can serve as tools to find useful resis-
tance genes in host and nonhost plants.
B. NIPl from Rhynchosporium secalis
Rhynchosporium secalis, the causal agent of leaf
scald on barley, rye and several other grass es,
secretes small necrosis-inducing proteins (NIPs)
into the culture filtrate that are toxie to leaves of
barley and other cereals (Wevelsiep et al. 1991).
Treatment of isolated plasma membrane vesicles
with NIP1 or NIP3 leads to the stimulation of the
K+-stimulated H+-ATPase, while NIP2 does not
affect this enzyme (Wevelsiep et al. 1993). In addi-
tion, NIP1 induces the rapid and transient expres-
sion of PR genes in plants carrying the resistance
gene Rrsl (Hahn et al. 1993). The Nipl gene is
present in a single copy in the genome of fungal
strains avirulent on Rrs1 plants. In contrast, most
virulent strains have completely deleted the gene,
but several retain an allele encoding an inactive
isoform of the protein (see below). Disruption of
the Nipl gene allows the fungal mutant strain to
grow on Rrs1 plants but at the same time reduces
symptom expression on rrs1 plants. This demon-
strates that NIP1 is the product of the fungal
avirulence gene AvrRrsl and that the pro tein,
although not essential, has a function in express-
ing fUll virulence in planta.
NIP1 is an 82 amino acid protein with a 22
amino acid signal sequence (Rohe et al. 1995), its
ten Cys residues forming five disulfide bonds
(unpublished results). Synthetic, overlapping
peptides spanning the primary sequence of the
pro tein were neither alone nor in any possible
combination elicitor-active, suggesting the 3D
structure of the molecule to be required for activ-
ity. Sequencing of Nipl alleles from several fungal
wild-type strains has so far revealed four NIP1 iso-
forms. Type I is the most active elicitor and toxin,
whereas type II, differing in three amino acid posi-
tions from type I, exhibits reduced activities. In
contrast, type III and type IV contain two differ-
ent single amino acid alterations in comparison
with type II and are inactive. The type-III- and
300 W. Knogge
type-IV-specific amino acids were introduced into
the type-I sequence and expressed in a heterolo-
gous system (Gierlich et al. 1999) yielding the
inactive proteins type III* and type IV* (unpubl.
results). This suggests that both amino acids alone
and independent of each other are essential for
the function of the protein (see Sect. VI).
c. AVR-Pita from Magnaporthe grisea
Crosses between strains of M. grisea exhibiting
differential virulence on various rice cultivars
have led to the identification of several avirulence
genes (Valent and Chumley 1994). The gene AVR-
Pita, previously referred to as AVR2-YAMO, pre-
vents the fungus from infecting rice cultivars
carrying the resistance gene Pi-ta (Jia et al. 2000).
The gene is located near the tip of chromosome 1
of M. grisea. Consequently, many spontaneous
gain-of-virulence mutants resulted from insertion
or deletion of DNA. AVR-Pita was isolated by
map-based cloning and found to encode a 223
amino acid protein with a putative secretory signal
peptide. The amino acid sequence shows similar-
ity with fungal Zn z+ metalloproteases (Valent
1997; Jia et al. 2000). Recent results support the
hypothesis that the protein is further processed to
a 176 amino acid active form,AVR-Pita176, corre-
sponding to the mature processed version of
known metalloproteases (Jia et al. 2000). Within
the putative active site, several point mutations
were found in virulent fungal strains suggesting
the enzyme activity to be required for elicitor
activity. However, biochemical evidence for pro-
tease activity is stilllacking. Surprisingly, apoplas-
tic application of AVR-Pita176 to rice leaves failed
to induce HR suggesting that the mode-of-action
of this elicitor of resistance differs from that of the
C. fulvum and R. secalis avirulence factors (see
Sect. VI).
D. Novel Avirulence Genes
The avirulence genes from C. fulvum and R.
secalis were cloned through areverse genetics
approach by first identifying fungal elicitors of
genotype-specific host defence reactions. Simi-
larly, the first two cultivar-specific elicitor proteins
were purified from the cowpea (Vigna unguicu-
Iata) rust fungus, Uromyces vignae (D'Silva
and Heath 1997). These proteins induce HR in a
resistant host cultivar. N-Terminal sequencing
revealed some similarity between the elicitors, but
no homology to other known proteins. Future
experiments are required to demonstrate whether
these pro teins are the products of fungal aviru-
lence genes that match resistance genes in the
host.
Genetic characterization of additional aviru-
lence genes is in progress from M. grisea (Mandel
et al. 1997; Dioh et al. 2000), from Leptosphaeria
maculans, the fungus causing black-leg on brassi-
cacean species (Ansan-Melayah et al. 1995), from
the flax rust fungus, Melampsora Iini (Ellis et al.
1997), from the wheat stem rust fungus, Puccinia
graminis f. sp. tritici (Zambino et al. 2000) and, in
addition, from the oomycete Phytophthora sojae
(Whisson et al. 1995). Future studies need to
answer the question as to whether the gene prod-
ucts share common traits at the structural and,
more likely, at the functionallevel.
VI. Perception of Avirulence Signals
Of the plant genes cloned to date that confer
resistance to fungal pathogens, several encode
membrane-anchored extracellular proteins
whereas others encode putative cytoplasmic pro-
teins (Hammond-Kosack and Jones 1997; Jones
and Jones 1997). Therefore, an important open
question aims at the mode of interaction between
avirulence and resistance gene products in the
process leading to the initiation of the plant
defence response. While extracellular resistance
proteins may directly bind the avirulence proteins,
intraceHular avirulence proteins require the trans-
duction of the avirulence signal into the host
cytoplasm or, alternatively, the translocation of the
avirulence proteins into the plant cell.
Binding studies using the AVR9 elicitor from
C. fulvum have shown that a high-affinity binding
site is present in plasma membranes of resistant
(Cf9) and susceptible (Cf0) tomato genotypes as
weH as of other solanaceous species (Kooman-
Gersmann et al. 1996). This suggests that the Cf9
gene does not encode the primary receptor of the
AVR9 pro tein. Several models have been pro-
posed to explain the AVR9/CF9 signal perception
system (Joosten and de Wit 1999). In analogy to
other signal transduction systems (Wciss and
Schlessinger 1998), ligand-induced dimerization
may occur. Binding of AVR9 to the high-affinity
 Avirulence Determinants and Elicitors 301

binding site may enable a specific association RLK R

with CF9. Only upon interaction with AVR91
CF9 is a conformational change induced in the
AVR
binding protein that leads to signal transduction
into the cytoplasm. This model would explain the
strict correlation between HR-inducing activity
and affinity to the high-affinity binding site of
LRR
AVR9 mutant proteins (Kooman-Gersmann et al.
1998). PM
Two other models assume that the high-affin-
ity binding site is the target of the so-far elusive
intrinsic function of AVR9. In addition, a low-
affinity binding site may exist that cannot be
detected in the presence of the high-affinity
binding site in isolated membran es. This low- Oefeneetaelions
affinity bin ding site may be the CF9 protein that
upon AVR9 binding interacts with a transducer
molecule. Since CF9 lacks a cytoplasmic signaling R
t
domain (Jones et al. 1994), the transducer should
contain a component that mediates the defence-
related signal transduction. Alternatively, the low-
affinity bin ding site may be a receptor-like kin ase
with an extracellular Leu-rich repeat (LRR) AVR
domain that may have structural similarity with
the Xa21 gene product conferring resistance of
rice to Xanthomonas oryzae pv. oryzae (Song et LRD
al. 1995). Dimerization of this transmembrane
kinase with CF9 may be required for AVR9
binding and generation of a cytoplasmic signal. Fig.15.1. Models for the interaction of fungal AVR pro-
teins and plant R proteins to initiate plant defence reac-
Alternatively, AVR9 may need to bind to the tions. Top A high-affinity binding site for the AVR9 protein
receptor-like kinase to allow the interaction from C. fulvum is present in the plasma membrane (PM)
with CF9 (Fig. 15.1). This latter model would be of tomato lines carrying or lacking the complementary R
analogous to the mechanism by which the gene, Cf-9. The CF9 protein consists of an extracellular
LRR domain, a transmembrane domain and a short cyto-
CLAVATA proteins mediate signal transduc- plasmic tai!. The signal transduction into the host cytoplasm
tion in A. thaliana (Joosten and de Wit 1999; may be mediated by an as-yet unknown receptor-like
Trotochaud et al. 1999). protein kinase (RLK). AVR9 may bind to the RLK allow-
Although the Rrsl gene conferring resistance ing the subsequent interaction of the complex with CF9,
which causes the activation of the kinase. Bottom The cyto-
of barley to R. secalis has not been cloned yet, plasmic R protein Pi-ta from rice contains aC-terminal
several lines of evidence point to a similar mode leucine-rich domain (LRD) that differs from the protein in
of action. Binding studies have revealed that a susceptible lines in one amino acid. The R protein from
resistant, but not from susceptible, plants is able to directly
NIP1-binding site is present in membranes iso- interact with the AVR-Pita protein from M. grisea. Activa-
lated from Rrs1 and rrs1 barley lines (unpublished tion of defence signaling may be caused by specific prote-
results). In addition, binding sites were found in olytic cleavage of the R protein. For simplicity, inactive and
other cereals but not in A. thaliana. Consequently, active effector domains are depicted in differing shapes
the resistance gene is again unlikely to encode the
primary receptor for the avirulence protein.
However, affinities of the four NIP1 isoforms to analysis it is known that the type-Ill- and type-IV-
this binding site do not correlate with their elici- specific amino acids are located in the same
tor activities. While the active type-I pro tein and domain of the protein (unpublished results). This
the inactive type-III* and type-IV* proteins have domain can therefore not be involved in the direct
the similarly low dissociation constants, the type- inter action with the binding protein. One of the
11 pro tein exhibiting reduced elicitor activity has three type-lI -specific amino acids is located on the
a 130-fold higher dissociation constant. From 3D opposite side of NIPl. If this domain is respon-
302 W. Knagge
sible for the reduced binding efficiency and, as a
consequence, for the lower elicitor activity, it may
indicate the binding domain of NIPl. Clearly,
binding of NIPI to the putative receptor does not
suffice for elicitor activity. It can be speculated
that the amino acids that are specifically altered in
the type-IH and type-IV isoforms may be involved
in attracting a third component into the receptor
complex. Hence, the inactivity of these isoforms
may result from a failure to complex with this spe-
cific signaling component.
Isolation, cloning and biochemical characteri-
zation of the plant plasma membrane proteins that
specifically bind fungal avirulence proteins will
eventually allow the inclusion of the primary
recognition process in the sequence of events
leading to plant defence gene activation. The
similarities between the signaling pathways medi-
ating the plant disease resistance response and the
animal innate immune response (Hultmark 1994;
Meyers et al. 1999; Aderem and Ulevitch 2000)
suggest that the latter may provide the models for
the mode-of-action of avirulence protein receptor
systems.
Although the mechanism underlying the
signal perception and transduction process has not
been fully elucidated for the avirulence gene prod-
ucts from C. fulvum and R. secalis, it is clear that
binding of the signals to their putative receptors
occurs on the extracellular side of the host plasma
membrane. In contrast, recent results provide
strong evidence for a direct intracellular inter ac-
tion between the products of rice-resistance gene
Pi-ta and the M. grisea avirulence gene AVR-Pita
(Jia et al. 2000). Different constructs were deliv-
ered into rice cells by particle bombardment. Only
AVR-Pita176, but not the intact AVR-Pita223 or a
shortened protein, AVR-Pita166, triggered the
hypersensitive response in plants expressing Pi-ta.
The deletion in AVR-Pita166 removes CYSS3 that is
supposed to be required for protease function. In
addition, fungal strains expressing alleles with a
CyssrGly substitution or two other mutations in
the protease motif lost full avirulence on Pi-ta
plants. Elicitor activity was only detected by intra-
cellular expression of AVR-Pita176 but not by its
extracellular application to rice leaves, indicating
the plant cytoplasm to be the site of action.
The resistance gene Pi-ta encodes a putative
cytoplasmic pro tein of 928 amino acids with a
centrally localized nucleotide-binding site (NBS)
domain and aC-terminal Leu-rich domain (LRD;
Bryan et al. 2000). The protein is most similar to
the NBS-LRR class of resistance gene products
(Hammond-Kosack and Jones 1997), but it does
not contain LRRs fitting to any previously
reported consensus. When N-terminal deletions of
the Pi-ta protein were expressed in the yeast two-
hybrid system to test their capacity to bind to the
avirulence protein, only the LRD portion inter-
acted with AVR-Pita176. In contrast, expression of
a pi-ta allele or of defined avr-pita alleles impaired
the inter action. This binding specificity was con-
firmed in a far-Western analysis (Chen and Evans
1995). AVR-Pita refolded on the membrane binds
specifically to the LRD domain of Pi-ta, whereas
a point-mutated LRD domain and a single amino
acid substitutedAVR-Pita176 (Met178-Trp) failed to
interact in vitra. This high specificity of the physi-
cal interaction between the LRD domain of Pi-ta
and AVR-Pita176 suggests that the avirulence gene
product is the fungal signal molecule that is
direct1y perceived by the resistance gene product.
Interestingly, during refolding of AVR-Pita176 as
well as during the inter action with the LRD
protein, Zn2+ was required to be present, a finding
that underlines the metalloprotein nature of the
avirulence gene product. Therefore, AVR-Pita176
being a putative protease may be a fungal tool to
release nutrients from host cells. The pro tein may
trigger the plant-resistance response by binding to
the resistance gene product, thus causing the con-
formation al alteration required for signal trans-
duction. Alternatively, AVR-Pita176 may cleave the
Pi-ta pro tein to an active form (Fig. 15.1). Future
experiments will need to show how the fungal
pro tein is translocated into the plant cytoplasm.
VII. Recognition-Based Enhancement
of Plant Disease Resistance
Gene technology-based strategies aimed at
improving plant disease resistance to fungal
pathogens can basically target any step in patho-
genesis and plant resistance biochemistry. Various
strategies to engineer plant disease resistance
have recently been reviewed (Dixon and Arntzen
1997; Shah 1997; Bushnell et al. 1998; Evans and
Greenland 1998; Salmeron and Vernooij 1998;
Honee 1999; Melchers and Stuiver 2000). The
technically least-demanding approach involves
the transfer of single genes to analyze their impact
 Avirulencc Determinants and Elicitors
on resistance expression. Transgenie plants that
constitutively express proteins with antifungal
activity, such as glucanases, chitinases, ribosome-
inactivating proteins, or other antimicrobial pep-
tides of different origin, alone or in combination,
showed a higher degree of resistance to various
pathogens. Also, the constitutive expression of an
H 20 2-generating fungal glucose oxidase in potato
resulted in an enhanced resistance to several
fungal pathogens (Wu et al. 1995, 1997). Further-
more, the secondary metabolism including low-
molecular-weight antifungal compounds can be
engineered (for reviews, see Dixon et al. 1996;
Dixon and Amtzen 1997). Alternative targets for
manipulation are plant genes that control the
expression of SAR. For example, transgenie A.
thaliana plants constitutively expressing the regu-
latory gene NPRl displayed nonspecific resistance
to oomycete and bacterial pathogens by constitu-
tively expressing the SAR pathway (Bowling et al.
1997; Cao et al. 1998).
An alternative strategy aims at extending the
pathogen range that can be recognized by a plant
through exploitation of different elicitor-receptor
interactions. To this end, several concepts are
conceivable that all confer novel recognition
specificities to plants. The different approaches
involve the deployment of single resistance genes,
the implementation of two-component sensor
systems, or the generation of novel recognition
specificities.
Genes controlling recognition at the species
level are probably targeting molecules essential
for the fungus that cannot easily be mutated. They
can therefore be expected to be more durable as
compared to race-specific resistance genes. Hence,
the transfer of these genes across the species
boundary has the potential to reestablish the
nonhost status in a plant species. In this context, it
will be very exciting to learn about the receptor in
parsley that recognizes a particular invariant
domain in a protein secreted by different Phy-
tophthora species (see Sect.IV.3). Once cloned,
transfer of the receptor-encoding gene may
improve resistance of a variety of plants to their
Phytophthora pathogens. These may include eco-
nomically important species such as potato and
soybean and their oomycete pathogens, P. infes-
tans and P. sojae.
Another example for the potential benefits
from a careful biochemical and genetic analysis of
a pathosystem is provided by the Cf-ECP2 gene
303
that confers resistance of tomato to strains of C.
fulvum expressing the Ecp2 gene (see Sect. V.l).
The Cf-ECP2 gene was detected in all tested
fungal isolates. In addition, Ecp2-deficient strains
were strongly inhibited in their development in
planta suggesting an essential function of the gene
product. Therefore, resistance of tomato through
transformation with the Cf-ECP2 gene may not
only allow tomato to recognize all C. fulvum
isolates, but also prove to be more durable than
"normal" single gene-based resistance.
Fungal genes encoding elicitor moleeules can
be transferred to plants expressing the matching
receptor or resistance gene, thus creating a two-
component sensor system that may trigger broad-
spectrum disease resistance (de Wit 1992). Crucial
to this approach is the availability of tightly regu-
lated plant promoters that are nonspecifically
activated upon pathogen attack to avoid major
damage to the plant by inducing systemic HR
(Honee et al. 1995). Along these lines, Cf9 tomato
plants were transformed with the Avr9 gene from
C. fulvum under the transcriptional control of a
promoter fragment from the potato gstl gene
(Strittmatter et al. 1996). Upon infection with
several different pathogens, the trans genie plants
showed a resistance response similar to the
response induced by Avr9-carrying C. fulvum
strains (Honee 1999). It should also be feasible to
transfer an Avr9-Cj9 cassette to a different plant
species. Prerequisite for this approach, however, is
the presence in the acceptor species of the signal-
ing cascade that res ponds to AVR9-CF9 interac-
tion. In a similar approach, the promoter of the
pathogen-inducible tobacco hsr203J gene (Pontier
et al. 1994) was fused to the gene encoding the
elicitin cryptogein from Phytophthora cryptogea
and the construct was transferred into tobacco
(Keller et al. 1999). The transgenie plants dis-
played enhanced resistance to P. parasitica and to
various fungal pathogens.
Once more details become known ab out the
signal perception process itself at the plant plasma
membrane and the genes encoding the participat-
ing pro teins have been cloned, novel recognition
specificities may be designed. For instance, the
signal perception domain of a plant protein may
be fused to the cytoplasmic domain of a trans-
membrane receptor. In such a way, the extracellu-
lar LRR and transmembrane domains of a
receptor kin ase involved in brassinosteroid sig-
naling in A. thaliana were fused to the Ser/Thr
304 W. Knogge
kinase domain of the product of the rice Xa21
resistance gene (He et al. 2000). As a consequence,
the chimeric receptor initiates plant defence re ac-
tions in rice cells upon treatment with brassino-
steroids. Provided that a suitable receptor kin ase
is available that feeds into the signaling pathway
leading to defence gene activation, this approach
may be generally applicable. Binding domains for
various pathogen-secreted molecules such as elic-
itors, toxins or "neutral" compounds could be uti-
lized in a strategy to implement enhanced plant
disease resistance.
VIII. Conclusions
The last years have seen the identification and
molecular characterization of a number of mole-
cules that are capable of triggering defence reac-
tions of plants. With the exception of the product
of avirulence genes and single genes determining
resistance at the species level, the biological rele-
vance of most elicitors has not been proven to
date. The role of general elicitors such as fungal
membrane lipids or chitin fragments during the
inter action is not understood, although the pres-
ence of highly sensitive perception systems for
these compounds suggests an important function.
Also, the contribution of cultivar-nonspecific elic-
itors such as the ß-glucans or the elicitins to the
expression of disease resistance in a host plant
needs to be assessed. If elicitors with different
specificities were active in a given inter action, it
may be possible that they trigger overlapping
subsets of the defence reactions rather than the
entire defence response. It is also possible that
general elicitors function to alert the plant and
additional specific elicitors have to come in to
launch the effective defence response. Alterna-
tively, the role of cultivar-nonspecific elicitors may
be to amplify the specific response. Unraveling of
the biochemistry of the primary signal perception
is expected to lead to a better understanding of the
processes that result in the activation of the plant
defence machinery. This will then be the basis for
designing novel strategies to improve plant dis-
ease resistance through genetic engineering.
Acknowledgements. I am grateful to Drs. Pierre
IG.M. de Wit and Thorsten Nürnberger who
kindly provided a preprint of an article in press
and unpublished data, respectively.
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16 Fungal Phytotoxins

DANIEL G. PANACCIONE,l RICHARD D. JOHNSON/ JACK B. RASMUSSEN,2 and T.L. FRIESEN2

CONTENTS I. Introduction
I. Introduction .......................... . 311
11. Nonribosomally Synthesized Peptide
Phytotoxins .......................... . 312 A variety of plant pathogenic fungi produce phy-
A. HCToxin ............................ . 313 totoxins that contribute to their ability to colonize
1. Biological Significance of HC Toxin ..... . 313
2. Biosynthesis of HC Toxin ............. . 314
their host plants or to cause specific disease symp-
B. Victorin ............................. . 315 toms. Some of these compounds are true toxins
1. Biological Significance of Victorin ...... . 315 in that they elicit an almost immediate necrotic
2. Biosynthesis of Victorin .............. . 317 response in affected cells. Others, though still
C. AMToxin ........................... . 317
1. Biological Significance of AM Toxin ..... . 317
called toxins, have more subtle effects on the host
2. Biosynthesis of AM Toxin ............. . 318 plant of the fungus resulting in inhibition of plant
D. Other Peptide Phytotoxins .............. . 319 processes that eventually leads to cell death or
III. Polyketide-Derived Phytotoxins .......... . 320 that allow the pathogen to colonize the plant
A. TToxin ............................. . 321
1. Biological Significance of T Toxin ....... . 321 resulting in disease symptoms.
2. Biosynthesis of T Toxin ............... . 322 Researchers in this field have traditionally
B. AK Toxin ........................... " 323 dassified fungal phytotoxins as either host-specific
1. Biological Significance of AK Toxin ..... . 323 toxins or nonspecific toxins. Host-specific toxins
2. Biosynthesis of AK Toxin ............. . 323
C. Cercosporin .......................... . 324 (also sometimes called host -selective toxins)
1. Biological Significance of Cercosporin ... . 324 delimit the host range of the producing fungus.
2. Biosynthesis of Cercosporin ........... . 325 These toxins affect only plant varieties or geno-
D. Other Polyketide-Derived Phytotoxins ..... . 326 types that are susceptible to the producing fungus.
IV. Proteinaceous Phytotoxins .............. . 326
A. Ptr Toxins ........................... . 326 Accordingly, the fungus is able to parasitize only
1. Biological Significance of Ptr ToxA ..... . 326 plants that are sensitive to the toxin. Often, only
2. Biosynthesis of Ptr ToxA ............. . 327 certain isolates within a fungal species will have
3. Biological Significance of Ptr ToxB ..... . 327
B. Cerato-ulmin ......................... . 328
the ability to produce a host-specific toxin. In
1. Biological Significance of Cerato-ulmin .. . 328 many cases, these toxin-producing isolates are
C. Other Proteinaceous Phytotoxins ......... . 329 given a race or pathotype designation to distin-
V. Miscellaneous Fungal Phytotoxins ........ . 329 guish them from toxin-nonproducing isolates that
A. Trichothecenes ........................ . 330
B. HS Toxin ............................ . 330
are given a different ra ce or pathotype designa-
C. Fusicoccin ........................... . 330 tion. In contrast, nonspecific toxins are gene rally
D. PCToxin ............................ . 331 toxic to numerous plants, even some outside the
VI. Conclusions .......................... . 331 host range of the producing fungus. These non-
References ........................... . 332
specific toxins indude fungal metabolites that
affect common processes in numerous plants and,
also, some fungal metabolites traditionally con-
sidered mycotoxins (primarily toxic to mam-
mals). The importance of some mycotoxins (e.g.,
the trichothecenes, discussed below) in specific
plant diseases has been carefully evaluated,
whereas evidence for a role in disease for others
1 Division of Plant and Soil Sciences, West Virginia Uni-
versity, Morgantown, West Virginia 26506, USA
is not as dear. Nonetheless, they are important
2 Department of Plant Pathology, N orth Dakota State Uni- economically because they affect the quality of
versity, Fargo, North Dakota 58105, USA food and feed.

The Mycota XI
Agricultural Applications
Kempken (Ed.)
© Springer-Verlag Berlin Heidelberg 2002
312 D.G. Panaccione et al.
Among the fungal phytotoxins are a variety of
molecules that often have at their core nonribo-
somally synthesized peptides or polyketide/fatty
acid derivatives. A few polypeptide toxins that are
primary gene products have been isola ted and
characterized. Still other phytotoxins are derived
from isoprenoid and carbohydrate components. In
the sections below, we cover some representative
examples in several of the major chemical classes.
Our objective is not to catalogue aB the fungal
phytotoxins, but rather to review examples within
the listed classes that have been the subject of
extensive re cent investigation and that therefore
may provide insight into the generalities (or the
diversity) of the phytotoxins within each class.
Particular emphasis has been placed on the role of
each featured toxin in the plant-fungus interac-
tion, including the mechanism of resistance (when
known), and the biochemistry and molecular
genetics of toxin biosynthesis. Our contribution is
one among several reviews on host-specific toxins
(Walton and Panaccione 1993; Otani et al. 1995;
Walton 1996; Yoder 1998) and nonspecific toxins
(Desjardins and Hohn 1997; Hohn et al. 1998)
published in recent years.
11. Nonribosomally Synthesized
Peptide Phytotoxins
A wide range of biologically active peptides are
synthesized, nonribosomally, by multifunctional
enzymes called peptide synthetases (Kleinkauf
and von Döhren 1990; von Döhren et al. 1997;
Marahiel et al. 1997). In contrast to ribosomally
synthesized polypeptides, which may contain only
the 21 proteinogenic amino acids, nonribosomally
synthesized peptides may contain a diverse array
of residues that include nonproteinogenic amino
acids (as found in HC toxin, victorin, and AM
toxin), N-methylated amino acids (as found in
enniatins and destruxins), D-amino acids (as found
in HC toxin and destruxins), and a-hydroxy acids
(as found in AM toxin and enniatins). In addition,
the peptide backbone can be linear, cyclic, or
cyclic-branched and may be further modified by
glycosylation, acylation, or heterocyclic ring for-
mation (Marahiel et al. 1997). These diverse bioac-
tive peptides range from 2 to 48 residues (to date)
and include antibiotics (e.g., penicillin), immuno-
suppressive agents (e.g., cyclosporine) and toxins
active against insects (e.g., destruxins, discussed
below), mammals (e.g., ergopeptines, discussed
below), and plants (e.g., HC toxin, victorin, and
AM toxin, discussed below). In many cases, the
genes encoding these peptide synthetases have
been cloned and characterized (e.g., Diez et al.
1990; Scott-Craig et al. 1992; Haese et al. 1993;
Weber et al. 1994; Bailey et al. 1996; Johnson et al.
2000a) and all share a common modular structure.
Without exception, peptide synthetases
are multifunctional enzymes of 100-1700 kDa
(Marahiel et al. 1997) that link amino acid (or
occasionally a-hydroxy acid) residues according
to the multiple-carrier thiotemplate mechanism
(Kleinkauf and von Döhren 1990; Stein et al. 1994;
Marahiel et al. 1997). A minimal module, of ab out
650 amino acids, has been defined (Marahiel et
al. 1997) that is composed of an amino-acid-
activating domain, a thiolation domain and a con-
densation domain on a single polypeptide chain.
The activation domain recognizes, specifically in
most cases, a substrate amino (or a-hydroxy) acid
and activates it as its acyladenylate by reaction
with ATP. The activated ester then becomes cova-
lently linked as a thioester to an enzyme-bound 4'-
phosphopantetheine cofactor located within the
module. Finally, the condensation domain medi-
ates transfer to another acylamino acid interme-
diate on the adjacent downstream module to form
a peptide bond. In some cases, modifications (such
as epimerization, N-methylation or cyclization)
are catalyzed by additional domains or by modi-
fied domains within a module. In all cases, the
number and order of repeating modules within the
enzyme corresponds to the number and order of
the residues in the peptide being made.
Many peptide synthetase enzymes were
initially identified biochemically using the ATP-
pyrophosphate exchange re action (Lipmann
1971) which takes advantage of the reversible acti-
vation of amino acid substrates to acyladenylates.
This technique, which requires the addition of
particular amino acid substrates, is also the most
commonly used method to determine the sub-
strate specificity of the adenylate-activating
domain. However, this technique is limited by the
availability of amino acid substrates and, as such,
is not feasible for the analysis of many peptides
containing nonproteinogenic residues.
The cloning and sequencing of peptide syn-
thetase genes has led to the identification of highly
conserved motifs (of 3 to 8 amino acids) within the
functional domains already discussed (Turgay and
Marahiel 1994; Marahiel et al. 1997). Degenerate
 Fungal Phytotoxins
oligonucleotides based on these core motifs
have been successfully used in polymerase chain
re action (PCR) experiments to identify several
genes encoding peptide synthetases (Turgay
and Marahiel1994; Nikolskaya et al. 1995; Panac-
cione 1996; Johnson et al. 2000a) and is widely
applicable.
The biosynthesis of several important peptide
phytotoxins and their role in plant-fungus inter-
actions are discussed below.
A. He Toxin
1. Biological Significance of HC Toxin
The leaf spot disease of maize (Zea mays L.)
caused by the HC toxin-producing race 1 of
Cochliobolus carbonum Nelson was first observed
in the late 1930s on certain inbred lines (Ullstrup
1941). The importance of the disease from an
agricultural perspective was short-lived, as most
of the maize planted at that time, and certainly
since, contained the HMI allele which provides
resistance to C. carbonum race 1 (Ullstrup and
Brunson 1947) and insensitivity to He toxin. A
lesser degree of resistance is provided by the dom-
inant allele HM2 at a second locus (Nelson and
Ullstrup 1964). The leaf spot disease caused by C.
carbonum race 1 appeared when both HMI and
HM2 had been unknowingly bred out of the sen-
sitive inbreds (Multani et al. 1998). In the years
since, HMI has provided durable resistance to the
pathogen. Despite the brevity of its importance in
the field, the disease has had enduring importance
from a scientific perspective. In the 60 years since
its discovery, the interaction of C. carbonum race
1 and maize has developed into one of the best-
understood plant-fungus interactions from
genetic, biochemical, and molecular biological
perspectives.
The host-specific nature of He toxin and its
significance to disease development on susceptible
maize were demonstrated by the research of
Scheffer and colleagues. Only isolates of the
fungus that produce HC toxin are capable of
causing the large necrotic lesions typical of ra ce
1 of C. carbonum (Scheffer and Ullstrup 1965;
Scheffer et al. 1967) and only maize lines that are
susceptible to C. carbonum race 1 are sensitive to
HC toxin (Scheffer and Ullstrup 1965). The addi-
tion of HC toxin to the infection court of a toxin-
nonproducing race 2 isolate of C. carbonum on
313
susceptible maize results in symptoms comparable
to those caused by race 1 of the fungus (Comstock
and Scheffer 1973).
Severallaboratories (Gross et al. 1982; Leisch
et al. 1982; Pope et al. 1983; Walton et al. 1982;
Kawai and Rich 1983) determined the structure of
He toxin as a cyclic tetra peptide of D-proline,
L-alanine, D-alanine, and L-Aeo (2-amino-9,l0-
epoxy-8-oxodecanoic acid). The terminal epoxide
of Aeo (Ciuffetti et al. 1983; Walton and Earle
1983) and the carbonyl group at position 8 (Kim
et al. 1987) of Aeo are both required for toxin
activity. Analogs containing glycine in pI ace of D-
alanine (Kim et al. 1985) and D-hydroxyproline in
pI ace of D-proline (Tanis et al. 1986) have also
been characterized.
HC toxin is unlike many other phytotoxins in
that it does not induce rapid degenerative effects
in host tissue. Some of the responses of plant
tissue to the toxin are stimulatory rather than
inhibitory. For example, exposure of susceptible
tissues to HC toxin has been shown to increase
dark CO 2 fixation (Kuo and Scheffer 1970) and
stimulate uptake of several ions and organic com-
pounds (Yoder and Scheffer 1973a,b). HC toxin
inhibits the growth of roots in vitro (Scheffet and
Ullstrup 1965) and the greening of dark-grown
seedlings (Rasmussen and Scheffer 1987) in a cul-
tivar-specific manner. It suppresses either the pro-
duction or activity of a diffusible substance that
appears to be involved in limiting the ingress
of potential pathogens (Cantone and Dunkle
1990a,b). All these data indicate that HC toxin
is cytostatic rather than cytotoxic, and exhibits
far more subtle effects than other host-selective
phytotoxins.
Based on the finding that the peptide trapoxin
(structurally similar to HC toxin and containing
the epoxide-containing amino acid Aeo) acts as
an inhibitor of mammalian histone deacetylase
(Kijima et al. 1993), Brosch et al. (1995) tested
whether maize histone deacetylase was sensitive
to HC toxin. All three isoforms of histone deacety-
lase from susceptible maize (but not from resistant
maize) were inhibited by He toxin. Susceptible
maize inoculated with race 1 C. carbonum accu-
mulated hyperacetylated histone H3 and histone
H4, whereas maize inoculated with a toxin-
nonproducing isolate of the fungus did not
(Ransom and Walton 1997). These data indicate
that inhibition of histone deacetylase by HC toxin
may promote a cytostatic state in which the
affected plant cell cannot make the changes in
314 D.G. Panaccione et al.
gene expression required for mounting an ade-
quate defense to the pathogen.
Surprisingly, the mechanism of resistance
to HC toxin is uncoupled from the site of action
of the toxin. Meeley and Walton (1991) demon-
strated that an enzyme, HC toxin reductase, detox-
ifies HC toxin by NADPH-dependent reduction
of the carbonyl group at position 8 in the Aeo side
chain to the corresponding alcohol, a compound
previously shown to be nontoxic (Kim et al. 1987).
Cell-free extracts of resistant maize catalyze this
re action whereas extracts from susceptible maize
do not (Meeley et al. 1992). Without exception,
HC toxin reductase activity segregates with HMI
in the F2 progeny of a cross between resistant and
susceptible maize (Meeley et al. 1992). Further-
more, mutants tagged at HMI by insertion of a
transposable element concurrently lose their HC
toxin reductase activity and their resistance to
C. carbonum. Somatic reversion in these tagged
mutants to C. carbonum resistance, due to excision
of the element, accompanies the return of HC
toxin reductase activity (Meeley et al. 1992).
DNA containing the site of insertion of the
transposable element from the tagged mutants, as
weIl as the corresponding locus from wild-type
maize, have been cloned and analyzed (Johal and
Briggs 1992). The deduced amino acid sequence of
the HMI allele has greater than 50% sequence
identity to the product of the Al locus of maize,
which encodes an oxidoreductase involved in the
synthesis of anthocyanins. This indicates that HMI
is the structural gene for HC toxin reductase.
Interestingly, the product of HMI appears to have
no other essential function as hml/hml strains
have no altered phenotype other than susceptibil-
ity to C. carbonum. Homologues of the maize
resistance gene have been found in other grasses,
including important crops such as rice, sorghum,
and barley (Han et al. 1997; Multani et al. 1998).
2. Biosynthesis of HC Toxin
The original genetic studies on HC toxin biosyn-
thesis indicated that the ability to produce the
toxin segregated as a single Mendelian locus
(Scheffer et al. 1967). That locus, now known as
Tox2, is a complex of biosynthetic and regulatory
genes, many of which are found in two or three
copies, and all of which are absent from HC toxin-
nonproducing isolates of C. carbonum (Walton et
al. 1998). The elucidation of the molecular nature
of the Tox2 locus began with the biochemical
experiments of Walton (1987), who hypothesized
that, based on the peptidic nature of HC toxin,
a nonribosomal peptide synthetase must be
involved in its synthesis. Characterization and iso-
lation of proteins catalyzing activities expected
from an HC toxin peptide synthetase (Walton
1987; Walton and Holden 1988) led to the identi-
fication of HTSI (Panaccione et al. 1992; Scott-
Craig et al. 1992), the first HC toxin biosynthetic
gene and the me ans of access to the Tox2 locus.
(Refer to Table 16.1 for relevant information on
HTSI and other fungal genes described in the
text.) HTSI encodes a four-module nonribosomal
peptide synthetase (Scott-Craig et al. 1992) that
catalyzes the assembly of the four constituent
amino acids into HC toxin (Panaccione et al.
1992). The essentiality of this gene for HC toxin
biosynthesis and disease development on suscep-
tible maize has been demonstrated through gene
knockout experiments (Panaccione et al. 1992).
Immediately 5' of HTSI is a transcribed open
reading frame named TOXA, which encodes a
putative HC toxin efflux pump (Pitkin et al. 1996).
Results of experiments designed to knock this
gene out indicate that to do so in an HC toxin-
producing strain is lethai (Pitkin et al. 1996).
Sequences homologous to HTSI and TOXA are
found exclusively in HC toxin-producing isolates
of C. carbonum (Panaccione et al. 1992). By chro-
mosome walking and knocking out additional
transcribed sequences physically linked to HTSI
and TOXA and that are unique to HC toxin-
producing isolates, Wal ton and colleagues have
identified several more HC toxin biosynthetic
genes. These include two genes that are likely to
be involved in the biosynthesis of Aeo: TOXe
which encodes a product similar to the ß subunit
fatty acid synthase (Ahn and Walton 1997); and
TOXF encoding a branched-chain amino acid
amino transferase (Cheng et al. 1999). A gene
(TOXG) for an alanine racemase has also been
identified (Cheng and Walton 2000). Finally, a reg-
ulatory gene named TOXE (Ahn and Walton
1998) has been identified that appears to encode
a bZIP-like positive regulatory factor that is
required for the expression of TOXA, TOXe,
TOXF, TOXG and an additional gene named
TOXD (Ahn and Walton 1998; Cheng and Walton
2000). TOXD is found as part of the Tox2 locus
though it is not essential for HC toxin production
(Walton et al. 1998).
Most of the genes at the Tox2 locus are found
in two or three copies (Panaccione et al. 1992; Ahn
 Fungal Phytotoxins 315

Table 16.1. Cloned genes described in the text associated with fungal phytotoxin production or resistance

Toxin Gene Gene function Producing fungus Sensitive host Ref

HC toxin HTSI Peptide synthetase Cochliobolus Maize Panaccione et al. (1992)

carbonum
TaXA Efflux pump Pitkin et al. (1996)
TaXC Fatty acid synthase Ahn and Walton (1997)
TaXE Transcription factor Ahn and Walton (1998)
TaXF Amino transferase Cheng et al. (1999)
TOXG Ala -racemase Cheng and Walton
(2000)
AM toxin AMT Peptide synthetase Alternaria alternata Apple Johnson et al. (2000)
Enniatin esynl Peptide synthetase Fusarium scirpia Multiple Haese et al. (1993)
T toxin PKSI Polyketide synthase Cochliobolus Maize (cmsT) Yang et al. (1996)
heterostrophus
DECI Decarboxylase Yoder (1998)
AK toxin AKTI Carboxyl-activating Alternaria Japanese pear Tanaka et al. (1999)
cnz. alternata
AKT2 Unknown Tanaka et al. (1999)
AKTR-I Transcription factor Tanaka and Tsuge (2000)
AKT3-I Hydrataselisomerase Tanaka et al. (1999)
AKTR-2 Similar to AKTR-I Tanaka and Tsuge (2000)
AKT3-2 Similar to AKTJ-I Tanaka and Tsuge (2000)
Cercosporin SaRI Toxin resistance Cercospora spp. Multiple Jenns and Daub (1995)
CRGI Toxin resistance Chung et al. (1999)
CFP Efflux pump Callahan et al. (1999)
PtrToxA ToxA N ecrosis-inducing Pyrenophora Wheat Ciuffetti et al. (1997)
peptide tritici-repentis
Cerato-ulmin cu Type II hydrophobin aphiostoma ulmi Elm Bowden et al. (1994)
Trichothecenesb TRI5 Trichodiene synthase Fusarium Parsnip Desjardins (1992)
sambucinum
TRI6 Transcription factor Proctor et al. (1995)
TRI12 Efflux pump Alexander et al. (1992)

"Different enniatins are also produced by several other Fusarium spp.

bTrichothecenes are produced by several different fungi and are toxic to a wide variety of organisms. The described genes
are part of a larger gene cluster (Hohn et al. 1998).

and Walton 1996; Walton et al. 1998). Ahn and
Walton (1996) showed that the Tox2 locus spans
at least 540kb of contiguous DNA on what is the
largest chromosome in most race 1 isolates of the
fungus. Though some race 1 isolates of C. car-
bonum have Tox2 on different sized chromo-
somes, when these isolates cross, the Tox2
chromosomes act as homologues (Canada and
Dunkle 1997). Genetic studies by Pitkin et al.
(2000) showed that when the Tox2 locus is on dif-
ferent sized chromosomes in crossed strains, it is
sensitive to physical breakage. Progeny from such
crosses produced strains with fragmented Tox2
loci. Those strains that still containcd at least one
of all the known Tox2-associated genes were still
pathogenic, though some had reduced virulence
indicating less HC toxin was produced. Scheffer et
al. (1967) may have observed similar strains when
they studied pro ge ny resulting from crosses of
race 1 isolates from different geographie areas.
Their conclusion, that the amount of toxin pro-
duced was conditioned by several genes, has
proven to be correct.
B. Victorin
1. Biological Significance of Victorin
The toxic peptide victorin and its producing
fungus Cochliobolus victoriae Nelson are named
as a result of their association with the oat (Avena
sativa L.) variety "Victoria" and its derivatives.
Oat varieties derived from "Victoria" were widely
planted in North America by the 1940s due to
their resistance to specific races of the crown rust
fungus, Puccinia coronata Corda. Widespread
dependence on this genotype made the emergence
of victorin-producing C. victoriae particularly dev-
astating (Meehan and Murphy 1946). The suscep-
tibility of these varieties of oats to C. victoriae was
due to their extraordinary sensitivity to victorin, a
316
peptide toxin produced by the fungus (Meehan
and Murphy 1947). Victorin is among the most
phytotoxic compounds ever discovered when
assayed on sensitive oat genotypes, but it is almost
harmless to any other plant.
Victorin is a partially cyclic pentapeptide
containing unusual, modified amino acids, some
of which are chlorinated. Victorin C, the form
of victorin accumulating most abundantly in cul-
tures of C. victoriae, contains residues of 5,5-
dichloroleucine, 3-hydroxylysine, chloroacrylic
acid, 3-hydroxyleucine and a novel amino acid
named victalanine (Wolpert et al. 1985; Fig.16.1).
As is the case with many fungal phytotoxins, a
number of variations of victorin can be isolated
from culture filtrates. These alternate forms of
toxin differ in the degree of chlorination and
hydroxylation of various amino acid side chains
or, in one case, an amino acid substitution
(Wolpert et al. 1986).
The observation that sensitivity to victorin,
and consequently susceptibility to C. victoriae, is
A
HN~O
~O HN
NH 0
O~
OH
o
B
c>~ 0
COOH
CHCI
NH2 ~o
OH
D.G. Panaccione et al.
dominant to toxin insensitivity indicated that sus-
ceptible oat varieties contained a specific receptor
for victorin. Wolpert and Macko (1989) used an
1125 -labeled derivative of victorin as a probe for
potential receptors and identified a 100-kDa
protein that bound their labeled victorin in sus-
ceptible oat varieties but not resistant varieties.
A 15-kDa pro tein also bound victorin in both
resistant and susceptible varieties. Though other
researchers have observed binding of victorin to
several proteins in resistant and susceptible culti-
vars (Akimitsu et al. 1992; Loschke et al. 1994),
continued research on the 100-kDa victorin-
bin ding protein has provided surprising informa-
tion on a possible site of action for the toxin.
Wolpert et al. (1994) cloned a cDNA for the
100-kDa victorin-binding protein from a library
enriched for this gene by using mRNA isolated
from in vitro translation complexes immunopre-
cipitated with antibody raised against the purified
lOO-kDa protein. DNA sequence analysis indi-
cated that the cDNA encoded the P pro tein
Fig. 16.1. Structures of several
peptide fungal phytotoxins. A
HC toxin I, B victorin C, and
C AM toxin I. Illustrations
represent chemical structures but
not necessarily bond angles or
conformations
 Fungal Phytotoxins
subunit of the mitochondrial glycine decarboxy-
lase complex. Subsequently, the 15-kDa protein
that bound victorin in a cultivar-nonspecific
manner was shown to be the H pro tein subunit of
the glycine decarboxylase complex (Navarre and
Wolpert 1995).
Although the glycine decarboxylase complex
may appear an unlikely site of action for a rapid
acting toxin like victorin, severallines of evidence
support the inter action of victorin with this mito-
chondrial enzyme complex. Susceptible leaf slices
treated with victorin have greatly reduced glycine
decarboxylase activity as compared to similarly
treated resistant leaf slices (Navarre and Wolpert
1995). Glycine decarboxylase is an integral co m-
ponent of photorespiration and victorin toxicity is
greatly reduced under conditions that eliminate
photorespiration (Navarre and Wolpert 1999a).
Pyrodoxal phosphate, a cofactor of the P pro tein
of glycine decarboxylase, competitively reduces
victorin binding to the 100-kDa protein and
reduces victorin-induced electrolyte leakage
(Wolpert et al. 1998). Moreover, to demonstrate
that victorin reduction of glycine decarboxylase
activity was not an indirect effect of organelle
or membrane disruption due to victorin toxicity
elsewhere in the cell, Navarre and Wolpert
(1995) compared the effects of victorin on glycine
decarboxylase with the effects of victorin on
light-dependent CO 2 fixation, another organelle-
localized cellular process that would be subject 10
similar indirect effects. Their data indicated that
glycine decarboxylase activity is 350 times more
sensitive to victorin than is light-dependent CO z
fixation.
In addition to its effects on glycine decar-
boxylase, victorin also caused premature senes-
cence of leaves by inducing an apoptotic response
in susceptible oats (Navarre and Wolpert 1999b).
Victorin induced a specific proteolytic cleavage of
the large subunit of ribulose bis-phosphate car-
boxylase/oxygenase (Rubisco), the critical enzyme
in photosynthetic CO z fixation and photorespira-
tion. Furthermore, DNA isolated from victorin-
treated leaf slices displayed pronounced apoptotic
laddering. Since glycine decarboxylase is a mito-
chondrial enzyme complex and mitochondrial
dysfunction is a hallmark of senescence, Wolpert
et al. (1998) have speculated on the potential con-
nection between the glycine decarboxylase effects
of victorin and the senescence-like response. This
potential connection is supported by the observa-
tions that glycine decarboxylase deficient mutants
317
of several species of plants senesce prematurely
(Wolpert et al. 1998).
2. Biosynthesis of Victorin
In Mendelian genetic analyses involving crosses
of victorin-producing isolates of C. victoriae with
nonproducing mutants of this species, or with com-
patible isolates of C. carbonum race 2, the ability
to produce victorin segregated as a single gene-
tic locus (Scheffer et al. 1967), now commonly
referred to as Tox3. Based on the structural co m-
plexity of victorin and on the molecular charac-
terization of the ToxI and Tox2 loci in other
Cochliobolus species (see descriptions under T
toxin and HC toxin, respectively), it is very likely
that Tox3 is a large tract of DNA containing genes
for several different biosynthetic enzymes.
The cloning of the Tox3 locus of C. victoriae
or individual genes within this locus has proven
problematic. The peptidic nature of victorin
indicates that one or more nonribosomal peptide
synthetases are involved in its synthesis. Genes
for fungal peptide synthetases contain enough
conserved amino acid residues for them to be
identified by PCR primed from degenerate
oligonucleotides (e.g., Nikolskaya et al. 1995;
Panaccione 1996; Johnson et al. 2000a). Attempts
to use this strategy for the isolation of a victorin-
associated peptide synthetase have been con-
founded by the several (perhaps many) peptide
synthetase genes in the genome of this fungus
(Nikolskaya et al. 1995; Yoder 1998).
Similarly, the strategy of biochemically identi-
fying a victorin peptide synthetase by assaying C.
victoriae for ATP/PPj exchange activities is co m-
plicated by the unavailability of the substrate
amino acids. Any plan for basing such assays on
the activation of likely precursors to the victorin
constituents (e.g., leucine for dichloroleucine) may
also be confounded by the large numbers of
potential peptide synthetases in Cochliobolus
spp., some of which are also likc1y to activate
common amino acids that would serve as substi-
tutes for the amino acids present in victorin.
C. AM Toxin
1. Biological Significance of AM Toxin
Alternaria blotch of apple (Malus domestica
Borkh.) is caused by the apple pathotype of
318 D.G. Panaccione et al.
Alternaria alternata (Fr.:Fr.) Keissler (previously
described as a virulent form of A. mali Roberts).
This disease is one of the most serious diseases
of apple in Japan (Sawamura 1966) and has been
increasing in incidence worldwide (Sawamura
1990; Filajdic and Sutton 1991). The causal fungus
pro duces multiple host-specific toxins named
AM toxin I (alternariolide), AM toxin II, and AM
toxin III, which selectively affect a narrow range
of susceptible apple cultivars (Kohmoto et al.
1974).
The chemical structure of AM toxin I has been
elucidated (Okuno et al. 1974; Ueno et al. 1975,
1977) and consists of a four-membered cyclic dep-
sipeptide, consisting of one standard amino acid,
L-alanine (L-Ala), and three unusual residues, L-a-
aminomethoxyphenylvaleric acid (L-amv), L-a-
hydroxyisovaleric acid and dehydroalanine (Fig.
16.1). AM toxins II and III were shown to be
derivatives of AM toxin I, and have either L-a-
aminophenylvaleric acid or L-a-aminohydrox-
yphenylvaleric acid in pi ace of L-amv. Of the three
derivatives, AM toxin I is the most abundant and
cytotoxic in spore germination fluids of virulent
isolates (Kohmoto et al. 1976).
Physiological and ultrastructural studies have
identified two primary sites of action for this toxin:
the cell wall/plasma membrane interface, where
the toxin causes plasma membrane invagination
and cell-wall degradation (Park et al. 1977); and
the chloroplast, where the toxin induces fragmen-
tation and vesiculation of grana lamellae (Park
et al. 1981). Dysfunction of either one of these
organelles, but especially the plasma membrane,
leads to suppression of the host defense reaction,
fungal penetration, and induction of disease
(Kohmoto and Otani 1991; Shimomura et al.
1991). Disease symptoms first appear on leaves
in late spring or early summer as round purpie to
black spots that gradually enlarge and coalesce.
When lesions occur on petioles, early leaf senes-
cence can occur, resulting in up to 60% defoliation
on susceptible cultivars (Filajdic and Sutton 1991).
Severe defoliation often leads to premature fruit
drop.
Apple-breeding experiments have suggested
that susceptibility to Alternaria blotch is con-
trolled by a single dominant gene, with most sus-
ceptible apple cultivars appearing heterozygous
and only the homozygous recessive state provid-
ing resistance (Saito and Niizeki 1988). This sup-
ports the presence of a receptor for AM toxin on
the plasma membrane and/or chloroplast of sus-
ceptible cells, but to date this receptor has not
been identified.
2. Biosynthesis of AM Toxin
Given the cyclic peptide structure of AM toxin,
nonribosomal synthesis by a peptide synthetase
seemed probable. Peptide synthetases are highly
conserved and there are numerous toxins that are
synthesized in a similar manner (including those
summarized in this chapter; for reviews, see
Kleinkauf and von Döhren 1990; Marahiel et al.
1997; von Döhren et al. 1997). Because of the
unusual nature of three of the constituent
residues, traditional methods to identify this
enzyme class (such as ATP/PP j exchange with
purified enzyme) were not feasible. However, the
highly conserved nature of core motifs within
peptide synthetase enzymes allows the corre-
sponding genes to be identified by the polymerase
chain re action (PCR; Nikolskaya et al. 1995;
Panaccione 1996). By analysis of a peptide syn-
thetase gene isolated through such a PCR-based
strategy, AM toxin was shown to be synthesized
on a nonribosomal peptide synthetase, AMT,
which has a predicted molecular mass of 479kDa
(Johnson et al. 2000a). AMT is coded for by AM
toxin synthetase (AMT), a gene 13.1 kb in length
which contains no introns and is present in multi-
ple copies, only one of which appears active
(Johnson et al. 2000a). Gene knockout experi-
ments have proven a crucial role for AMT in the
biosynthesis of AM toxin (Johnson et al. 2000a)
and the gene is required and present in all AM
toxin-producing strains (Johnson et al. 2000b).
Peptide synthetase genes share a common
modular structure with conserved core motifs
whose functions have been extensively reviewed
(Turgay and Marahiel 1994; Stachelhaus and
MarahieI1995a,b; von Döhren et al. 1997) and are
also discussed at the beginning of this section.
Analysis of AMT identified four catalytic domains
(designated A, B, C and D) responsible for the
adenylation and thioesterification of each con-
stituent residue during synthesis of AM toxin
(Johnson et al. 2000a). The first two core motifs
conserved within other peptide synthetases
appear to be missing in module D of AMT which,
to date, is a unique observation (Johnson et al.
2000a).
The unusual nature of three of the amino acid
constituents of AM toxin indicates that genes
in addition to AMT are required for AM toxin
 Fungal Phytotoxins
biosynthesis. Based on analogy with other toxin
biosynthetic gene clusters (Keller and Hohn 1997;
see HC toxin and AK toxin, below), it is likely that
AMT and additional AM toxin biosynthetic genes
are part of a larger gene cluster. The finding that
AMT is part of a large duplication that extends at
least 10kb to either side of the gene (Johnson et
al. 2000a) is reminiscent of the HC toxin biosyn-
thetic locus (Tox2, described above) and supports
the hypothesis of an AM toxin gene cluster.
D. Other Peptide Phytotoxins
The three phytotoxins described in detail above
are all host-specific toxins. There are nnmerous
other peptide toxins produced by plant pathogenic
fungi. With the possible exception of destruxin B
(described below), these toxins do not delimit the
host range of their producing organisms like the
well-studied toxins described above. However,
they are still agriculturally significant because of
their role in virulence on their respective hosts,
or toxicity to humans or livestock who consume
affected plants. Three examples, the destruxins, the
enniatins, and the ergopeptines, are discussed
briefty below.
Destruxin B is a cyclic depsipeptide consist-
ing of D-hydroxymethylvaleric acid, L-proline, L-
isoleucine, N-methyl-L-valine, N-methyl-L-alanine,
and ß-alanine. This peptide is the most abundant
and best studied of a family of similar peptides
produced by Alternaria brassicae (Berk.) Sacc., the
"black spot" pathogen of crucifers (Ayer and
Pena-Rodriguez 1987; Bains and Tewari 1987) and
also by Metarhizium anisopliae (Metsch.) Sor., a
pathogen of numerous insects (Gupta et al. 1989).
Destruxins cause necrosis and chlorosis on a
variety of plant species and thus do not necessar-
ily fit the definition of host-specific toxins defined
above. However, among the plants affected by
destruxins, species of Brassica are approximately
tenfold more sensitive (Buchwaldt and Green
1992). Arecent report indicates that radiolabeled
destruxin B is detoxified to less phytotoxic
hydroxy forms at greater rates in plants resistant
to A. brassicae than in susceptible plants (Pedras
et al. 1999).
Enniatins are cyclohexadepsipeptides pro-
duced by various plant pathogenic and ento-
mopathogenic species of Fusarium. Different
Fusarium species produce a complex of different
enniatins that contain alternating residues of an
319
N-methylated branched-chain L-amino acid
(leucine, isoleucine, or valine) and D-2-hydroxy-
isovaleric acid, repeated three times to make a
hexidepsipeptide (Deol et al. 1978). Enniatin syn-
thetase is a two-module peptide synthetase that
has all the activities required to synthesize enni-
atins (Zocher et al. 1982). This is exceptional
among nonribosomally synthesized peptides,
which often require additional enzymes for amino
acid activation or modification. In contrast to most
toxin-nonproducing isolates of other fungal
species, some enniatin-nonproducing isolates of
Fusarium spp. have homologues of the enniatin
peptide synthetase gene, esyn1 (Herrman et al.
1996a).
Enniatins act as ionophores (Benz 1978) and
it is hypothesized that they contribute to the viru-
lence of producing fungi on several species of
plants (Burmeister and Plattner 1987; Herrman
et al. 1996a). The role of enniatins in increasing
virulence of Fusarium avenaceum (Fr.:Fr.) Sacc.
to potato (Solanum tuberosum L.) has been well
documented. Strains of F. avenaceum in which
the enniatin synthetase gene had been inactivated
by gene knockout still infected potato tubers
but caused significantly less disease compared to
control isolates of the fungus (Herrman et al.
1996b).
Ergopeptines (e.g., ergotamine and ergova-
line ) are a family of four-component peptides con-
taining lysergic acid and three amino acids that
vary among different members of the ergopeptine
family (Panaccione 1998). These peptide forms of
ergot alkaloids are produced by several plant-
pathogenic and plant-mutualistic fungi in the
family Clavicipitaceae. Although ergopeptines are
not toxie to the plants that serve as hosts for these
fungi, their toxicity to mammals that consume
affected plants makes them significant toxins from
an agricultural perspective. Ergopeptines are
highly vasoconstrictive (Clark et al. 1978) and also
act as agonists of serotonin and dopamine
(Rutschman and Stadler 1978), resulting in a
pleiotropic condition known as ergotism or St.
Anthony's fire in people who consume these
toxins. Symptoms of ergotism include increased
blood pressure and body temperature, destruction
of the nervous system, reduced reproductive capa-
bility, and gangrene of the extremities. Histori-
cally, the consumption of ergopeptines (primarily
ergotamine) produced by Claviceps purpurea
(Fr.:Fr.) Tul., pathogenic on rye (Secale cereale L.),
led to significant human suffering (Matossian
320 D.G. Panaccione et al.
1989). Recognition of C. purpurea as the source of
ergotism, a better diet, and mechanical means of
removing the fungus and its associated toxins from
grain have essentially eliminated human ergotism
(Matossian 1989; Schumann 1991). However, the
accumulation of ergopeptines (primarily ergova-
line ) in forage grasses infected with endophytic
fungi from the genus Neotyphodium causes sig-
nificant problems in grazing animal production
(Schardl and Philips 1997; Siegel and Bush 1997).
Biochemical evidence indicates that in C. pur-
purea the enzyme responsible for adenylating and
thiolating lysergic acid is a separate polypeptide
from the one catalyzing similar activities for the
three typical amino acids found in ergotamine
(Riederer et al. 1996). This division of activities
among polypeptides makes the ergopeptine
peptide synthetase unlike most other eukaryotic
peptide synthetases, in which adenylation and
thiolation activities for each constituent residue
are contained on a single polypeptide (Marahiel
et al. 1997; von Döhren et al. 1997). A peptide syn-
thetase gene required for ergovaline biosynthesis
has recently been cloned from Neotyphodium lolii
(Latch, Christensen & Samuels) Glenn, Bacon &
Hanlin and its function demonstrated by gene
knockout (Panaccione et al. 2001). A similar gene
has been cloned from C. purpurea (Panaccione
1996; Tudzynski et al. 1999; Panaccione et al.
2001). As is the case with most toxin-producing
fungi, isolates or closely related species that do
not produce ergopeptines have no homologue of
the peptide synthetase gene (Panaccione et al.
2001). The peptide synthetase from C. purpurea is
very tightly linked to an additional ergot
alkaloid biosynthesis gene (Tudzynski et al. 1999;
Panaccione et al. 2001), which is typical of fungal
toxin biosynthetic loci.
111. Polyketide-Derived Phytotoxins
Polyketides are naturally occurring compounds
that are commonly produced by plants and actin-
omycetes in addition to fungi. They are highly
diverse in structure, ranging from simple aromatic
compounds like 6-methylsalicylic acid (Jordan and
Spencer 1993) to complex polycyclic compounds
like maitotoxin (Murata and Yasumoto 2000),
which is the largest known secondary metabolite
characterized to date. Other examples of polyke-
tides include antibiotics (e.g., tetracycline), anti-
fungal agents (e.g., griseofulvin), immunosuppres-
sive agents (e.g., rapamycin), mycotoxins (e.g.,
aflatoxin) and phytotoxins (see T toxin, cer-
cosporin and AK toxin, below). In addition, many
other metabolit es (e.g., cyclosporine and HC
toxin) that are synthesized via an independent
pathway also contain polyketide-derived moieties.
Although polyketides are diverse in structure,
they all share a common biosynthetic mechanism
involving polyketide synthases (PKSs). PKSs are
large multifunctional enzymes that synthesize the
carbon backbones common to this class of mole-
cule (reviewed in Hutchinson and Fujii 1995; Tsoi
and Khosla 1995). PKSs are closely related to fatty
acid synthases (FASs), and, like them, catalyze
repeated condensations of acyl CoA esters to
build a linear polyketide chain. Linear fatty acids
are essentially fully reduced polyketides derived
exclusively from acetate and malonate precursors.
AK toxin (described below) from Alternaria alter-
nata (Fr.:Fr.) Keissl. Japanese pear pathotype
contains a fatty acid backbone, most probably syn-
thesized by a FAS. It will therefore be considered
here, under the subheading of polyketide-derived
phytotoxins.
Unlike FASs, which use acetate and malonate
as starter and extender units, respectively, PKSs
can use a variety of chain starter units (e.g.,
acetate, propionate, benzoate, cinnamate, and
amino acids), and a range of extender units (e.g.,
malonate, methylmalonate, or ethylmalonate).
Both PKSs and FASs typically have an acyl carrier
domain (ACP) containing a phosphopantetheine
moiety. Synthesis is initiated by an acyl transferase
(AT) domain that transfers the starter unit, as a
thiol ester, to the phosphopantetheine moiety on
the ACP domain. Each condensation can be fol-
lowed by a cycle of ketoreduction (catalyzed by
ß-ketoacyl reductase), dehydration (mediated by
adehydratase) and enoyl reduction (via enoyl
reductase; Wakil 1989; Hopwood and Sherman
1990; Hopwood and Khosla 1992). The polyketide
chain is typically released from the PKS by cleav-
age of the thiol ester, usually accompanied by
cyclization. Not all PKSs have or use all of the
catalytic domains mentioned above and it this,
together with variation in chain length, reductive
modification, stereochemistry, and the mode of
cyclization, that generates the structural diversity
seen for this group of compounds.
There are two major classes of PKS enzymes.
Modular type I PKSs contain separate catalytic
domains for each reaction catalyzed in the biosyn-
 Fungal Phytotoxins
thetic pathway. The active sites for each round of
processing are encoded by a single open reading
frame containing DNA modules ordered as they
are required biochemically. Type II PKSs contain
fewer active domains, which are encoded only
once in the DNA open reading frame. These
domains are therefore used repeatedly as
required. Filamentous fungi, like those discussed
here, contain PKSs that structurally resemble type
I enzymes. However, enzymatically, they resemble
type II enzymes since they have only one domain
per catalytic activity, which must be repeatedly
used, for each catalytic cyde (Tkacz 2000).
A. TToxin
1. Biological Significance of T Toxin
T toxin was the central molecule in the southem
com leaf blight epidemic of 1970. This epidemic
caused substantial losses in the com crops of the
United States and resulted in critical assessment
of genetic uniformity in our major crops. The
southem com leaf blight epidemic was caused
by Cochliobolus heterostrophus (Drechs.) Drechs.
ra ce T, which pro duces T toxin. More common,
toxin-nonproducing isolates of C. heterostrophus
are referred to as race 0. Whereas race 0 isolates
produce small necrotic lesions on a wide range
of maize varieties, race T isolates produce large
necrotic lesions with chlorotic streaking specifi-
cally on maize varieties carrying the Texas-type
male sterile cytoplasm (ernsT). As described in
detail below, the inter action between T toxin and
cmsT mitochondria results in the increased viru-
lence of C. heterostrophus race T.
T toxin is the general name given to a family
of partially reduced linear polyketols ranging from
35 to 45 carbons in length (Kono and Daly 1979;
Fig.16.2). The distantly related fungus Phyllosticta
maydis Amy & Nelson pro duces a similar family
of compounds called PM toxins (chain lengths
ranging from 33 to 35 carbons) that have similar
activity and confer identical host specificity (Kono
et al. 1983; Danko et al. 1984). Phyllosticta maydis
was restricted to the cooler northem regions of the
US and never had the impact on maize production
that the more widely distributed and southem C.
heterostrophus race T did (Rhoads et al. 1998).
Sensitivity or insensitivity to T toxin, with
resulting susceptibility or resistance to ra ce T of C.
heterostrophus, is due to the presence or absence
321
000
H
0
A
OH
o
o
H
o 0
c o
H
o
Fig. 16.2. Structures of several polyketide or fatty acid
derived fungal phytotoxins. A T toxin band I, B AK toxin
I, and C cercosporin. Illustrations represent chemical struc-
tures but not necessarily bond angles or conformations
of a small chimeric protein in the inner mitochon-
drial membrane (Levings 1990). This protein,
URF13 (Dewey et al. 1987; Wise et al. 1987), is the
product of a novel gene (T-urf13) that apparently
arose from recombination of mitochondrial DNA
containing the promoter of the gene for ATPase
subunit 6 joined with fragments of the 26S ribo-
somal RNA gene and additional sequences with
unspecified original function. URF13, in addition
to conferring sensitivity to T toxin, appears to be
the factor responsible for Texas-type cytoplasmic
male sterility (ernsT), a desirable agronomic trait
(Levings 1990). Because of the reduced cost and
labor associated with male sterility, cmsT was
incorporated into most maize germplasm, making
the appearance of C. heterostrophus race T excep-
tionally devastating.
The critical nature of the interaction ofT toxin
with URF13 to toxin activity has been demon-
strated by expressing T-urf13 in organisms that are
not sensitive to T toxin and converting them to
toxin sensitivity. This was first done in Escherichia
322 D.G. Panaccione et al.
eali that, after expression ofT-urJ13, responded to
T toxin in a manner typical of cmsT mitochondria.
URF13-containing E. eali exposed to T toxin
showed a dramatic decrease in glucose-driven res-
piration, coupled with leakage of small molecules
and spheroplast swelling, whereas a control strain
was insensitive to the toxin (Dewey et al. 1988;
Braun et al. 1989).
In yeast (Saeeharomyees eerevisiae Hansen)
transformants, URF13 had to be targeted to mito-
chondria in order for cells to become sensitive
to T toxin, which was demonstrated by greatly
reduced growth relative to control strains on
medium containing T toxin (Huang et al. 1990).
However, in transgenic tobacco (Nieatiana
tabaeum L.) plants, expression of URF13 con-
verted plants to T toxin sensitivity, demonstrated
by light-dependent bleaching on exposure to T
toxin, regardless of its cellular localization (von
Allmen et al. 1991). The ability of URF13 to
confer sensitivity to T toxin in organisms ranging
from bacteria to fungi to plants, suggests that this
protein alone is sufficient to confer the toxin sen-
sitive phenotype.
Oligomers of URF13 are hypothesized to
form pores in mitochondrial membranes that are
gated by binding T toxin (or PM toxin). In support
of this hypothesis, tritiated toxin binds directly to
URF13 in a specific and saturable manner in cmsT
mitochondria and in E. eali expressing T-urJ13
(Braun et al. 1990). URF13 has been immunocy-
tochemically localized in the inner mitochondrial
membrane (Hack et al. 1991; Korth et al. 1991).
Binding of T toxin to URF13 occurs in a co-
operative manner, which is typical of oligomeric
pro teins (Braun et al. 1990). Protein cross-linking
studies (Korth et al. 1991; Kaspi and Siedow 1993)
provide further evidence that URF13 acts as an
oligomer and indicate that four molecules of
URF13 associate and form an open pore upon
binding T toxin or PM toxin resulting in ion
leakage and mitochondrial debilitation (Levings
1990; Korth et al. 1991; Rhoads et al. 1998).
2. Biosynthesis of T Toxin
Similar to the genetics of the peptide toxin-
producing Caehliabalus species described above,
the ability of C. heterastraphus to produce T toxin
was originally defined as a single Mendelian locus
called Taxi (Lim and Hooker 1971; Leach et al.
1982). Molecular analysis has revealed that Taxi
contains at least two genes that are located on two
separate chromosomes associated with a translo-
cation in toxin-producing isolates and a significant
deletion in toxin-nonproducing isolates (Kodama
et al. 1999). As predicted from the structure of T
toxin, one of the Taxi-associated genes is a type I
PKS (Yang et al. 1996). This gene was originally
tagged by the restriction enzyme mediated inte-
gration (REMI) procedure (Lu et al. 1994).
Sequencing of the vector integration site revealed
a 7.8-kb open reading frame that, in addition to
encoding the domains required for synthesis of
the primary polyketide (ß-ketoacyl synthase, acyl
transferase, and acyl carrier protein), also encodes
domains for all the keto-processing functions
(Yang et al. 1996), which makes this PKS unique
among fungal PKSs (Yoder 1998). This PKS gene,
PKSi, is present in all race T isolates and absent
from all race 0 isolates of C. heterostraphus. Dis-
ruption of this gene in a wild-type race T isolate
resulted in a T toxin minus phenotype, with
virulence reduced to the level of T toxin-
nonproducing ra ce 0 isolates of the fungus (Yang
et al. 1996). PhyUastieta maydis, which produces
the polyketide PM toxin, contains a similar PKS
(62% identical at the nucleotide level) that is
required for PM toxin production and pathogenic-
ity to cmsT maize (Yoder 1998; Yun et al. 1998).
A second Taxi-associated gene, DECi, has
also been cloned and shown to be required for
T toxin biosynthesis and full virulence on cmsT
maize (Yoder 1998). Based on DNA sequence
analysis, it is hypothesized that D ECi encodes a
decarboxylase that removes the terminal car-
boxylic acid group from the polyketide precursor
resulting in chains with odd numbers of carbon
atoms (Yoder 1998). DECi and a reductase
gene [REDi, also found exclusively in T toxin-
producing isolates but not required for T toxin
production (Yoder 1998)] are part of a large
region of unique DNA associated with Taxi.
Inactivation of DECi by gene disruption leads to
lack of toxin production and reduced virulence
(Yoder 1998).
Since both PKSi and DECi segregate with
Taxi in crosses of race T and race 0 isolates of
C. heterostraphus, it was surprising that they
are clearly located on different chromosomes
in Southern hybridization of pulsed field gels
(Kodama et al. 1999). This apparent contradiction
is resolved by the association of Taxi with a
translocation break point (Tzeng et al. 1992;
Chang and Bronson 1996; Kodama et al. 1999).
P KSi, D ECl, and the sequences Banking these
 Fungal Phytotoxins
genes are all unique to race T isolates of C.
heterostrophus and, in crosses with race 0 iso-
lates, these Tox1-associated sequences segregate
together as part of a four-armed linkage group.
In crosses between race T isolates, the loci can
segregate independently because homologous
sequences are present along the lengths of the
translocated chromosomes (Kodama et al. 1999).
B. AK Toxin
1. Biological Significance of AK Toxin
The Japanese pear pathotype of Alternaria alter-
nata (Fr.:Fr.) Keissl. produces the polyketide or
fatty acid derived metabolite AK toxin and causes
black spot on susceptible cultivars of Japanese
pear (Pyrus pyrifolia Nakai), particularly the
important commercial cultivar, Nijisseiki (Tanaka
1933; Nakashima et al. 1985; Otani et al. 1985).
This disease is a critical problem in Japan, and first
became established in the early 1900s (Tanaka
1933; Nishimura and Kohmoto 1983). The disease
occurs on the fruit, young leaves and young shoots,
but never on old leaves and branches. On fruits,
infection first appears as small black specks that
expand into round brown spots, often with black
concentric rings. Under wet conditions the spots
rapidly expand, causing uneven growth, cracking
and eventually rotting of the fruit. On leaves, small
dark-brown or black/brown specks appear in early
summer and slowly enlarge. Later, concentric
rings appear on the lesions and severely infected
trees can become defoliated.
Although most host -specific toxins are diverse
in structure, AK toxin shares a common moiety
with AF toxin (Otani et al. 1972) and ACT toxin
(Kohmoto et al. 1979) of the strawberry and
tangerine pathotypes of A. alternata, respec-
tively. This moiety, 9,10-epoxy-8-hydroxy-9-
methyldecatrienoic acid, was identified as a
precursor to all three toxins (Feng et al. 1990;
Nakatsuka et al. 1990; Kohmoto et al. 1993) and
exhibits a structure typical of polyketide or long
chain fatty acid metabolites. AK toxin occurs as
two related molecular species, AK toxin I (Fig.
16.2) and AK toxin 11 (Nakashima et al. 1982,
1985). AK toxin I is the most abundant and active
species, exhibiting toxicity to only a very narrow
range of susceptible pear cultivars (Otani et al.
1985). This all-or-nothing specificity matches
exactly the host or nonhost response to the
323
pathogen. AK toxin 11 is a dimethyl derivative of
AK toxin I and shows the same specificity, but with
an activity 1I20th that of AK toxin 1.
The site of action for AK toxin is on the
plasma membrane, near to plasmodesmata of sus-
ceptible cells (Park et al. 1987, 1992). An early
effect includes the depolarization of membrane
electropotential, within 5 min of toxin treatment
(Namiki et al. 1986; Otani et al. 1989). This is fol-
lowed by electrolyte loss and plasma membrane
invagination within 1 h of exposure to the toxin
(Otani and Kohmoto 1992). The dysfunction of
the plasma membrane is a critical event for induc-
tion of accessibility by the pathogen and a specific
recognition site (receptor) on the plasma mem-
brane of susceptible cultivars has been proposed
(Otani et al. 1995), but not yet identified.
2. Biosynthesis of AK Toxin
Genes required for biosynthesis of AK toxin were
initially identified by insertion al mutagenesis by
the REMI technique (Tanaka et al. 1999). Toxin-
deficient REM I transformants were further char-
acterized and two genes, AKT1 and AKT2, were
cloned (Tanaka et al. 1999). Gene disruption
experiments showed that these genes are involved
in AK toxin biosynthesis. Gene homologues have
also been identified in the strawberry and tanger-
ine pathotypes of A. alternata (Tanaka et al. 1999;
Masunaka et al. 2000; Tanaka and Tsuge 2000),
suggesting that these genes are involved in the
biosynthesis of the polyketide derived 9,10-epoxy-
8-hydroxy-9-methyldecatrienoic acid precursor.
AKT1 encodes a carboxyl-activating enzyme
(Aktl) and is likely required to activate an earlier
precursor of 9,l0-epoxy-8-hydroxy-9-methyldeca-
trienoic acid for further modification by other
enzymes (Tanaka et al. 1999). AKT2 encodes a
pro tein (Akt2) of unknown function (Tanaka et al.
1999). Both of these genes are present in single
copies but multiple nonfunctional homologues
have also been identified. AKT1 and AKT2 are
located within a gene cluster and two additional
genes, AKTR -1 and AKT3-1, have been identified
downstream of AKT2 (Tanaka and Tsuge 2000).
AKTR-1 and AKT3-1 are present in multiple
copies in the genome and homologues have been
identified in both AF toxin and ACT toxin
producers, indicating that these genes are also
involved in the biosynthesis of the 9,10-epoxy-
8-hydroxy-9-methyldecatrienoic acid precursor
(Tanaka and Tsuge 2000).
324 D.G. Panaccione et al.
AKTR -1 encodes a 444 amino acid protein
(AktR-1) that contains a zinc binuclear cluster
DNA-binding domain in the amino terminal
region and an internal leucine zipper domain
(Tanaka and Tsuge 2000). The presence of a zinc
binuclear cluster domain suggests this pro tein
is involved in DNA binding and regulates the
expression of other pathway genes.
AKT3-1 encodes a 296 amino acid pro tein
with similarity to the hydratase/isomerase enzyme
family and also terminates with a type 1 peroxiso-
mal targeting signal (PTS1; Tanaka and Tsuge
2000). This signal is also present in Aktl and Akt2
(Tanaka et al. 1999), suggesting that 9,l0-epoxy-8-
hydroxy-9-methyldecatrienoic acid is synthesized
in peroxisomes.
Two other genes,AKTR-2 andAKT3-2, which
share high similarity with AKTR-l and AKT3-1,
respectively, have also been identified (Tanaka
and Tsuge 2000). Unlike AKT3-1 and AKTR-1,
these genes are present in single copies and
AKTR-2 appears unique to the Japanese pear
pathotype. This suggests that AKTR-2 plays a role
in the biosynthesis of the AK toxin specific moiety
in the A. alternata Japanese pear pathotype, but
gene disruption experiments indicate it is also
required for biosynthesis of the 9,l0-epoxy-8-
hydroxy-9-methyldecatrienoic acid moiety.
AK toxin biosynthesis is therefore controlled
by a strncturaIly and functionally complex gene
cluster that likely also includes a multifunctional
fatty acid synthase enzyme. All of the identified
genes and their homologues have been shown to
be located on a 4.1-Mb chromosome in the A.
alternata Japanese pear pathotype by pulsed field
gel electrophoresis (Tanaka and Tsuge 2000).
C. Cercosporin
1. Biological Significance of Cercosporin
Cercosporin is a secondary metabolite, deep red
in color, produced by several plant pathogenic
species of the genus Cercospora. Biologically, this
perylenequinone compound is considered to be
host-nonspecific, meaning that the compound is
toxic to many or all plant species, including those
that are not hosts of the fungus. Cercosporin is a
photosensitizing compound. Thus, it is not toxic to
cells in the dark. Biological activity results when
cercosporin absorbs light energy and, in an elec-
tronically excited state, reacts with oxygen to
produce active oxygen species. In the case of
cercosporin, both superoxide (Oz-) and singlet
oxygen eOz) are produced, but 10Z is thought to
be responsible for the most toxicity at the infec-
tion court (Daub and Hangarter 1983).
Cercosporin was first isolated from dried
mycelium of the soybean pathogen Cercospora
kikuchii Majsumoto & Tomoyasu (Kuyama and
Tamura 1957). Lousberg et al. (1971) and
Yamazaki and Ogawa (1972), working indepen-
dently, determined the structure of cercosporin to
be 1,l2-bis(2-hydroxypropyl)-2,1l-dimethoxy-6,7-
methy lenedioxy-4,9-dihydroxyperylene-3, 10-
puinone, with a molecular weight of 534.
Cercosporin has been shown to be toxic in
many laboratory models including mice, bacteria,
many species of fungi, human tumor cells, and
several species of plants (Foote 1976; Macri and
Vianello 1979; Chung et al. 1999). The compound
has been shown to inactivate pro tein kinase C and
to be a potent anti-viral agent (Tamaoki and
Nakano 1990; Hudson et al. 1994; Diwu 1995).
Cercosporin-produced 102 and O 2- result in oxi-
dation of fatty acids, sugars, cellulosic materials,
guanine and several amino acids. This action
results in damage to DNA, inactivation of
enzymes and destruction of cell membranes
(Foote 1976). In diseased plants, the effect of cer-
cosporin on host cell membranes is thought to be
critical. Cercosporin has been demonstrated to
cause lipid peroxidation of host cell membranes
within minutes of exposure to light (Daub 1982).
This changes the structure and composition of
host membranes (Daub and Briggs 1983) and
increases electrolyte leakage from the host (Daub
1982).
Light has been demonstrated to be essential
for disease in the pathosystem, an observation that
suggested cercosporin is necessary for disease
development. This conclusion was more firmly
supported when Upchurch et al. (1991) demon-
strated that mutants of the fungus deficient in
cercosporin production were nonpathogenic.
Whether cercosporin plays a critical role in
diseases caused by other species of Cercospora,
such as Cercospora beticola Sacc. on sugarbeet
(Beta vulgaris L.), for example, has not been weH
investigated.
As outlined above, cercosporin appears to be
toxic to nearly aH laboratory models tested.
Despite this, Cercospora species that produce
the toxin are unaffected. The basis of this auto-
resistance has received considerable attention in
 Fungal Phytotoxins
the past two decades. Daub and associates (sum-
marized in Daub et al. 1998) concluded that resis-
tance to cercosporin in cercosporin-producing
fungi cannot be explained by differences in mem-
brane fatty acids (targets of active oxygen) or in a
number of antioxidant activities (superoxide dis-
mutase, catalase, carotenoids, and so forth). It was
eventually demonstrated that a major component
of cercosporin auto-resistance is based on the
transient and reversible reduction of cercosporin
by mycelium of the producing fungus (Daub et al.
1992; Sollod et al. 1992).
The genetic basis for resistance to cercosporin
in Cercospora nicotianae Ellis & Everh. was inves-
tigated by a mutation al approach in the fungus,
followed by genetic complementation through
transformation. Six mutants of the fungus were
isolated that were sensitive to cercosporin and
lacked the ability to grow in the presence of
the toxin (Jenns and Daub 1995). Five of the six
mutants were similar and apparently harbored
mutations in the same gene. The sixth mutant,
which had physiological characteristics different
from the first five (Jenns and Daub 1995), was
found to possess a mutation in a second gene
(described below). All five mutants in the first
group were sensitive to low levels of cercosporin
(0.1,uM) and sensitivity to toxin increased with
light intensity (Jenns and Daub 1995). Functional
complementation experiments resulted in the
cloning of a gene, designated SORi (singlet
oxygen resistance 1), from a wild-type library of
the fungus. SORi restored wild-type resistance
to cercosporin in each of the five mutants. In
other experiments, targeted disruption of wild-
type SORi resulted in cercosporin sensitivity
(Ehrenshaft et al. 1998). Homologues of SORi
were detected in four kingdoms of organisms, but
the function of the gene initially was not known.
Ehrenshaft et al. (1999) later demonstrated that
SORi encoded a gene involved in pyridoxine
(vitamin B6) biosynthesis. This revelation uncov-
ered a second, distinct pathway for pyridoxine
biosynthesis previously unknown, and demon-
strated for the first time that pyridoxine possessed
singlet oxygen quenching activity.
The sixth cercosporin-sensitive mutant of C.
nicotianae described by Jenns and Daub (1995)
and not complemented by SORi was used to
clone a second gene for cercosporin resistance.
This gene, designated CRGi (cercosporin resis-
tance gene 1), was also cloned by functional com-
plementation from the wild-type library (Chung et
325
al. 1999). CRG], a single copy gene found only in
Cercospora species, encodes a 550 amino acid
protein with four putative trans-membrane helical
domains. No homology was found between the
deduced product of CRGi and other proteins in
databases. Targeted disruption of this gene results
in cercosporin sensitivity (Chung et al. 1999).
2. Biosynthesis of Cercosporin
In addition to being light-activated, production of
cercosporin itself is induced by light (Jenns et al.
1989). This observation led to the hypothesis that
fungal genes involved in cercosporin biosynthesis
and/or secretion might be regulated by light. Using
the soybean pathogen C. kikuchii as a model,
Ehrenshaft and Upchurch (1991) identified six
cDNAs that were up-regulated by light. One of
these, designated LE6, was targeted for additional
studies because its corresponding transcript was
shown to be induced 20-fold by light. Callahan et al.
(1999) demonstrated that the C. kikuchii gene CFP
corresponding to this highly light-induced cDNA
encodes a 65.4-kDa protein with homology to the
ToxA HC toxin transporter described above and
with other members of the major facilitator super-
family of membrane transporters known to be
involved in antibiotic and toxin secretion. Thus, it
was hypothesized that the gene is a transporter of
cercosporin. Targeted disruption of this gene in C.
kikuchii resulted in reduced accumulation of cer-
cosporin, reduced virulence on soybean [Glycine
max (L.) Merrill], and increased sensitivity of the
fungus to exogenously supplied cercosporin.
Based on the polyketide nature of cer-
cosporin, Upchurch and Eweida (2000) have
undertaken a PCR-based approach to identify the
PKS involved in cercosporin biosynthesis. PCR
amplification primed from degenerate oligonu-
cleotides designed to anneal to conserved
sequences in the acyl transferase and ketoacyl syn-
thase domains yielded a fragment of the predicted
size that hybridizes specifically to mRNA from
cercosporin-producing cultures. The identity of
this particular candidate gene as the cercosporin
PKS remains to be tested. However, Daub and
associates have recently used the REMI technique
to produce 38 mutants of C. nicotianae with
altered levels of cercosporin production (M.E.
Daub, pers. comm.). Integrated plasmid and flank-
ing genomic sequences were recovered from some
of the mutants and used as transformation vectors
designed to produce knockout mutants in the
326 D.G. Panaccione et al.
wild-type C. nicotianae. Five such vectors pro-
duced cercosporin-deficient mutants in transfor-
mation experiments. Sequence analysis of the
flanking genomic DNA in two of those vectors
revealed high homology with the acyl/malonyl
transferase domain of PKSs from other organisms.
Disruption of the PKS gene in C. nicotianae
resulted in cercosporin-deficient mutants. This
work provides the first direct evidence that cer-
cosporin is produced through the polyketide
pathway.
D. Other Polyketide-Derived Phytotoxins
A variety of other fungal polyketides are pro-
duced by plant-associated fungi. Some of these
additional important polyketides are summarized
briefly below.
AAL toxins I (Ta) and II (Tb) are produced
by Alternaria alternata f. sp. lycopersici (A. alter-
nata tomato pathotype) which causes stern canker
disease on susceptible tomato (Lycopersicon escu-
lentum Mill.) cultivars (Gilchrist and Grogan 1976;
Kohmoto and Otani 1991). AAL toxin shows dis-
tinct structural similarity to the mammalian toxin
fumonisin, which is produced by the maize
pathogen Gibberella jujikuroi (Sawada) Ito in Ito
& Kimura, and both toxins exhibit cross-toxicity
to plant and mammalian cells (Mirocha et al. 1992;
Wang et al. 1996). In addition, both toxins appear
to inhibit ceramide synthase, which is involved
in sphingolipid biosynthesis (Wang et al. 1991).
Given the structural similarity of these two toxins,
the mode of biosynthesis is likely to be similar.
Biochemical analysis suggested that fumonisins
and AAL toxins were products of either a polyke-
tide or fatty acid synthase. peR from degenerate
primers based on PKS domains and template from
fumonisin-producing strains of G. jujikuroi, facil-
itated the cloning of a fumonisin biosynthesis
gene, FUM5 (Proctor et al. 1999). This gene con-
tains a 7.8-kb open reading frame predicted to
encode a pro tein with high similarity to bacterial
and fungal type I PKSs. Gene knockout experi-
ments provided strong evidence that FUM5 is
required for fumonisin biosynthesis (Proctor et al.
1999). This gene is part of a larger gene cluster,
which has been previously identified in G. juji-
kuroi fumonisin-producing isolates (Desjardins et
al. 1996a). The biosynthesis of AAL toxin is
hypothesized to involve a similar gene cluster in
A. alternata apple pathotype.
Aßatoxins are mycotoxins produced by
several Aspergillus species. Although not con-
sidered factors in plant pathogenesis, they are
agriculturally important as contaminants of food
and feed. The biosynthesis of aflatoxins has been
extensively studied and pro vi des a good model for
polyketide biosynthesis by filamentous fungi. The
biosynthesis of aflatoxins is controlled by at least
20 clustered genes and has been recently reviewed
(Brown et al. 1999).
Although aflatoxins are not considered phy-
totoxins, the structurally related compound doth-
istromin produced by Dothistroma pini Hulbary
has been implicated as the major pathogenicity
factor in Dothistroma ne edle blight, a major
disease of pine trees in New Zealand (Bradshaw
et al. 1997, 2000). Several genes involved in the
biosynthesis of this dothistromin have been cloned
from D. pini on the basis of homology to genes in
the aflatoxin biosynthetic pathway (R. Bradshaw,
pers. comm.).
IV. Proteinaceous Phytotoxins
Traditionally, fungal toxins have been thought of
as secondary metabolites. However, during the
past decade, several proteinaceous toxins, either
known or presumed to be encoded directly by
fungal genes, have been discovered and studied.
These toxins are pro teins in the classical sense
because they are primary gene products with
amino and carboxy termini and are translated
on ribosomes. With the re cent establishment
that fungal phytotoxins may be proteinaceous in
nature, it is likely that additional proteinaceous
toxins will be identified and characterized in the
near future. All of the toxins described below are
secreted and two of the four are rather sm all for
pro teins (less than 15kDa). Otherwise, the group
apparently shares little else in common. Because
they are primary gene products, these toxins are
especially weIl suited for molecular biological
investigation.
A. Ptr Toxins
1. Biological Significance of Ptr ToxA
Pyrenophora tritici-repentis (Died.) Drechs. is the
causal agent of tan spot of wheat (Triticum aes-
 Fungal Phytotoxins
tivum L.) and other grasses. Five races of the
fungus have been described based on host range
and lesion symptoms on host leaves. Race 1, the
most prevalent form of the fungus in commercial
wheat fields in the United States (Lamari and
Bernier 1991; Ali and Francl 1998), pro duces
necrotic lesions (nec+) surrounded by extensive
areas of chlorosis (chl+). Race 2 (nec+chl-) is vir-
ulent on wheat but is extremely rare in nature.
Races 3 and 5 are both nec- chl+, but can be dif-
ferentiated by wheat cultivars affected. Race 4 is
avirulent on wheat (nec-chl-), but has been iso-
lated as a pathogen of wild grasses.
Ptr ToxA, formerly known as Ptr toxin and
Ptr necrosis toxin (Ciuffetti et al. 1998), is a host-
selective, necrosis-inducing toxin produced by
races 1 and 2 of P tritici-repentis. The toxin was
first detected in culture fluids of the fungus by
Tomas and Bockus (1987). In the initial report, all
tan spot susceptible wheat cultivars examined
were shown to be sensitive to the toxin; all tan spot
resistant cultivars and a resistant barley cultivar
were insensitive to the culture filtrates. Since then,
several toxin-insensitive lines have been discov-
ered. Lamari and colleagues (Lamari and Bernier
1989, 1991) developed host populations that seg-
regated for sensitivity to Ptr ToxA and suscepti-
bility to tan spot. A perfect correlation was found
between toxin sensitivity and disease susceptibil-
ity or, conversely, between toxin insensitivity and
disease resistance. Further, Lamari et al. (1995)
used antibodies against Ptr ToxA to detect toxin
in the intercellular wash fluids (IWF) of wheat
plants inoculated with races 1 and 2. The toxin was
not detected in the IWF of wheat plants inocu-
lated with races 3 and 4, forms of the fungus not
known to produce toxin in culture. Based on these
observations, it was proposed that Ptr ToxA is a
host -selective toxin critical for tan spot develop-
ment. Toxin sensitivity in wheat is controlled by a
single dominant gene that maps to chromosome
5BL (Faris et al. 1996).
Four different research groups have iso la ted
and characterized Ptr ToxA. Ballance et al. (1989)
reported that the toxin was a heat-labile protein
of 13.9kDa with an average minimum active con-
centration of 0.2 nM. Tomas et al. (1990) described
the toxin as a heat-stable protein of 14.7kDa
with an average minimum active concentration of
90 nM. Although differences were found in
average minimum active concentration and heat
stability, only minor differences were found in
amino acid composition. Tuori et al. (1995) puri-
327
fied a heat-stabile necrosis-inducing toxin of 13.2
kDa with an average minimum active concentra-
tion of 60nM. Most recently, Zhang et al. (1997),
using the same P. tritici-repentis isolate as Ballance
et al. (1989), have demonstrated that the toxin is
a 13.2-kDa protein. The deduced primary amino
acid sequence, with the exception of the extreme
N-terminus, predicted the secondary structure to
be composed mostly of ß-sheets. Based on these
independently collected data, it appears that Ptr
ToxA is a 13.2-kDa protein that requires a con-
centration of 60-90 nM for the induction of necro-
sis in sensitive wheat.
The site of action for Ptr ToxA has not been
identified, but Kwon et al. (1996) demonstrated
that the toxin induces electrolyte leakage, and
developed a bioassay for the toxin on that basis.
The bioassay was then used to demonstrate that
the toxin causes a form of programmed cell death
in the host that requires host metabolism includ-
ing de novo gene expression (Kwon et al. 1998).
2. Biosynthesis of Ptr ToxA
Ballance et al. (1996) and Ciuffetti et al. (1997)
both cloned cDNAs for TaxA, the gene directly
encoding Ptr ToxA. Interestingly, sequences from
the two clones differed by only a single nucleotide
even though they were derived from different
races isolated in different years and in different
parts of North America. Ballance et al. (1996)
obtained a 900-bp cDNA clone encoding a 19.7-
kDa pro tein precursor of the toxin from a race
2 isolate. Southern hybridizations revealed that
TaxA was present only in toxin-producing isolates
of the fungus. Ciuffetti et al. (1997) isolated both
cDNA and genomic clones ofToxA from race 1 of
P tritici-repentis. The genomic clone was used to
transform a toxin-deficient, avirulent race 4 isolate
of the fungus to a toxin-producing isolate virulent
on wheat. This provided conclusive evidence that
production of Ptr ToxA in an otherwise avirulent
P tritici-repentis background is sufficient to make
P tritici-repentis virulent on wheat.
3. Biological Significance of Ptr ToxB
Orolaza et al. (1995) have shown that a second,
apparently unrelated, proteinaceous toxin, named
Ptr ToxB, is responsible for the host -specific
chlorosis produced by race 5 (nec- chl+) of P
tritici-repentis. Ptr ToxB was detected from spore
germination fluid and cell-free culture filtrates of
328 D.G. Panaccione et al.
the fungus and from intercellular wash fluids of
infected leaves. Race 5 appears to be quite rare in
nature but has been found at low frequency in the
northern Great Plains of the United States (Ali
and Francl 1998).
Orolaza et al. (1995) partially purified the
toxic compound from culture filtrates and showed
that it was a hydrophilie moleeule stable when
exposed to organic solvents. Progenies of a cross
between a resistant and a susceptible wheat line
were used to show that a single dominant gene
in the host controlled resistance to race 5 of the
fungus and insensitivity to the partially purified
toxin. Strelkov et al. (1999) demonstrated that Ptr
ToxB is a heat-stable 6.61-kDa protein that is
active at concentrations as low as 14nM.
Although Ptr ToxA and Ptr ToxB are both
proteinaceous in nature, several observations indi-
cate that the two Ptr toxins are not closely related.
Ptr ToxA and Ptr ToxB differ substantially in size
and in the symptoms incited on sensitive wheat.
Moreover, there is a distinct lack of DNA
sequence hybridization between the Ptr ToxA
gene and genomic DNA of races 3 and 5, which
produce only Ptr ToxB.
B. Cerato-ulmin
1. Biological Significance of Cerato-ulmin
Dutch elm disease (DED), caused by the
ascomycetes Ophiostoma ulmi (Buisman.) Nannf.
and the more aggressive Ophiostoma novo-ulmi
C. Brasier (Brasier 1991), derives its name from
the fact that it was first identified on elm in
Holland in 1921. All species of elm are susceptible
to the disease, but American elm (Ulmus ameri-
canus L.) has been especially hard hit by the
disease. Both pathogens cause a wilt disease of
elm and are transmitted to elm by beetles that
complete their life cycle by laying eggs in the bark
of dead or stressed trees, including those affected
by DED. Juvenile beetles pick up spores of the
fungus as they emerge from infected trees.
The spores are transmitted to healthy trees that
the young beetles feed on.
Researchers have long examined the culture
fluids of the DED pathogens for evidence of wilt
toxins (e.g., Zentmyer 1942). Several candidate
moleeules have been identified, but cerato-ulmin
(CU), first described by Takai (1974), has received
the most attention by far. CU is now known to be
a 75 amino acid (MW 7618.4) pro tein (Yaguchi et
al. 1993) whose structural features pI ace it in a
group of pro teins known as type 11 hydrophobins
(Wesseis 1994). Type II hydrophobins are abun-
dantly secreted into culture fluids and the cell
surface of mycelium and are partially soluble in
water, alcohol, and sodium dodecyl sulfate (SDS).
Features common to these proteins are an amino-
terminal signal for secretion to the fungal cell wall
and eight cysteine residues at conserved sites of
the protein (Stringer and Timberlake 1993).
A considerable amount of circumstantial
physiological evidence collected during the 1970s
and the early 1980s suggested that CU may be a
wilt toxin closely associated with DED. American
elm, the susceptible host, was shown by Takai
(1974) to develop typical wilt symptoms when sub-
jected to cell-free preparations of Cu. Scanning
electron microscopy was then used to reveal that
the effects of CU on the host were the same as
those caused by the fungus (Takai and Hiratsuka
1984). Claims were made that CU caused
increased host respiration and cellular electrolyte
leakage, features characteristic of DED (Takai
and Richards 1978). None of these effects were
found in the more DED-resistant Siberian elm.
Further, it was demonstrated that more virulent
strains of the fungus produce greater quantities
of CU than do less virulent strains (Takai 1980;
Svircev et al. 1988; Brasier 1991).
Brasier et al. (1995) provided some of the
first evidence to question the role of CU in DED.
They found two naturally occurring strains of 0.
novo-ulmi that exhibited a flat, waxy colony mor-
phology that were simultaneously deficient in pro-
duction of aerial mycelium and Cu. However,
these strains showed typical virulence toward elm,
suggesting that CU production may not be
required for pathogenicity. Molecular analyses of
CU production have supported this conclusion.
Bowden et al. (1994) cloned the gene encoding
CU (designated cu), then, in a subsequent paper,
used the clone to create site-directed knockout
mutants of 0. novo-ulmi deficient in CU pro duc-
tion (Bowden et al. 1996). Bioassays revealed that
the knockout mutants were fully pathogenic on
elm, despite their in ability to produce Cu. Tegli
and Scala (1996) supported this claim when they
isolated UV-induced mutants of 0. novo-ulmi that
showed wild-type virulence on elm, despite their
reduced ability to produce Cu.
 Fungal Phytotoxins
Temple et al. (1997) constructed a transfor-
mation vector, containing a wild-type copy of cu
obtained from avirulent strain of 0. novo-ulmi,
designed for overproduction of CU in trans-
formation recipients. This vector was used to
transform nonaggressive isolates of 0. ulmi.
Transformants were shown to produce copious
quantities of CU, yet no significant increase in vir-
ulence or pathogenicity on elm was detected. In
other experiments, the same vector was used to
transform the CU -deficient mutants (described
previously in Bowden et al. 1996) that lacked
aerial mycelium and grew with a flat, waxy habit.
Transformants were shown to produce CU and the
growth habit was restored to the wild-type condi-
tion. The authors postulated that CU may act as
a parasitic fitness factor that helps the fungus
produce mycelium and propagules better able
to disseminate, adhere to the host, and survive
desiccation.
In contrast to the abundant data indicating
that CU is a dispensable factor in DED, arecent
molecular study suggests that in some fungal
backgrounds CU production may be important
for DED symptom development. DeI Sorbo
et al. (2000) demonstrated that Ophiostoma
quercus (Georgen.) Nannf., a pathogen of oak
(Quercus spp.) but not of elm, was able to cause
DED symptoms on elm when the 0. novo-
ulmi gene for CU production was introduced by
transformation.
Collectively, these data raise the peculiarity
that CU is sufficient for DED symptoms in a
rather artificial 0. quercus-elm interaction, even
though it is not required in the natural interactions
of other Ophiostoma spp. with elm. This apparent
contradiction could be the manifestation of
CU being functionally redundant in the 0.
novo-ulmi and 0. ulmi backgrounds but not so
in 0. quercus. This hypothesis has not yet been
investigated.
C. Other Proteinaceons Phytotoxins
One other proteinaceous toxin, AB toxin pro-
duced by Alternaria brassicicola (Schw.) Wilts., the
causal agent of black spot of cultivated Brassica
species, has recently been identified as a host-
specific toxin (Otani et al. 1998). Though several
host-selective toxins have been reported from
various pathotypes of A. alternata (e.g., AM toxin
329
and AK toxin, discussed above), this is the first
re port of a host-selective toxin from A. brassici-
cola. AB toxin is also the first proteinaceous toxin
reported from any Alternaria spp.
AB toxin is a protein with a reported MW of
about 35 kDa and is almost certainly a primary
gene product. The toxin was shown to be active
against several hosts of the fungus, including
B. campestris (canola), B. carinata (Abyssinian
mustard), B. juncea (leaf mustard), B. nigra (black
mustard), and B. olearacea (cabbage), but no activ-
ity was found against cucumber, pea, tomato, or
eggplant. Probably the most unique feature of this
toxin is that its production, as weH as its toxicity,
is host-specific. AB toxin was detected in spore
germination fluids (SGF) obtained 24h after inoc-
ulation of hosts (Brassica spp.), but was absent
in SGF of the same fungus inoculated onto leaves
of tomato or other nonhost plants of the fungus
or on plastic plates. In other experiments, AB
toxin was produced in SGF on plastic plates when
the fluids contained hot-water extracts of cabbage.
Thus, it appears that host plants possess a hot-
water-soluble compound or compounds that
induces the production of AB toxin. Although
most other host-selective toxins can be obtained
from culture fluids of the producing fungus grown
on a modified Fries medium, AB toxin is not pro-
duced under these conditions. Experiments are
underway to characterize AB toxin and to identify
the host compound(s) responsible for induction of
toxin production by the host.
V. Miscellaneous Fungal Phytotoxins
In this review we have covered several of the
better-studied phytotoxins under subheadings
according to their major structural class. The struc-
tural classes were chosen because each contained
more than one toxin for which there was extensive
re cent data on their biosynthesis, role in disease,
or both of these topics. There are other toxins that
do not fit precisely within the three structural
classes described above but are still noteworthy
because of their importance in the disease inter-
actions in which they occur. Most notable are the
trichothecenes, two glycosylated terpene deriva-
tives (fusicoccin and HS toxin), and an unusual
peptide derivative, PC toxin.
330 D.G. Panaccione et al.
A. Triehothecenes
Trichothecenes are a family of related sesquiter-
penoid compounds that differ from the toxins
described above in that they are produced by
several fungi, including species of Fusarium (and
their Gibberella teleomorphs), Myrothecium, and
Stachybotrys, and affect a wide variety of organ-
isms, including animals and fungi, in addition to
plants (Hohn et al. 1998). Among the tri-
chothecenes are deoxynivalenol (DON), diace-
toxyscirpenol (DAS), and T-2 toxin. The
trichothecenes in general are potent inhibitors of
protein synthesis (Hohn et al. 1998).
Evidence of a role for trichothecenes in a
variety of plant-pathogen interactions has been
provided by gene knockout analyses. Disruption
of the trichodiene synthase gene TRI5 (also called
Tox5) of Fusarium sambucinum Fuckel resulted in
astrain of the fungus that could no longer produce
its major trichothecene, DAS (Hohn and Des-
jardins 1992). This DAS-deficient mutant had
significantly reduced virulence on parsnip roots
but retained full virulence on potato tubers
(Desjardins et al. 1992). Disruption of the homol-
ogous gene in the economically important wheat
sc ab pathogen, Fusarium graminearum Schwabe,
resulted in the loss of its major trichothecene,
DON, and reduced virulence to wheat in the
greenhouse (Proctor et al. 1995a) and in the field
(Desjardins et al. 1996b). Virulence was restored
by transformation with the wild-type trichodiene
synthase gene (Proctor et al. 1997). DON-
deficient mutants of F. graminearum also were less
virulent on maize when inoculated by a silk
channel method or a direct kernel puncture
method (Harris et al. 1999).
The various trichothecenes are the products
of a very complex pathway. Ten genes that are
likely to be involved in trichothecene synthesis
have been identified within a 23-kb cluster in F.
sambucinum (Hohn et al. 1998). These include
genes for biosynthetic enzymes, a transcriptional
regulator encoded by TRI6 (Proctor et al. 1995b),
and a potential transporter or pump encoded
by TRI12 (Alexander et al. 1999). A gene that
appears to be involved in auto-resistance to tri-
chothecenes, by acetylation, in Fusarium sporotri-
chioides Sherbakoff has been found outside the
major trichothecene gene cluster (Kimura et al.
1998). A cluster of several homologous bio-
synthetic genes has been identified in another
trichothecene producer, Myrothecium roridum
Tode:Fries, but the relative positions and orienta-
tions of some genes differ from that in F. sam-
bucinum (Trapp et al. 1998).
B. HS Toxin
HS toxin, also known as helminthosporocide,
is a family of three isomerie host-specific toxins
produced by Bipolaris sacchari (E.I Butler)
Shoemaker (Duvick et al. 1984; Livingston and
Scheffer 1984a), making the fungus pathogenic on
specific clones of sugar cane (Saccharum spp.
hybrids). The toxin displays great host specificity
in that only sugar cane clones sensitive to the toxin
are susceptible to the pathogen. Little is known
about the biosynthesis of the toxin or its mode of
action, except that it induces electrolyte leakage
(Livingston and Scheffer 1984a) and inhibits CO2
uptake (Duvick et al. 1984) in a host-specific
manner. The biologically active forms of HS toxin
contain a sesquiterpenoid moiety with four
galactofuranose residues attached (Duvick et al.
1984; Livingston and Scheffer 1984a). Isomeric
forms differ by the position of a double bond with
the sesquiterpene group. HS toxin metabolites
(toxoids) with less than four galactofura-
nose residues are nontoxic and protect sensitive
sugar cane from the biologically active forms of
the toxin, presumably by competition for a
common receptor (Livingston and Scheffer
1984b). Interestingly, B. sacchari produces a ß-
galactofuranosidase that removes galactofuranose
residues from HS toxin (Livingston and Scheffer
1983).
C. Fusieoccin
Fusieoccin is another glycosylated terpene-
derived moleeule, though it is not otherwise struc-
turally similar to HS toxin. Fusicoccin is an
a-glucoside of a highly oxygenated tricyclic diter-
pene produced by Fusicoccum amygdali Delacr., a
canker pathogen of almond [Prunus du leis (Mill.)
Webb] and peach (Prunus persica L.) trees. Fusic-
occin is transported through the transpiration
stream of affected trees and is responsible for the
systemic wilt symptoms seen in diseases caused by
the pathogen (Graniti et al. 1995). Although the
pathogen is limited in its host range in the field,
fusicoccin is active against a wide variety of plants.
Fusicoccin is not directly toxic since it does not kill
 Fungal Phytotoxins
affected cells. Rather it causes wilting by stimu-
lating plasma membrane H+/ ATPases (e.g., Marre
et al. 1974; Marre 1979), resulting in sustained
opening of stomata (Turner and Graniti 1969) and
water loss by uncontrolled transpiration. Fusicoc-
ein does not bind directly to H+/ATPases but asso-
ciates indirectly through bin ding 14-3-3 type
pro teins (Marra et al. 1994), found in the mem-
branes of many plants (Aducci et al. 1995).
D. PC Toxin
PC toxins, or peritoxins, are very unusual
dipeptide-derived moleeules containing an addi-
tional chlorinated 10-carbon carboxylic acid co m-
ponent (Wolpert and Dunkle 1980; Macko et al.
1992). They are discussed here rather than under
the peptide section because little is known ab out
their biosynthesis. It appears likely that a peptide
synthetase is involved in assembling the peptide
portion of the toxins, but this has not been
demonstrated.
pe toxin is a host-specific toxin produced by
some isolates of the soil-borne fungus Perieonia
circinata (M. Mangin) Sacc. and is critical to path-
ogenicity of these isolates in the milo disease of
sorghum [Sorghum bieolor (L.) Moench.; Dunkle
and Macko 1995]. They are true host-specific
toxins in that only a single genotype of the host
plant is sensitive to the toxin and susceptible to the
pathogen, and only pe toxin-producing isolates of
the fungus are pathogenic. The plant-pathogen
interaction is unique in that pathogenic isolates of
P. eireinata do not occur near the center of origin
for sorghum (Africa), though nonpathogenic iso-
lates of the fungus can be isolated from that area.
Pathogenic isolates of the fungus are limited to
sorghum-growing regions of the United States;
areas in which sorghum was not grown until the
1800s. In the 1920s, outbreaks of milo disease
occurred at various locations over this region
and the pathogenieity of these isolates was later
assoeiated with their ability to produce pe toxin
(Scheffer and Pringle 1961). The toxin does not
otherwise appear to benefit the producer because,
in the absence of the host, 24 of 32 isolates lacked
the ability to produce pe toxin (Scheffer and
Pringle 1961). Moreover, isolates of P circinata
from many locations (including Africa, the center
of origin for sorghum) lack the ability to produce
the toxin (Odvody et al. 1977; Dunkle and Macko
1995).
331
Insensitivity to pe toxin and resistance to the
pathogen are conferred by the homozygous reces-
sive condition at the pe locus (Schertz and Tai
1969). The heterozygous state at this locus confers
intermediate resistance, whereas the homozygous
dominant condition makes plants fully sensitive to
the toxin and susceptible to the pathogen (Schertz
and Tai 1969; Dunkle 1979). These data indicate
that the dominant allele, Pe, encodes a toxin
receptor or site of action (Dunkle 1979). RNA and
pro tein synthesis are required for pe toxin activ-
ity (Gardner et al. 1972; Wolpert and Dunkle
1983), and a family of four slightly aeidic 16-kDa
pro teins is expressed at higher rates in toxin-
treated sorghum in a host-specific manner
(Wolpert and Dunkle 1983). Additional studies on
the 16-kDa protein indicate that it is found in
many monocots (Ransom et al. 1994), its expres-
sion can be induced by a variety of treatments in
resistant as weIl as susceptible genotypes (Traylor
et al. 1988), and its production is not responsible
for or directly linked with symptom development
(Ransom et al. 1992). These findings indicate that
the 16-kDa protein is not critical to pe toxin activ-
ity though some other proteinaceous receptor
must be involved. A model for toxin activity in
which the product of the Pe allele is a membrane-
bound receptor that, upon interaction with pe
toxin, triggers a signal transduction cascade
leading to symptom development has been pro-
posed and is supported by studies with specific
inhibitors (Dunkle and Macko 1995).
VI. Conclusions
The fungal phytotoxins summarized above repre-
sent examples of different biochemical classes and
contribute in various ways to the outcomes of the
plant-fungus interactions in which they are pro-
duced. When viewed as a wh oIe, these phyto-
toxins have more in common relative to their
biosyntheses than they do by their site of action or
the mechanism of resistance to them in plants.
In cases where genes involved in toxin biosyn-
thesis have been cloned, clustering of two or more
biosynthetic genes is common. In the examples
summarized above, this is true for He toxin, the
ergopeptines, T toxin (though the cluster is dis-
placed by a translocation), AK toxin, aflatoxins,
fumonisins, and trichothecenes. The potential for
clustering of biosynthetic genes in several other
332 D.G. Panaccione et al.
examples, such as AM toxin, the enniatins, cer-
cosporin, Ptr ToxA, and cerato-ulmin, cannot be
assessed because either their biosynthesis requires
only a single gene or only one biosynthetic gene
had been described in the literature at the time of
writing. For the remaining toxins, no toxin biosyn-
thetic genes have been published to date. These
limitations are likely to change as this volume
makes its way to print.
Another remarkable attribute of the toxin
biosynthetic genes that have been cloned is the
lack of homologues in closely related fungi that do
not produce that particular phytotoxin. With one
exception, this appears to be the general rule
for all toxin biosynthetic genes studied; the sole
exception being some enniatin-nonproducing iso-
lates of Fusarium spp. that contain homologues of
esynl, the gene encoding the peptide synthetase
that assembles enniatins (Herrman et al. 1996a).
This combination of clustering of biosynthetic
genes and lack of homologues in related toxin
nonproducers has prompted hypotheses relating
to the acquisition of toxin biosynthetic capabilities
via horizontal transfer from other microorganisms
(e.g., Walton and Panaccione 1993; Nikolskaya et
al. 1995; Yoder 1998; Tanaka et al. 1999). With the
continually mounting evidence that toxin biosyn-
thetic capability is a derived trait, the hypothesis
appears quite reasonable. However, no source for
any of the potentially horizontally transmitted
toxin biosynthetic gene clusters has as yet been
discovered.
The similarities among the various toxins are
more obvious in their means of biosynthesis than
in their roles in fungus-plant interactions. The
activities and roles of phytotoxins in plant-
microbe interactions within each class are highly
varied, even for very similar molecules. Several of
the described phytotoxins, such as AM toxin,
the enniatins, T toxin, and cercosporin, exert
their effects on membran es, as toxins are often
generalized to do. However, even in these exam-
pIes, the actual mechanism of membrane damage
or perturbation of membrane function differs
drastically. Other toxins, such as HC toxin and
fusicoccin, do not directly kill cells but leave
affected plants unable to respond to the attacking
pathogen or otherwise debilitate the plant. The
activities for some toxins have been discovered as
our knowledge of cell biology has expanded. This
is true for the induction of the apoptotic responses
in sensitive host cells by victorin and Ptr ToxA.
The mechanisms by which plants establish
resistance to toxin-producing fungi vary signifi-
cantly among the different pathosystems exam-
ined. Contrary to the situation with many
plant-fungus interactions, resistance in plants to
pathogens that rely upon phytotoxins as patho-
genicity or virulence factors may be a recessive
trait. Such is the case with oats and victorin-
producing C. victoriae and maize with T toxin-
producing C. heterostrophus. In such cases it has
been postulated that resistance is due to the lack
of a specific receptor for the toxin in the resistant
plant. Interaction in which the heterozygous state
provides an intermediate level of resistance (e.g.,
sorghum and Periconia circinata) is viewed as an
extension of the receptor hypothesis, with the
toxin receptor present at a lower frequency than
in the homozygous dominant state. Dominant
resistance, as in the HC toxin-detoxifying enzyme
in resistant maize, has also been described. More-
over, mechanisms of auto-resistance (e.g., for HC
toxin, cercosporin, and trichothecenes) have been
identified in several phytotoxin-producing fungi.
It may be possible to exploit these mechanisms to
make potential host plants insensitive to particu-
lar phytotoxins.
Acknowledgements. We thank Erin Zervos for
typing and formatting the references section.
Work in the senior author's laboratory was sup-
ported by USDA NRI grants 98-35303-6663 and
2001-35319-10930. Published with the approval of
the Director of the West Virginia Agricultural and
Forestry Experiment Station as Scientific Artic1e
#2782.
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17 The Contribution of Cell Wall Degrading Enzymes to Pathogenesis
of Fungal Plant Pathogens
ARJEN TEN HAVE,1 KLAUS B. TENBERGE,2 JACQUES A.E. BENEN,3 PAUL TUDZYNSKI,2 JAAP VISSER,4
and JAN A.L. VAN KAN 1
CONTENTS
I. Introduction ........................ .
11. Structure of Plant Cell Walls ........... .
111. Classification and Characteristics
of Cell Wall Degrading Enzymes (CWDEs) .
IV. Prerequisite Properties of CWDEs
for a Role in Pathogenesis ............. .
A. Environmental Conditions
at the Host-Pathogen Interface ......... .
B. Substrate Specificity and Concerted
Action of CWDEs ................... .
v. Evidence for the Involvement of Fungal
CWDEs in Pathogenesis ............... .
VI. Case Study: Claviceps purpurea, a Highly
Specialized Biotroph .................. .
A. Biology and Life Cyc1e ................ .
B. Cell Wall Degrading Enzymes:
General Aspects ..................... .
C. Pectinases .......................... .
D. Cellulases .......................... .
E. Xylanases .......................... .
F. ß-1,3-Glucanase ..................... .
G. Perspectives ........................ .
VII. Case Study: Botrytis cinerea,
an Opportunistic Necrotroph ........... .
A. Biology and Infection Strategy .......... .
B. Pectinases .......................... .
C. Other CWDEs ...................... .
VIII. Inhibitors of CWDEs and Polymer
Fragments: Friend or Foe? ............. .
A. Oligogalacturonides as Inducers
of Plant Defense ..................... .
B. Polygalacturonase-Inhibiting Proteins .... .
IX. The Role of CWDEs in Pathogenesis,
a Matter of Fungal Lifestyle? ........... .
X. Conc1uding Remarks and Prospects ...... .
References ......................... .
1 Wageningen University, Laboratory of Phytopathology,
Binnenhaven 5, 6709 PD Wageningen, The Netherlands
2 Westfälische Wilhelms-Universität Münster, Institut für
Botanik, Schloß garten 3,48149 Münster, Germany
3 Wageningen University, Laboratory of Microbiology,
section Fungal Genomics, Dreijenlaan 2, 6703 HA
Wageningen, The Netherlands
4 FGT, PO Box 396, 6700 Al Wageningen
I. Introduction
341
341
The plant cell wall functions as a barrier to biotic
343 and abiotic agents. Plant pathogenic bacteria
and fungi produce cell wall degrading enzymes
344
(CWDEs) which are believed to degrade this
345 barrier, thereby facilitating both inter- and intra-
cellular growth and providing nutrients to the
345 invader. A pectate lyase from the bacterium
Erwinia chrysanthemi was the first CWDE that
346
was shown to be required for full virulence
347 (Roeder and Colmer 1985). Subsequent molecu-
347 lar genetic studies have shown that many other
348 bacterial CWDEs are virulence factors (reviewed
348 by Hugouvieux-Cotte-Pattat et al. 1996). It took
349 many years before similar evidence was obtained
349 for the involvement of fungal CWDEs in patho-
350
350 genesis, in spite of several efforts (reviewed by
Walton 1994; Annis and Goodwin 1997). Eventu-
350 ally, an endopolygaIacturonase from Aspergillus
350
351
flavus was shown to playa role in the invasion of
352 cotton bolls (Shieh et al. 1997). Since then, more
evidence for the function of fungal CWDEs has
352 appeared (ten Have et al. 1998; Rogers et al. 2000)
352
and these enzymes have regained the attention of
353 plant pathologists. In this chapter we review the
current knowledge on the functions that fungal
353 CWDEs have in plant pathogenesis. In addition
354
355 to overviews of the plant cell wall structure
and fungal CWDE classification, we discuss the
research on CWDEs of several plant pathogenic
fungi, with emphasis on two case studies of fungi
in which CWDEs have unequivocally been shown
to act as virulence factors. This chapter provides
an extension and an update of previous reviews by
Annis and Goodwin (1997) and Walton (1994).
11. Structure of Plant Cell Walls
The plant cell wall is highly dynamic and its overall
structure and composition vary depending on the
The Mycota XI
Agricultural Applications
Kempken (Ed.)
© Springer-Verlag Berlin Heidelberg 2002
342
.,...------
MIDDLE
LAMELLA
PAIMARY
CEll WALL
Fig.17.1. Extremely simplified and schematic representation of the spatial arrangement of celluloses, hemicelluloses and
pectins in a cell wall. Scale bar 50,um. (McCann and Roberts 1991 with permission)
plant species, the type of cell it surrounds, the
stage of differentiation of a cell and the stage of
development of the plant itself. Nonetheless,
certain features of the plant cell wall are common.
Here we will present a general overview of the
plant cell wall architecture of dicotyledonous
plants.
The main function of the plant cell wall is to
provide mechanical strength and intercellular
adhesion, meanwhile allowing cell elongation. The
plant cell secretes various polysaccharides and
proteins that form the building blocks of an intri-
cate complex network called the cell wall or
apoplast. From one cell to the next the following
regions can often be distinguished: a primary cell
wall, the middle lamella and again a primary cell
wall. Figure 17.1 shows a representation of a
primary cell wall after the model of McCann and
Roberts (1991). Each region is characterized by
the dominance of (a) certain type(s) of polysac-
charides/compounds. The middle lamella has a
high content of pectin, which forms a matrix. The
primary cell wall of dicotyledons typically consists
of a cellulose-hemicellulose (often xyloglucan)
network embedded in the pectin matrix (McCann
and Roberts 1991; Carpita and Gibeaut 1993).
Arrest of cell elongation is accompanied by the
formation of the secondary cell wall. The sec-
ondary cell wall contains less pectins and the
celluloses are notably longer than in the primary
cell wall (Taylor et al. 1999). This two-section
(in young growing tissue) or three-section (older
A. ten Have et al.
tissue) model is a simplification of a cell wall with
a gradual transition of chemical composition and
structural complexity (Bateman and Basham
1976). However, electron microscopy studies
often distinguish these three layers (McCann and
Roberts 1991). Lignification in older cell walls
around the xylem results in cell wall thickening
and woodification of the secondary cell wall
(Carpita et al. 1996; Taylor et al. 1999).
Cellulose consists of ß-1-4-linked D-glucose
chains. The chains are associated by strong hydro-
gen bonds into crystalline cellulose microfibrils.
The hemicelluloses comprise a diverse group of
polysaccharides. Like cellulose, the major hemi-
cellulose xyloglucan consists of ß-1-4-linked D-
glucose chains. However, D-xylose is a-1,6-linked
to glucose residues in a more or less orderly
fashion: repeats of four glucose residues either
carry three xylose residues (XXXG type; gymno-
sperms and angiosperms) or two xylose residues
(XXGG type; solanaceous plants; Vincken et al.
1997). Some of the xylose residues can be sub-
stituted either with a-1,2-L-rhamnose, ß-1,2-
D-galactose or a-1,2- L-fucosyl-ß-1,2-D-galactose
(McNeill et al. 1984). Xyloglucan chains are
tightly linked to cellulose fibrils by hydrogen
bonds. The chain length is such that connections
between different fibrils are possible thus forming
the basis of the network that provides physical
strength to the cell wall (Albersheim et al. 1996).
Other types of hemicelluloses in the primary cell
wall, such as (arabino )xylan and galacto(gluco)
 The Contribution of Cell Wall Degrading Enzymes to Pathogenesis of Fungal Plant Pathogens
mannan, represent only a small fr action of the
polysaccharides in dicotyledons.
The (arabino)xylan backbone consists of
ß-1,4-linked D-xylose. Modification can occur by
various substitutions: a-1,2- and a-1,3-L-arabi-
nose, a-1,2-D-glucuronic acid and a-1,2-4-0-
methyl-D-glucuronic acid (Wilkie 1979; Brillouet
and Joseleau 1987). Acetylation of xylose at 02
and 03 is common and the arabinose residues can
be esterified with ferulate (Ishii 1991). Ferulate
can form di-ferulic acid bridges between xylan
chains and can also cross-link xylan and pectin
(see below).
The galactomannan backbone consists of
ß-1,4-linked D-mannose which can be substituted
with D-galactose by an a-1,6-linkage (Aspinall
1980). In galacto(gluco )mannan, the mannan
backbone contains ß-1,4-linked glucose residues.
Acetylation occurs at 02 and/or 03 of the
mannose or glucose (Timell1967).
The pectin network is not only confined to the
middle lamella, but also extends into the primary
wall and to a lesser extent into the secondary cell
wall. Pectin is a very complex heteropolysac-
charide (De Vries et al. 1982). The backbone
is formed by a-1,4-linked D-galacturonic acid.
The galacturonic acid residues can either be
methylesterified at 06 or exist as free acid. Gen-
erally, 70% of the galacturonic acid residues in
plants are methylesterified. Acetylation can occur
at 02 and or 03 of the galacturonic acid. This part
of the pectin molecule is known as homogalac-
turon an. The homogalacturonan is interrupted
by rhamnogalacturonan I (O'Neill et al. 1990),
which has a backbone of as many as 100 repeats
of the disaccharide a-1,2-L-rhamnosyl-a-1,4-D-
galacturonic acid. In addition, there are regions in
rhamnogalacturonan I where the frequency of
rhamnose residues in the backbone is lower and
regions where no rhamnose is present. In the
latter regions, known as xylogalacturonan, galac-
turonic acid is highly substituted with xylose
(Schols et al. 1995). Arabinosyl and galactosyl side
chains can be attached to 04 of the rhamnose in
rhamnogalacturonan I (O'Neill et al. 1990). Side
chains can vary in length from 1 to 50 residues. The
a-1,5-linked arabinan chains can again have ara-
binan side chains (a-1,3-linked). Terminal arabi-
nose residues can be esterified at 02 with ferulic
acid, allowing cross-linking with other polysac-
charides (i.e., arabinoxylan, pectin; Guillon and
Thibault 1989). Galactan consists of ß-1,4-linked
D-galactose residues. The terminal residues can
also be esterified with ferulate at 06. In rhamno-
343
galacturonan I the galacturonic acid residues can
again be methylesterified at 06 or acetylesterified
at 02 and/or 03 (Schols and Voragen 1994).
Rhamnogalacturonan II is yet another com-
ponent of the pectin network (Whitcombe et al.
1995). It is a highly conserved structure with a
backbone of nine a-1,4-linked D-galacturonic acid
residues. Four short complex side chains are
attached to 02 or 03 of only four galacturonic
acid residues. The complex side chains contain a
number of rare sugars (Whitcombe et al. 1995).
In addition to polysaccharides, the plant cell
wall contains proteins. The major protein is
extensin, which is very rich in hydroxy-proline
residues. It is highly decorated with arabinose
residues and, to a lesser extent, galactose residues
(Sadava and Chrispeels 1973). Extensin molecules
form a network by intermolecular isodityrosyl
bridges (Fry 1982). Arabinogalactan proteins are
also found in the cell wall, primarily associated
with the plasma membrane (Albersheim et al.
1996).
III. Classification and Characteristics
of Cell Wall Degrading Enzymes
(CWDEs)
In view of the complexity of the carbohydrate
composition of the cell wall, it is logical that path-
ogenic rnicroorganisms have a vast repertoire of
polysaccharidases at their disposal. It is beyond
the scope of this chapter to describe all polysac-
charidases in detail. A full listing of enzymes
which inc1udes hydro las es, lyases, transferases and
esterases is provided by Coutinho and Henrissat
(1999) in a website database.
Polysaccharidases can be divided into two
c1asses: exo-acting enzymes and endo-acting
enzymes. The exo-acting enzymes can be specific
for the nonreducing end or the reducing end of
the polysaccharide. Generally, exo-acting enzymes
release monomeric or dimeric glycosyl moieties
during each catalytic event, thereby providing the
microorganism with low molecular mass com-
pounds that can easily be taken up. Exolytic activ-
ity only slowly decreases the average chain length
of a polysaccharide solution. True endo-acting
enzymes c1eave a polysaccharide randomly along
the chain, resulting in a very rapid decrease in
average chain length. Generally, both exo- and
endolytic enzymes attack the chain only once
every encounter. However, some enzymes repeat-
344 A. ten Have et al.
edly attaek the ehain onee they have encountered
it. These enzymes are called processive enzymes.
This proeess is also known as "multiple attack on
a single chain".
In principle, only the two moieties of which
the connecting bond will be broken need to bind
to the enzyme. Rowever, it turns out that more
moieties bind to the enzyme. Each site where such
a moiety is accommodated is called a subsite; as
many as ten subsites have been reported far a
single enzyme (All an and Thoma 1976). Far an
endo-acting enzyme, the catalytic site can be
located anywhere around the middle of the array
of subsites. For an exo-acting enzyme, the catalytic
site is located between the first and the second or
the second and the third subsite when monomers
ar dimers are cleaved off, respectively.
Several plant cell wall polysaccharides like the
pectins and the xylans can be acetylesterified.
Acetyl esters must be removed by specific
acetylesterases before the main chain can be
cleaved by depolymerizing enzymes. In addition,
the presence of side chains often requires rem oval
before the backbone can be cleaved. As an
example, the enzyme spectrum aetive on pectin
will be discussed.
As mentioned in Section H, pectin consists of
homogalacturonan, rhamnogalacturonan I and
rhamnogalacturonan H. As yet, no enzymes have
been identified that are able to degrade rhamno-
galacturonan 11. Romogalacturonan appears to be
the simplest part of the pectin moleeule: only
methylesterifieation of the carboxylie funetion
and, to a lesser extent, acetylesterification at 02
and/or 03, are known. Nonetheless, batteries of
enzymes exist in a single organism to attack this
seemingly simple substrate. Pectin methylesterase
and pectin acetylesterase deesterify the homo-
galacturonan. Aspergillus niger has at its disposal
six pectin lyases for the methylesterified homo-
galacturonan (Harmsen et al. 1990) and seven
endopolygalacturonases for nonmethylesterified
homogalacturonan (Bussink et al. 1992). The pR
optima of the lyases are above pR 6 whereas the
pR optima of the endopolygalacturonases are
below pR 6. The strict substrate specifieity sug-
gested by their names is only partly true: both
the pectin lyases and the endopolygalacturonases
are also (Iess) aetive on partly methylesterified
homogalacturonan. There are even endopoly-
galacturonases that are more aetive on partly
methylesterified substrate than on polygalactur-
onic acid (Pai'enicova et al. 2000a). The existenee
of so many isozymes, with a specifie aetivity dif-
fering by two to three orders of magnitude on
model substrates, strongly indicates that the
specific substrate for some of these enzymes is
not homogalaeturonan but an as-yet unidentified
structure of the pectin molecule. Peetate lyase
completes the currently identified ensemble of
enzymes active towards homogalacturonan. It is
specific far low or nonmethylesterified homo-
galacturonan and requires Ca2+ ions far catalysis.
Rhamnogalacturonan I is structurally more
complex than homogalacturonan. The arabinan
and galactan side chains must be removed by a-
endoarabinanases and arabinofuranosidases, and
ß-galactanases and ß-galactosidases, respectively,
before the backbone depolymerizing enzymes ean
act. Also, acetyl esters must be removed by the
rhamnogalacturonan acetylesterase. A methyl-
esterase specifie for the rhamnogalacturonan I
has not yet been identified. When all decorations
have been removed, the endo-acting rhamno-
galacturonan hydrolase ar rhamnogalacturonan
lyase can cleave the alternating rhamnose-
galacturonic acid stretches (Mutter et al. 1996,
1998b). Rhamnogalacturonan fragments gener-
ated by the rhamnogalacturonan hydrolase action
are further degraded by the exolytic rhamno-
galacturonan-rhamnohydrolase that liberates
rhamnose from the nonreducing end and the
exolytic rhamnogalacturonan-galacturonohydro-
lase which liberates galacturonic acid from the
nonreducing end (Mutter et al. 1994, 1998a). The
xylogalacturonan is cleaved by the endolytic
xylogalacturonase (Van der Vlugt-Bergmans et al.
2000). Exopolygalacturonase is able to liberate
xylogalacturonic acid from xylogalacturonan and
this enzyme may be involved in the further degra-
dation of the xylogalacturonan fragments gener-
ated by the xylogalacturonase (Kester et al. 1999).
Although it has been suggested that the
enzymes act sequentially, a concerted action may
be required as the action of one enzyme may be
hindered by the presence of side ehains or back-
bone which have not yet been removed.
IV. Prerequisite Properties of CWD Es
for a Role in Pathogenesis
In order to degrade a structural component of the
cell wall, either fully or partially, a CWDE must be
expressed and active under conditions prevailing
 The Contribution of Cell Wall Degrading Enzymes to Pathogenesis of Fungal Plant Pathogens
in host tissue. In addition, the substrate for the
CWDE of interest should be located in the vicin-
ity of the pathogen. When those criteria are ful-
filIed, the biochemical function of the enzyme can
be performed. The degradation of plant cell walls
by a fungal pathogen may be dissected into five
possible actions, either operating alone or in
combination:
1. Facilitating inter- and intracellular growth
through a physical barrier (general pathologi-
cal function).
2. Facilitating formation of feeding structures
(pathological function of certain biotrophs).
3. Providing the fungus directly with nutrients
(saprophytic function).
4. Decreasing wall strength resulting in plant cell
death (necrotrophic function).
5. Releasing elicitors of cell death (necrotrophic
function).
The classification and characteristics of CWDEs
have been discussed in Section III. Here, we
discuss the properties and functions of these
enzymes in relation to pathogenesis.
A. Environmental Conditions
at the Host-Pathogen Interface
Conditions in the host tissue must be appropriate
for CWDEs to be enzymatically active. Tempera-
ture and ambient pH are important factors in
this respect. Most endopolygalacturonases are
optimally active at low pR. Some plant patho-
gens, especially from the family of Sclerotiniaceae,
acidify their environment predominantly by oxalic
acid (reviewed by Dutton and Evans 1996), stim-
ulating the activity of endopolygalacturonases. In
contrast, pectate Iyases are inactive at low pH. The
secretion of pectate lyase by Colletotrichum
gloeosporioides in liquid cultures only occurs at an
ambient pH of 5.8 and higher (Yakoby et al. 2000).
The pH of ripe pericarp tissue of an avocado
cultivar resistant to C. gloeosporioides was low
«5.8), whereas the pH of ripe pericarp of a
susceptible cultivar was 6.3. The effect of ambient
pH on the regulation of enzyme production by
the pathogen thus seems to be an important
factor in determining host resistance in this
pathosystem.
A second environmental parameter is tem-
perature. Many pathogens infect at moderate
temperatures. Botrytis cinerea, however, can even
345
infect plants at temperatures as low as 2 oe.
B. cinerea endopolygalacturonase activity is
readily detectable at this temperature (ten Have,
unpubl.).
B. Substrate Specificity and Concerted
Action of CWDEs
Endopolygalacturonase cannot cleave highly
methylated pectin, while pectin methylesterase
can demethylate pectin without affecting the
polymer chain. Each enzyme alone will be insuffi-
cient to degrade highly methylated pectin. Con-
certed, sequential action of the two enzymes is,
however, devastating and will cleave the polymer
chain into oligomers and ultimately into di- or
monomers. In Uromyces viciae-fabae, an obligate
parasite of bean, the secretion of pectin
methylesterase and pectate lyase accompanies the
onset of host wall penetration by the haustorial
mother cell (Deising et al. 1995). The secretion of
these pectic enzymes during the infection process
is highly regulated, depending on the differen-
tiation stage of the pathogen as well as the avail-
ability of substrate. There is also an increase
in apoplastic pR in order to enhance enzyme
activity during the penetration process (Deising et
al. 1995).
Pectinase activity from the host plant itself
mayaiso contribute to the concerted action.
Ripening of tomato fruits is often accompanied by
a decrease in the degree of methylation of pectin,
which possibly increases their susceptibility to
saprophyt es (Koch and Nevins 1989).
Concerted action is important for several
combinations of enzymes involved in cell wall
degradation, both at the level of enzyme activity
and the regulation of gene expression. In
Aspergillus niger, there is evidence for coregula-
tion of CWDEs degrading two different types of
cell wall constituent, namely xylanases and cellu-
lases. The transcriptional activator XlnR is induced
by xylose, enzymatically released from xylan
present in the hemicellulose network (van Peij et
al. 1998). Not only does the XlnR gene product
activate genes involved in xylan degradation, it
also induces cellulolytic genes. Thus, xylose simul-
taneously regulates the breakdown of cellulose
and hemicellulose. A different type of concerted
regulation is the induction of rhamnogalacturonan
hydrolase gene expression in Aspergillus aculeatus
by the combination of galacturonic acid and rham-
346 A. tell Have et al.
nose, both end-products of the enzyme actlvlty
(Suykerbuyk et al. 1996). There are only a few
examples of such seemingly complex regulation
of CWDE gene expression in plant pathogenic
fungi. Arabinose, not present in polygalacturonic
acid, induces endopolygalacturonase gene ex-
pression in Colletotrichum lindemuthianum
(Hugouviex et al. 1997).
The compIexity of pectins (see Sect. II) implies
that they can only be degraded by an enzyme with
broad substrate specificity or by a set of isozymes
with different, overlapping specificities (as dis-
cussed for Aspergillus niger in Sect. III). This could
account for the presence of CWDE gene families
that are found in several plant pathogenic fungi.
There is as yet no information about the sub-
strate preferences and biochemical properties of
CWDEs of any plant pathogenic fungus. A combi-
nation of a phylogenetic analysis, as performed for
endopolygalacturonases by Wubben et al. (1999),
and a biochemical characterization might reveal
whether enzymes in the same monophyletic group
share biochemical properties.
An endopolygalacturonase will have more
impact on the integrity of a pectin polymer than
an exopolygalacturonase (see Sect.III). However,
nonprocessive endopolygalacturonase activity
will contribute less to nutrient provision than
an exopolygalacturonase activity. A processive
endopolygalacturonase (see Sect. III) can fulfill
both tasks. The most appropriate moment of
expression of a particular CWDE therefore
depends on its biological properties. When a fungal
pathogen attempts to breach a cell wall in order to
get access to the host tissue (action 1, see above),
the expression of an endopolygalacturonase seems
more functional than expression of an exopoly-
galacturonase. In a later stage, when the host tissue
is largely colonized, the pathogen must acquire
nutrients (action 3, see above), presumably by the
activity of exopolygalacturonase and/or processive
endopolygalacturonase. Although this strict sepa-
ration of pathological and saprophytic functions is
somewhat artificial, it might be useful in unravel-
ing the way in which CWDEs act during plant
pathogenesis.
v. Evidence for the Involvement
of Fungal CWD Es in Pathogenesis
Unti11997 there was ample evidence that fungal
CWDEs do not playa significant role in infection.
This was in sharp contrast to what had been pub-
lished for CWDEs from a number of bacterial
species. There is now growing evidence that fungal
CWDEs can also be involved in pathogenesis.
Shieh et al. (1997) were the first to report that an
endopolygalacturonase from Aspergillus flavus is
involved in the infection of cotton bolls. Although
this fungus is generally considered a saprophyte
rather than a pathogen, it is beyond doubt that it
pro duces a CWDE that is required for full growth
on living plant tissue. One year later, an endopoly-
galacturonase from the necrotrophic pathogen
B. cinerea was also shown to be required for
full virulence (ten Have et al. 1998; see Sect. VII).
In both these cases a reduced virulence was
found.
Endopolygalacturonase genes with more than
99% nuc1eotide sequence homology were isolated
from Alternaria citri and A. alternata rough lemon
pathotype. Gene replacement in A. citri resulted
in a mutant that lost almost all virulence, whereas
the mutant in A. alternata was equally virulent as
its wild-type counterpart (Isshiki et al. 2001). The
A. citri gene was expressed in planta. Expression
of the A. alternata gene was not studied. The
important difference between these two strongly
related fungi is in the production of a host-
selective toxin. A. alternata rough lemon patho-
type pro duces ACR-toxin (Akimitsu et al. 1989)
whereas A. citri seems to lack any toxin produc-
tion. It can be envisaged that the ACR-toxin over-
ruIes the function of an endopolygalacturonase
and other CWDEs in A. alternata. It would be
worthwhile studying the effect of the A. alternata
rough Iemon pathotype endopolygalacturonase
gene in a toxin-deficient mutant. Pectate lyases,
which are active at entirely different pH (see Sect.
III), can also be involved in pathogenesis. It has
been reported that a mutant of Nectria haemato-
cacca, deleted in two pectate lyase genes, virtually
lost all virulence (Rogers et al. 2000). N haema-
tococca contains a family of at least four pectate
lyase genes. pelA is induced in vitro by growing
the fungus on pectate. pelD is not inducible in
vitro, but it is expressed during fungal growth in
planta. Mutants in either pelA or pelD alone show
no distinct phenotype whereas the double mutant
has a drastically reduced virulence. This example
of functional overlap illustrates the difficulty of
mutational studies on fungal CWDEs.
A second example of a pectate lyase that is
important in virulence is pelB from C. gloeospori-
oides. Replacement of this gene yielded a mutant
with approximately 40% reduced virulence on
 The Contribution of Cell Wall Degrading Enzymes to Pathogenesis of Fungal Plant Pathogens
avocado fruits when compared to the wild-type.
Both pectate and pectin lyase activities were
reduced in the mutant, indicating that the pelB
gene product is active on partially methylated
pectin (Yakoby et al. 2001).
More evidence that fungal CWDEs are
involved in pathogenesis in other fungi was
obtained for endopolygalacturonases of Claviceps
purpurea (Oeser et al. 2002; see Sect. VI) and
xylanases of Magnaporthe grisea (Wu, Zhao,
Darvill and Albersheim, 6th International Con-
gress of Plant Molecular Biology).
Cochliobolus carbonum is the fungus for
which the most thorough analysis of the potential
functions of CWDEs has been performed. Walton
and colleagues have performed knockout muta-
genesis in numerous genes (Scott-Craig et al. 1990,
1998; Schaeffer et al. 1994; Apel-Birkhold and
Walton 1996; Gärlach et al. 1998) and all mutants,
including double knockout mutants, showed wild-
type levels of virulence. All mutants retained
residual enzyme activity, mediated by other
isozymes. A mutation in the regulatory gene
ccsnfl, an orthologue of the Saccharomyces cere-
visiae SNFl gene, resulted in reduced virulence
(Tonukari et al. 2000). The activity of most
CWDEs in the ccsnß mutant was reduced, but it
remained above the levels that are found in indi-
vidual knockout mutants in the CWDE encoding
genes themselves. On the one hand, this suggests
that virulence can only be reduced by interfering
with the concerted action of multiple CWDEs, on
the other hand, the ccsnfl mutation obviously
affects other functions in primary metabolism, as
the growth of ccsnß mutants on simple sugars was
impeded (Tonukari et al. 2000). It therefore
remains uncertain to wh at extent CWDEs are
involved in the infection process of Cochliobolus
carbonum. The genus Cochliobolus occupies a
special position among fungal pathogens, as it
consists of weakly pathogenic species as weIl as
species that are highly pathogenic on a very spe-
cific host (Turgeon and Lu 2000). There are strong
indications for multiple convergent gains of
pathogenicity traits in the genus. It is hypothesized
that there is a pool of "low general virulence"
germplasm containing basal genes for pathogenic-
ity (Turgeon and Lu 2000). Gene clusters encod-
ing highly specific virulence factors were acquired
by some of these weak pathogens, probably by
horizontal gene transfer. Cochliobolus heterostro-
phus, C. victoriae and C. carbonum are believed to
have acquired genes involved in the production
of toxins that are required for pathogenicity, as
347
discussed in detail in Chapter16. The CWDEs of
Cochliobolus spp. might therefore be regarded as
basal virulence factors whose function must be
evaluated in toxin-deficient strains, as performed
for ccsnfl by Tonukari et al. (2000) or in a weakly
virulent relative.
Genes encoding CWDEs have been subjected
to mutational analysis in three other fungi:
Glomerella cingulata (Bowen et al. 1995),
Cryphonectria parasitica (Gao et al. 1996) and
Fusarium oxysporum (Garcia-Maceira et al. 2000,
2001). No effect on virulence was found. Since in
these studies only one gene was investigated, it is
too early to conclude that these CWDEs do not
play a significant role in pathogenesis. It is
conceivable that knockout mutation of a second
isozyme would result in a strong decrease in
virulence, as was shown in Nectria haematococca
(Rogers et al. 2000).
VI. Case Study: Claviceps purpurea,
a Highly Specialized Biotroph
A. Biology and Life eycle
Claviceps purpurea (Fr. ex Fr.) Tul. causes ergot
disease in ab out 400 species of cereals and grasses
worldwide (Tab er 1985). The homothallic asco-
mycete parasitizes mainly rye, wheat and barley
as weIl as numerous forage grasses. C. purpurea
is organ-specific, exclusively attacking young
ovaries and replacing these gynoecia with its
own propagative and reproductive structures, the
sphacelia and sclerotia (for review, see Tudzynski
et al. 1995; Tenberge 1999).
In nature, the parasitic life cycle starts with
wind-borne ascospores in spring (Fig. 17.2).
Spores attach and germinate on the pistil of
blooming florets. Hyphae invade and colonize the
ovary, grow down to the tip of the ovary axis
and establish a persisting host-pathogen frontier.
At this site, tapping of the vascular traces by inter-
and intracellular hyphae coincides with the exu-
dation of a syrupy fluid, the honeydew (Fig. 17.2b).
The fungus never invades any part further down
in the host but proliferates above this boundary.
A sphacelial stroma grows profusely in the ovary,
producing masses of anamorphic spores. These
conidiospores are exuded into the honeydew,
which serves as nutrition and as dispers al vehicle
in the field. The formation of sclerotia (Fig. 17.2c)
during autumn leaves aspike instead of a caryop-
348 A. ten Have et al.
Fig.17.2. a Drawing of different primary infection sites
(long arrows) of Claviceps purpurea and infection paths, all
of which are terminated at the vascular tissue in the rachilla
(ra) tip, in a rye pistil: dotted Une spore germination and
infection via stigma (si) and style (sy) corresponding to the
pollen tube path up to the micropylar region (m);plus Une
infection at the base of the ovary wall (ow); dashed Une
spore germination on the stigma; infection point is at the
base of the ovary wall; double-arrow lodicule or filament
basis; ad adaxial; Ir furrow region. The routes in the histo-
logically heterogeneous pistil usually lead through several
different tissues: 1 a cuticularized epidermis of the ovary or
2 rachilla, 3 ovary wall mesophyll, 4 transmitting tissue,
5 integuments surrounding the ovule (ov), 6 rachilla corti-
cal parenchyma, 7 vascular tissue with xylem and phloem
elements and parenchyma. b Honeydew exudation from
infected rye florets. Scale bar 5mm c Mature rye ear with
sclerotia. Scale bar lOmm. (Tenberge et al. 1998 with
permission)
sis. Ergot is thus a typical tissue replacement
disease (Luttrell 1980). The next spring, sclerotia
germinate and produce ascospores representing
the new primary inoculum.
As the fungus draws nutrition from the living
ovary tissue, it causes only limited host cell death
and is consequently a biotroph. The biotrophic
nature of the interaction becomes apparent by the
honeydew exudation that depends on a continu-
ous flow of assimilates from the living host. The
interaction of Claviceps species with grass hosts is
discussed in Chapter 18. Here, we focus on the
role of CWDEs in the interaction of C. purpurea
with rye.
B. Cell Wall Degrading Enzymes:
General Aspects
An important characteristic of C. purpurea is the
growth in the apoplast of the (histologically het-
erogeneous) ovary. The pathogen is well adapted
to the monocotyledonous cell wall habitat since it
is able to split the middle lamella zones and breach
host cell walls in numerous grasses. Both fea-
tures point to the use of secreted CWDEs in a
controlled manner. Grasses have developed a
special cell wall, containing low amounts ofpectins
and high amounts of glucurono-arabino-xylans
(GAX) in addition to the predominant constituent
cellulose (Carpita and Gibeaut 1993). It was
therefore anticipated that especially xylanases and
cellulases, but also pectinases, are necessary for
breaking down the major cell wall components
during infection. The cell wall material is thought
to be important for nutrition during colonization
of the ovary because cell wall extracts of ears
stimulated growth in culture (St. Garay 1956).
Actually, these three types of CWDEs are crucial
for ergot pathogenicity, as will be described
below.
The molecular architecture of the host-
pathogen interface has been studied at the elec-
tron microscope level in rye, with emphasis
on interaction-specific reactions, e.g., polymer
alterations and protein secretion. This mole-
cular-cytological study was accompanied by a
molecular-genetic approach in order to elucidate
the roIe of CWDEs in ergot pathogenesis. In the
following sections we present a short update of
the information available for the major CWDE
classes.
C. Pectinases
Shaw and Mantle (1980) demonstrated pectolytic
enzyme activity in culture, in honeydew and in
parasitic tissue extracts. The observation of host
cell wall loosening during subcuticular and inter-
cellular growth of C. purpurea (Tudzynski et al.
1995) indicated that pectolytic enzymes playa role
in parasitism. Unexpectedly, the two major types
of pectin, methylesterified and nonmethylesteri-
 The Contribution of Cell Wall Degrading Enzymes to Pathogenesis of Fungal Plant Pathogens
fied homogalacturonan, were simultaneously pre-
se nt in the cell walls along the infection path in
healthy carpels, as visualized by immunogold
labeling with mono clon al antibodies (Tenberge et
al. 1996). During infection of rye a local molecu-
lar modification as well as adegradation of pectin
have been demonstrated in situ at the interface of
subcuticularly or intercellularly growing hyphae
and the host cell wall. Galacturonan was com-
pletely absent in late infection phases, providing
further evidence for the secretion of pectinolytic
enzymes in planta. Thus, endopolygalacturonase
activity seems a proper tool to enable both epi-
dermis penetration and an entry into the middle
lamella from the apoplastic space, which is not
continuous along the infection route.
Two C. purpurea endopolygalacturonase
genes were cloned and characterized (Tenberge et
al. 1996). They are closely linked in a head-to-tail
arrangement and show 95% identity, pointing to
arecent gene duplication event. Both genes are
expressed throughout the first three weeks of
infection, i.e., during the colonization phase and
the early sclerotium development. The head-to-
tail arrangement allowed a one-step gene inacti-
vation of both genes by areplacement approach,
using a linear DNA fragment containing a
phleomycin-resistance cassette fianked by the 5'-
part of the first gene and the 3' -part of the second
gene, respectively. Two independent double
mutant strains were significantly impaired in
virulence. Complementation by wild-type copies
of the endopolygalacturonase genes restored
the wild-type virulence. This proves that pectin
degradation has an essential role in successful
colonization of rye tissue by C. purpurea
(Oeser et al. 2002).
D. Cellulases
The ergot fungus actively and directly penetrates
plant cell walls (Tenberge and Tudzynski 1994).
ß-1,4-Glucan was not detectable at the position
where intracellular hyphae had penetrated the
host cell wall (Tudzynski et al. 1995), or on the
hyphal surface. Additionally no ß-1,4-glucan was
detected at host-pathogen interfaces of intercellu-
lar hyphae, pointing to the enzymatic action of
cellulases in ergot infection (Müller et al. 1997).
Cellulolytic activity could only be detected on
solid medium but never in liquid culture (Müller
1997), suggesting a strict regulation of cellulase
349
activity. So far one gene has been cloned that
is probably involved in cellulose degradation
(Müller et al. 1997): cpcell probably encodes a
cellobiohydrolase, lacking a cellulose-binding
domain. The gene was expressed during the first
days of infection of rye. Therefore, this putative
cellobiohydrolase may be involved in the initial
degradation of host cell walls. However, deletion
of the gene by transformation with areplacement
vector showed no effect on pathogenicity (u.
Müller and P. Tudzynski, unpubl.), suggesting the
presence of (an) additional cellulase gene(s) in
C. purpurea.
E. Xylauases
The hemicellulose xylan is not only a major cell
wall component of grass leaves, but also a struc-
tural compound in ovary cell walls throughout the
infection route (Giesbert et al. 1998). ß-1,4-Xylan
presumably represents the backbone of GAX
in ovary cell walls, which is decorated with
side chains including arabinofuranosyl epitopes
(Giesbert et al. 1998). The alteration of xylan early
in infection and its absence in late infection stages
were visualized in TEM and after silver enhance-
ment in LM (Heidrich and Tenberge, 5th Euro-
pean Conference on Fungal Genetics, 25-29
March 2000, Arcachon, France), strongly suggest-
ing the secretion of xylanolytic activity by the
fungus. Xylanase activity could be detected in
axenic culture and the secretion of ergot xylanases
during infection of rye has been localized in situ,
using heterologous antibodies in tissue printing
experiments (Giesbert et al. 1998).
Two putative endo-ß-l,4-xylanase genes were
cloned from C. purpurea and characterized: cpxyll
and cpxyl2, probably representing members of
two distinct enzyme families (Giesbert et al. 1998).
Both genes are expressed in planta during the
whole infection period. Using a gene re placement
approach, single mutants for both genes and
double mutants were obtained. Deletion of cpxyll
had no significant effect on virulence. Develop-
ment of cpxyl2 null mutants and double mutants
in planta was, however, significantly retarded
indicating that cpxyl2 is necessary for normal
pathogenesis, although the effect on virulence
was not as pronounced as with the polygalactur-
onase mutants (Giesbert et al. 1998; J. Scheffer, A.
Fleissner, P.M. Heidrich, B. Oeser, K.B. Tenberge
and P. Tudzynski, unpubl.).
350 A. ten Have et al.
F. {3-1,3-Glucanase
Plant synthates directed to the ovary are the main
nutrition source for the fungus (Mower and
Hancock 1975). Several enzymes are secreted to
exploit this natural sink (Luttrell 1980). In sharp
contrast to uninfected ovaries, common phloem
callose was not found in infected ovaries at all or
was distinctly reduced (Tudzynski et al. 1995). This
unblocking of sieve elements may facilitate hon-
eydew exudation due to increased flow of assimi-
lates to the infected floret. The current opinion on
the mechanisms is that ergot fungi enzymatically
degrade the phloem callose by secreting {3-1,3-
glucanases. The enzyme activity has been purified
from axenic cultures of C. purpurea (Brockmann
et al. 1992). ß-1,3-Glucanase has been localized
throughout the colonization phase and in the
fungal secretory system, proving the fungal origin
of the enzyme activity found in infected ovaries
and honeydew (Tenberge et al. 1999). Immuno-
gold electron microscopy documented that the
secreted enzyme is diffusing into the host
apoplast. The distribution of gold labelover host
periplasmic spaces showed that the enzyme was
able to reach the typical deposition sites of callose.
The host phloem was colonized inter- and intra-
cellularly. Hyphae penetrated into the pectic
middle lamella of sieve plates. Intense immunola-
beling for ß-1,3-glucanase was observed in this
area, supporting the phloem unblocking hypothe-
sis. Recently, a putative mixed-link (ß-1,3/1,6)-
glucanase gene was identified within an EST
library of in planta expressed genes of C.purpurea
(E. Oeser, unpubl.), enabling a functional analysis
of this enzyme system.
G. Perspectives
While in necrotrophs the release of cell wall
degrading enzymes results in tissue maceration
and host cell death ahead of the invading hyphae
(Parbery 1996), secretion of these enzymes by the
biotroph C. purpurea causes only limited damage
to the host during colonization. This requires a
strict regulation of the synthesis and/or activity of
hydrolytic extracellular enzymes in this system.
Strict regulation is supported by the extremely low
enzymatic activity in axenic culture, which is in
sharp contrast to the situation in necrotrophic
fungi like Botrytis cinerea (see Sect. VII). In addi-
tion, the fungus might control the physicochemi-
cal properties of the interface to restrict the enzy-
matic action to an adequate but limited area
(Tenberge et al. 1996). This balanced, subtle
interaction might explain why the inactivation of
polygalacturonase and, to a lesser extent, xylanase
genes has such a dramatic effect. In conclusion,
these two types of CWDEs appear to be essential
for the establishment of infection.
VII. Case Study: Botrytis cinerea,
an Opportunistic Necrotroph
A. Biology and Infection Strategy
Botrytis cinerea Pers.: Fr. Botryotinia fuckeliana
(de Bary) Whetz. is a necrotrophic ascomycete
that causes disease in at least 235 plant species
(Jarvis 1977) resulting in considerable economic
losses. The fungus does not only infect many plant
species, but can also infect various organs of a par-
ticular host. Leaf and petal blight, fruit and stern
rot, as weIl as infection of potato tubers (Ramsey
1941), have been reported. Various infection
strategies have been reported (Prins et al. 2000)
and B. cinerea is therefore often regarded a truly
opportunistic plant pathogen. Infection of ripe
fruits often results in direct rot, while the infection
of blossom is often initially followed by aperiod
of quiescence (Williamson 1994). Only upon fruit
ripening does aggressive infection occur. Infec-
tions of leaves in the laboratory typically result in
an infection pattern that is described in three
phases (Benito et al. 1998):
1. Primary lesion formation phase. B. cinerea
penetrates the epidermis and the plant
responds with the formation of a necrotic
lesion that restricts the fungus (0-16 HPI).
2. Quiescent phase. No fungal growth or plant
decay can be observed.
3. Lesion expansion phase. The fungus starts to
invade surrounding tissue from a sm all pro-
portion of the primary lesions, subsequently
colonizing the whole leaf.
How plant cells are actually killed is unknown.
It has been suggested that phytotoxins are
involved (Rebordinos et al. 1996) but this remains
to be supported by the construction of toxin-defi-
cient mutants. Direct penetration using appresso-
rium-like structures has been reported but it is
unlikely to play an important role (reviewed in
 The Contribution of Cell Wall Degrading Enzymes to Pathogenesis of Fungal Plant Pathogens 351

Table 17.1. Gene expression of Bcpg gene family in medium shift experiments a

Bcpgl Bcpg2 Bcpg3 Bcpg4 Bcpg5 Bcpg6

Glucose +++ ++ ++ + +
PGA +++ ++ ++~+ + ++
GA +++ ++ ++~+ ++
Pectin +++ +++ ++~+++ +~++ +++~+ ++~+++
Glucose+PGA +++ ++ ++ ++
Glucose+GA ++ ++ ++ +
Glucose (pH4b ) +++ +++ + +
GA (pH4b ) +++ ++ +++ + ++ +
Glucose (pH6b ) +++ ++ + ++ + ++
GA (pH6 b ) +++ ++ + ++ ++
Glucose (pH8b ) + ++ +
GA (pH8 b) + ++ +++

Data are derived from Wubben et al. (2000) and Wubben (unpubl. results).
aIndicated are the proportional transcript levels: ( +++ ) signifies the highest level of mRNA, (-) indicates that mRNA was
not detectable. An arrow (~) indicates a change in expression in time.
bCultures strongly buffered at indicated pH.

Prins et al. 2000). Many reports on B. cinerea
describe CWDEs and they are presumably
involved in all steps of the infection process. Pen-
etration of the epidermis often occurs at the anti-
clinal position and it is frequently accompanied by
a swelling of the epidermal cell wall as a result of
CWDE action (Mansfield and Richardson 1981;
Elad and Evensen 1995). CWDE activity is found
throughout the infection process and massive
tissue decomposition occurs in later stages.
B. Pectinases
Pectinases of B. cinerea have been the subject of
research for several decades. When the fungus
penetrates the anticlinal wall it subsequently
grows into and through the middle lamella, which
consists mostly of pectin. The host range of B.
cinerea, albeit wide, is restricted to plants with high
pectin contents in the cell wall, i.e., dicotyledons
and corroliferous monocot species. Graminaceae
contain low amounts of pectins and they are
typically nonhosts. Both exo- and endo-acting
polygalacturonases have been reported since the
1970s. At least 14 polygalacturonase isozymes
(Van der Cruyssen et al. 1994), as well as a pectin
lyase and pectin methylesterase (Movahedi and
Heale 1990), have been reported.
In recent years, a molecular genetic analysis of
an endopolygalacturonase gene family has been
performed and analysis of other pectinase genes
was initiated. B. cinerea has at least six endopoly-
galacturonase-encoding genes (Wubben et al.
1999), one pectin lyase gene, one pectate lyase
gene, one pectin methylesterase gene and one
rhamnogalacturonan hydro lase gene. Here we
focus on the results obtained with the endopoly-
galacturonase gene family. The expression of this
gene family has been studied in detail and a
knockout mutant has been obtained in the Bcpg1
gene. Table 17.1 shows a summary of data
obtained in gene expression studies in liquid cul-
tures (Wubben et al. 2000; IP. Wubben, unpubl.).
Four regulatory mechanisms were postulated
based on these data:
1. Basal expression is observed for Bcpg1 and
Bcpg2, with Bcpg1 being the major transcript
(10 to 100 higher than all other transcripts).
2. Induction by galacturonic acid is observed for
Bcpg4 and Bcpg6.
3. Glucose repression is observed for Bcpg4 only.
4. Induction by low ambient pH is observed for
Bcpg3, largely irrespective of the carbon source
present.
Other sugars like rhamnose, arabinose and galac-
tose do not notably induce the expression of any
of the genes. Regulation of Bcpg5 expression
remains unclear. It is induced when grown on
apple pectin, by an as yet unidentified compound
or combination of compounds.
Altogether this endopolygalacturonase gene
family equips the fungus with a flexible pectate-
degrading machinery. This can be advantageous
for a fungus with such a broad host range. The
expression pattern of the encoding genes (Bcpg1-
6) was therefore studied on four hosts: tomato,
352 A. ten Have et al.
broad bean, apple and courgette (ten Have et al.
2001). All gene family members are differentially
expressed, depending on the stage of infection and
on the host. Bcpg1 is expressed in all tissues tested
although differences in transcript levels occur.
Bcpg2 is expressed early in the infection of
tomato, broad bean and courgette, but not in
apple. In broad bean, the level of Bcpg1 transcript
is very low when compared to infection on other
hosts, whereas Bcpg2 transcript levels are very
high. Bcpg2 seems to be strongly induced in
necrotic tissue, but hardly in tissue where soft rot
occurs, suggesting that tissue necrosis or the
accompanying oxidative burst (Govrin and Levine
2000) might act as an inducing factor. Bcpg3 and
Bcpg5 are mainly expressed in apple fruit tissue,
which is in correspondence with the in vitro
inducibility of Bcpg3 at low pH and of Bcpg5
by apple pectin. Bcpg4 and Bcpg6 are mostly
expressed in later stages of infection, when exten-
sive tissue maceration occurs. This expression
pattern is in agreement with the inducibility of
Bcpg4 and Bcpg6 by galacturonic acid. Whether
the observed expression patterns reflect the need
for a specific isozyme at a particular stage of the
infection process remains to be elucidated. The
functionality of such a seemingly flexible enzy-
matic pectate degradation machinery must be
tested with targeted deletion mutants.
Bcpg1 was the first gene that was deleted and
the resulting knockout mutant showed reduced
virulence on tomato leaf and fruit, as well as on
apple fruit (ten Have et al. 1998), broad bean
leaves (A. ten Have, unpubl.) and Arabidopsis
thaliana leaves (1.A.L. van Kan, unpubl.). The
constitutive Bcpg1 expression can explain this
seemingly universal reduction of virulence. The
transcript level is, however, not always indicative
for functionality. Expression of Bcpg1 is high
in tomato leaves but low in broad bean leaves
(ten Have et al. 2001). However, the reduction in
virulence for the Bcpg1 deletion mutant in both
hosts is similar, around 20-30%. Deletion of other
endopolygalacturonase genes is in progress.
Unfortunately, not much is known on the
expression of other B. cinerea pectinases. A
rhamnogalacturonan hydro lase gene has been
cloned and limited expression data have been
obtained (Chen et al. 1997). The gene is induced
by rhamnogalacturonan I; it is expressed when the
fungus is grown on apple pectin and is subject
to glucose repression. Given the number of
endopolygalacturonases found in B. cinerea, it can
be envisaged that a pectin methylesterase plays an
important role. The cloning of a gene encoding
pectin methylesterase (0. Valette, P. Reignault, C.
Levis and M. Boccara, XIIth International Botry-
tis Symposium, 3-7 July 2000, Reims, France) will
enable a study of its function by RNA expression
analysis and knockout mutation. Sequences are
also available for a pectin lyase (w. Mulder and
1. Visser, unpubl.) and a pectate lyase (1.P. Wubben
and 1. Visser, unpubl.).
C. Other CWDEs
Although B. cinerea is often regarded as a pec-
tolytic fungus, nonpectolytic CWDEs have also
been reported. Verhoeff et al. (1983) showed
glucanase and cellobiohydrolase activity, both in
vitro and following inoculation of petioles and
fruits of tomato. Drawert and Krefft (1978) also
describe these enzymes, as well as ß-galactosidases
and ß-glucosidases. Xylanases and arabinase have
also been described (Urbanek and Zalewska-
Sobczak 1984). ESTs with homology to both
endo- and exo-glucanases, cellobiohydrolases
and xylanases have been identified. Cloning of
these genes is in progress (1.P. Wubben, T.W. Prins,
A. ten Have, unpubl.). The EST database (Bitton
et al. 1999) was made from RNA extracted from
an axenic culture subjected to nitrogen starva-
tion. It is therefore likely that substrate-induced
genes are not represented within this database.
This is indeed true for endopolygalacturonase
genes: 39 ESTs correspond to Bcpg1, which is
constitutively expressed, whereas other endopoly-
galacturonase genes are not represented. This
would imply that ESTs that show homology to
other CWDE-encoding genes also reflect consti-
tutively expressed genes. It is conceivable that
genes encoding substrate-inducible CWDEs
remain to be identified.
VIII. Inhibitors of CWD Es
and Polymer Fragments:
Friend or Foe?
A. Oligogalacturonides as Inducers
of Plant Defense
Some genes encoding CWDEs are induced by the
end-product of the enzyme itself or by the end-
 The Contribution of Cell Wall Degrading Enzymes to Pathogenesis of Fungal Plant Pathogens
product of a related enzyme. Clearly, sensing the
environment is an important aspect in the survival
of pathogens. The sensing of a particular degrada-
tion product of a cell wall constituent in the envi-
ronment points to the presence of substrate and
hence serves as a signal for recruiting additional
CWDEs (e.g., the induction of Bcpg4 and Bcpg6
by galacturonic acid, see Sect. VILB). However,
some cell wall degradation products, mainly oli-
gogalacturonides, also serve as elicitors of defense
responses by plants (Cervone et al. 1989). This
type of elicitor is referred to as endogenous elici-
tor and it is believed to act in a nonspecific
manner. Cell wall degradation by a polygalactur-
onase from a pathogen, as well as damage inflicted
by insect feeding or mechanical wounding, results
in an increase of oligogalacturonides in tomato
(Bergey et al. 1996). These molecules induce a
number of responses that are active against
several predators and pathogens. Galacturonides
with a degree of polymerization (DP) of 10 to 13,
believed to be the products of pectinase activity,
are the most effective elicitors in soybean (Noth-
nagel et al. 1983). In tomato, oligogalacturonides
with a DP of 4-6 have been shown to induce eth-
ylene production (Simpson et al. 1998). Biotrophic
pathogens must avoid causing host defense
responses and it seems likely that they will benefit
from a cell wall degrading system that does not
release oligogalacturonides. Processive activity
and concerted action of exo- and endo-polygalac-
turonases are therefore expected to be important
for fungi like U. viciae-fabae. For necrotrophs and
saprophytes the opposite can be envisaged. The
release of oligogalacturonides from galacturonan
by action of an endopolygalacturonase might
trigger plant cell death, which is beneficial to
necrotrophs.
B. Polygalacturonase-Inhibiting Proteins
Polygalacturonase-inhibiting proteins (PG IPs)
are proteins found in many plant species. They can
inhibit certain microbial endopolygalacturonases,
depending on their specificity (Albersheim and
Anderson 1971). In Phaseolus vulgaris, PGIPs are
expressed constitutively but they are also induced
during infection by Colletotrichum lindemuthi-
an um (Bergmann et al. 1994; Devoto et al. 1997).
Within one plant species and between different
species, PGIPs possess differential inhibitory
activity towards endopolygalacturonases from a
353
number of fungi (Desiderio et al. 1997; Cervone
et al. 1998; Stotz et al. 2000). Inhibition of an
endopolygalacturonase will resuIt in reduced
galacturonan degradation, which could slow down
microbial ingress and impede nutrient supply to
the pathogen. PGIPs can successfully be applied
as plant protection agents. A PGIP gene from pear
was introduced into tomato and the obtained
transgenic lines showed increased resistance
against B. cinerea (Powell et al. 2000).
It can be envisaged that the pattern of oli-
gogalacturonides produced in the interaction is
altered by PGIPs. They might affect the amount
and length distribution of oligogalacturonides that
act as elicitors of defense responses. There might
be a co evolution of endopolygalacturonases from
pathogens and PGIPs from plants (Stotz et al.
2000), as described for many gene-for-gene re la-
tionships between plants and pathogens (de Wit
1997). PGIPs contain leucine-rich repeat (LRR)
domains (de Lorenzo et al. 1997), which are also
present in many plant-resistance gene products
that comply with the gene-for-gene model
(Toubart et al. 1992; Jones et al. 1994).
IX. The Role of CWDEs
in Pathogenesis, a Matter
of Fungal Lifestyle?
To date, there have been six re ports on fungal
CWDEs that are involved in pathogenesis (Shieh
et al. 1997; ten Have et al. 1998; Rogers et al. 2000;
Isshiki et al. 2001; Yakoby et al. 2001; Oeser et al.
2002), as weIl as a few unpublished studies. The
fungi involved (A. flavus, B. cinerea, N haemato-
cocca, C. gloeosporioides, A. citri, C. purpurea
and M. grisea) show very distinct lifestyles, as
described in detail for C. purpurea and B. cinerea
in Sections VI and VII. Fungal CWDEs are appar-
ently common virulence factors. We postulated
five different, probably overlapping, roles for
microbial CWDEs in pathogenesis (Sect. IV). We
hypothesize that there is a correlation between the
function of a particular group of CWDEs and the
lifestyle of the pathogen of interest.
Biotrophs depend on living plant cells for
their survival. Rust and mildew fungi take up their
nutrients from the host cell by haustoria, special-
ized structures that invaginate the plant cell. Such
pathogens must breach the cell wall very precisely
and locally without disturbing the viability of the
354 A. ten Have et al.
host cell and without causing defense responses.
Cladosporium fulvum penetrates its host through
stomata and subsequently resides in the apoplas-
tic space where it depends on the available nutri-
ents, predominantly sucrose and nitrate (Joosten
et al. 1990). No cell wall degradation is required
in this interaction and no CWDE activity has been
found. Finally, as discussed in detail in Section VI,
C. purpurea has an adequately regulated CWDE
production that allows it to act as a biotroph, yet
eventually replace the entire ovary tissue by its
own biomass. The difference in lifestyle, even
among biotrophic pathogens, thus demands a dif-
ferent approach of cell wall degradation.
A regulated concerted action of CWDEs is
most probably an important strategy for many
plant pathogenic microbes. In this respect the bio-
chemical properties of the enzyme(s) are very
important. It makes a significant difference
whether a biotroph expresses a processive or a
nonprocessive endopolygalacturonase. Processive
endopolygalacturonases produce monogalactur-
onic acid (i.e., nutrients) whereas nonprocessive
endopolygalacturonases initially produce oligo-
galacturonides. Intercellular growth through
the middle lamella is probably facilitated best by
distortion of the pectin network (the general
pathological function, action 1 in Sect. IV). An
exopolygalacturonase presumably distorts the
walliess than a nonprocessive endopolygalactur-
onase. A fungus that grows through the middle
lamella, like B cinerea, would benefit most
from the sequential and/or concerted action of
a nonprocessive endopolygalacturonase and a
processive endopolygalacturonase or an exopoly-
galacturonase. Access to the underlying tissue is
first established by disturbing the pectin network
and killing the host cells (actions 4 and 5 in Sect.
IV), followed by the complete degradation of
pectin, providing nutrients to the fungus (action 3
in Sect. IV).
x. Concluding Remarks and Prospects
Evidence that fongal CWDEs can contribute to
pathogenesis has been accumulating since 1997.
This area is now regaining broader interest, after
all the experiments that failed to confirm a func-
tion of these enzymes in pathogenesis, especially
from the extensive efforts of Wal ton and co-
workers. We are only beginning to understand
how microbial CWDEs function in pathogenesis.
Many aspects remain to be studied. No general
conclusions can be drawn, each plant-fungus inter-
action and the function(s) of each individual
CWDE in this inter action need(s) to be studied
independently. Such studies require detailed
information on three aspects of the interaction:
1. The plant cell wall: its spatial, developmental
and cell type-specific composition; the amount
of cross-linking before and after attempted
invasions by pathogens.
2. The fungal CWDEs: the regulation of gene
expression and enzyme activity (both in time
and space) by substrates, reaction products and
environmental factors; the biochemical charac-
teristics of isozymes.
3. The interaction between the plant cell wall and
CWDEs: the chemical nature of products that
can be released from a plant cell wall as a con-
sequence of CWDE action; the effect of these
cell wall fragments on the induction of defense
responses and on fungal gene expression.
Molecular genetics provide a helpful tool in
elucidating the functions of individual CWDEs.
Transformation systems are commonly available
for many fungal pathogens, thus far including
only one obligate pathogen, the barley powdery
mildew Erisyphe graminis (Christiansen et al.
1995; Chaure et al. 2000). Functional overlap
between two CWDEs can significantly hin der
functional studies, as was elegantly shown in the
mutagenesis studies in N haematococca (Rogers
et al. 2000). Also, concerted action is difficult to
study. Multiple mutants in appropriate combina-
tions of genes should be made in order to study
this in detail. Unfortunately, a multiple mutagen-
esis strategy based on ura genes (d'Enfert 1996) is
laborious.
With the accumulating knowledge ab out the
synthesis and construction of plant cell walls,
defined plant cell wall mutants will be available.
One mutant in lignin biosynthesis has been
reported (Sederoff 1999) but its susceptibility to
biotic agents was not evaluated. A better und er-
standing of plant cell walls and their degradation
by pathogens will eventually contribute to a ratio-
nal and targeted design of crop protection
methodologies, involving molecular and conven-
tional breeding and the application of novel pro-
tecting agents.
 The Contribution of Cell Wall Degrading Enzymes to Pathogenesis of Fungal Plant Pathogens
Acknowledgements. The research described in
Sections VI and VII was financially supported by
the Deutsche Forschungsgemeinschaft (DFG,
Germany) and the Dutch Technology Foundation
(STW), respectively.
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18 Coevolution of Pathogenic Fungi and Grass Hosts
JACQUES MUGNIER
CONTENTS
I. Introduction ..........................
H. Reconstruction Grass Phylogeny .........
IH. Reconstruction Stramenopilal
and Fungal Phylogeny ..................
A. Stramenopila .........................
B. Ascomycota ..........................
1. Necrotrophic Fungi ..................
2. Hemibiotrophic Fungi ................
3. Biotrophic Fungi ....................
C. Basidiomycota ........................
IV. Reconstructing Grass and Pathogen
Phylogenies ..........................
A. Hemitrophic Fungi and Grasses ..........
1. Helminthosporium ..................
2. Fusarium ..........................
3. Rhizoctonia ........................
B. Biotrophic Fungi and Grasses ............
1. Clavicipitaceous Fungi ...............
2. Rust Fungi ........................
3. Erysiphales ........................
4. Smut and Bunt Fungi ................
V. Conclusions ..........................
References ...........................
I. Introduction
Grasses and pathogenic fungi have evolved
over a considerable period of time. The grass
pathogens are found amongst the Plasmodio-
phoromycota (Ligneria, Polymyxa), Oomycota
(Pythium) , Ascomycota (clavicipitaceous fungi,
Heiminthosporium, Septoria, Fusarium, powdery
mildew, take-all, eye spot, etc.) and Basidiomycota
(rust, smut, bunt, Rhizoctonia, Typhuia, Agari-
caies). The long history of the relationship
between grass and these pathogens may lead to
reciprocal adaptations (co evolution or cospecia-
tion). Gene-for-gene interactions (Flor 1955)
suggest also that biotrophic plant pathogens
Laboratoires Agroservices Aventis Crop Science France, 11
avenue Albert Einstein, 69100 Villeurbanne, France
should have similar phylogenetic histories to their
. 359 hosts.
. 360 A molecular phylogenetic approach was
adopted to address questions of fungal evolution
. 360
. 360 and to determine how fungi have coevolved with
. 361 grass es. This approach enables study of an entirely
. 362 new domain of topics that could not be explored
. 362 with nonmolecular data. There are many advan-
. 363
. 363 tages to using DNA sequeuces data in studies of
host-parasite iuteractions. Among these is the fact
. 364 that the characteristics being compared have a
. 364
. 364
common genetic basis. With DNA sequence data
. 364 we are able to compare homologous sequences
. 365 in the hosts and parasites, thereby avoiding the
. 365 problem of comparing nonhomologous morpho-
. 365
. 368 logical characteristics.
. 369 The analysis of the host-parasite system
. 371 involves the following steps: host tree building,
. 372 parasite tree building, comparison of the two trees,
. 372
and estimation of the divergence or convergence.
Because further analyses are dependent on the
quality of these trees, the trees must be weIl
resolved and consistent.
The DNA sequences analyzed here represent
relatively short regions (500-700 base pairs) of the
ribosomal DNA (Internal Transcribed Spacers)
as defined by primers ITSl and ITS4 (White
et al. 1990). The sequences were compared with
those obtained by other researchers (see
National Center for Biotechnology Information
on the Internet (http://www2.ncbi.gov/genbank/
query_form.html) and were aligned with the
program CLUSTALW in the PHYLIP package
(version 3.5c, Felsenstein). The neighbor-joining
trees produced are shown in Figs.18.l-18.3 and
18.5 and the KITSCH trees in Figs.18.4 and 18.6.
We examined the pattern of association
between the plant pathogens and their hosts
by comparing host phylogeny (based on ITS
sequences) with that of their pathogens (based on
ITS sequences). One might expect to find phylo-
genetic evidence for coevolution between plants
and their pathogens.
The Mycota XI
Agricultural Applications
Kempken (Ed.)
© Springer-Verlag Berlin Heidelberg 2002
360 J. Mugnier

11. Reconstruction Grass Phylogeny analysis supported the view that the subfamily
is polyphyletic. Separate lineages are resolved:
Evolutionary relationships among the Poaceae the tribe Danthonieae, the tribes Aristideae and
have been particularly difficult to resolve. Many Arundineae are sister taxa.
c1assifications have been proposed. Most of them The monophyly of Panieoidae is supported
recognize seven or eight major tribes: Bam- by the molecular analysis. The common mor-
busoideae (Orysoideae), Panicoideae, Cento te- phological characteristies are paired spikelets
coideae, Stipoideae, Chloridoideae, Arundinoidae, with a single female fertile floret. The tribes
and Pooideae. Oryzoid genera were placed within Andropogoneae (Sorghum, Zea) and Paniceae
the Bambusoideae in many c1assifications. The (Panic, Pennisetum) are sister groups.
system of c1assification proposed by Watson The subfamily Bambusoidae traditionally
and Dallwitz (1998) is the most widely used has been the focus of speculation concerning the
today. Comparative data are available for a wide most primitive members of the grass family. They
selection of characteristics inc1uding molecular share apparently plesiomorphic features, such as
characteristies (see Grass Phylogeny Working spikelet-like branches ("pseudospikelets").
Group 2000). Most molecular analyses regarding
grass phylogeny are based on data from the
chloroplast genome (rbcL sequence data). Hsaio 111. Reconstruction Stramenopilal
et al. (1995, 1998) sequenced the ITS, focusing
and Fungal Phylogeny
on Pooideae and on Arundinoideae. Recent
phylogenetic studies (phytochrome B) provide
resolution of evolutionary relationships within A. Stramenopila
Poaceae; Matthews et al. (2000) divided the grass
family in two c1ades: the first comprises bambu- Different species of Pythium (Oomycota) are par-
soid, oryzoid, and pooid genera (the BOP group); asites of the grass root systems. With the advance
the second comprises panieoid, arundinoid, chlo- of molecular techniques, hypotheses of phylogeny
ridoid, and centothecoid genera (the PACC among the various groups of fungi changed radi-
group). cally. Although there was already a great deal of
Interrelationships among the grass family evidence that the Oomycota and 'true' fungi were
inferred from the ITS sequence data generally different, analysis of the ITS sequences by Lee and
agree with the traditional c1assification (Fig.18.4). Taylor (1992) demonstrated that they are very dis-
The results highly supported resolution of tantly related. The Oomycota are a c1ass placed
two groups: (1) Panicoideae, Arundinoideae, in the kingdom Stramenopila (Straminipila for
Centothecoideae (PAC group), and (2) the some authors). The diversity of this kingdom is
Pooideae-Stipoideae group. Bambusoideae (Ory- the appearance of tripartite tubular hairs (stra-
zoideae) are placed as an additional lineage menopiles) during the life cycle of its members
among the early-diverging elements. Monophyly (inc1uding the chromophytic algae). Despite the
of the Centhecoideae remains undetermined apparent extreme diversity of Alga and Oomy-
because just Chasmanthium was sampled. cota, the monophyly of the Stramenopila has been
The placement of Chloroideae in our results strongly supported by several molecular system-
remains problematic. Based on the phytochrome atie studies.
B (Matthews et al. 2000) and chloroplast (Grass The principal, distinguishing characteristics of
Phylogeny Working Group 2000) phylogenies, the the genus Pythium are usually considered to be
Chloroidoideae are inc1uded in the PAC group. the sporangia: they are filamentous (nematospon-
The monophilies of Panieoideae and Poideae were gia), spherical (sphaerosporangia), and proligerat-
confirmed, whereas the unity of the Arundi- ing. The results from the analysis of the ITS data
noideae and the Panieoideae is refused. The tribes set (Grosjean 1992; Mugnier 2000) support the
Tritieeae (Triticum) and Bromeae (Bromus) are traditional view of three monophyletie groups
sister groups. The presence of many heteroge- (Fig.18.1). The evolution of Pythium examined by
neous morphological characteristics within the comparing the cytochrome oxidase gene (Martin
Arundinoideae cause taxonomie difficulty and 2000) corresponds well with our ITS results. One
the boundaries of the subfamily are uncertain in of the filamentous lineages consisted of the species
current grass classifications. The ITS phylogenetic Aristosporum, Arrhenomanes, Graminicola,
 Coevolution of Pathogenic Fungi and Grass Hosts 361

(1) P. irregu/are
P. paroecandrum (3) P. iwa yamai
P. sy/va/icum P. ohanoganense
P. mamilla/um 1 2 P. macrosporum
P. spinosum
(5) P. vic/ae
4 P. <llIimum
P. sporangiif&rum
6 P. rostra/um

(7) P. mas/ophoron
P. po;ymarrum

8 P. nodosum
P. midd/etonii 5 P. nunn
P. p&rpl&xum
P. om8Carpum 24
P. erinaceum

P /leliccides 23
P. marSlp,um "-"::::=:=====~;;;",.-.,.....--l<

P. oedoc/lilum
P. os/rscodss 22 9 Saprolegnia ferax
P. megscsrpum

12 P. co/ara/um
P. diclinum
14 P. roru/osum ~ dissOlfum
P ca/snu/s/um . 8Qua/1 e
P.monospermum P. flevoense
(15) P. perii/um
P. infls/um (13) P sulca/um
P. mynatylum
P z;ngtberum

16 P. echinu/a/um
(17) P. acanthicum P periplocum
P. h ydnosporum P ollgandrum

Fig. 18.1. Phylogenetic relationships among Pythium gial morphology: sphaerosporangia (1 to 8, and 24 to 28),
species using ITS sequence data on a neighbor-joining tree. nematosporangia (9 to 19), proliferating sporangia (20 to
There are three major phyla corresponding to the sporan- 23)

Volutum, which are weH characterized by being
parasitic to monocots. The taxa iwayamai-
okanoganense is composed of species that cause
diseases of cereals and grasses at low temperature.
B. Ascomycota
Over the last decade, mycologists have con-
centrated on delimiting monophyletic orders
within the Ascomycota (Reynolds and Taylor
1993; Taylor et al. 1994; Mugnier 1998). A journal,
Systema Ascomycetum, was published by
Ericksson and Hawsworth (1986-1999) and is
continuing in Myconet on the Internet. A new and
radical c1assification of families and high er taxa of
ascomycetes was published by Eriksson (1999)
based on the small subunit ribosomal DNA. Here,
the Erikson system of c1assification using
the internal transcribed spacers (Fig.18.2) is used.
The names for the suprageneric taxa are based on
a generic name, e.g., Sordariomycetes, Doth-
ideomycetes, Leotiomycetes, not Pyrenomycetes,
Loculoascomycetes, Discomycetes.
Early diverging Ascomycota have been
grouped into the Pneumocystidiomycetes, Taphri-
nomycetes, Saccharomycetes and Pezizomycetes.
The Saccharomycetes comprises the yeasts (ex.
Saccharomyces cerevisiae). The Pezizomycetes
("cup fungi") contain the MorcheHaceae (moreis),
the Tuberaceae (truffles), and the HelveHaceae
(false morels and saddle mushrooms). The cup
fungi (Helotiales and Pezizales) are, however,
a paraphyletic unit. Euascomycetes contain
weH over 90% of Ascomycota. Among them we
find some serious plant pathogens. The Artho-
362 J. Mugnier

Leotiomycetes
, Helotlales h P,eudoeefcosp«eIJa harpotm:f'I!OIICJe.s
2 Erys,ptlales I RhVnchO$poflum lI.ecohs
3 Rhyhsmatales J Ery"phe g<amln ..

Lecanoromycetes
lecanorales
Dothideomycetes 11
Oolh idea les

Eurotiomycetes
1 Onygenales
e 2 _ Eurotlales
e .A.spergIHLr3, P'BlbCllhUrf

Dothideomycetes I
1 Oothideales
2 Pleosporales
Septona nodorum
Orec:hIHtra 5pp
m Exs.,rnhdum.pp
n CUJVUlanII .pp c Sordariomycetes
o 81PQ1ans spp , Hypocreales
p AH~ 2 . Ooaporthales
G EptCCOCUm 3 Sordarlales
4 Microascales
Pezizomycetes 5 Magnaporthales
Pez,zales 6 . Opl1ios!omatales
7 Phyllacorales
8 Xylariales
• Collelotrd1um. Phy'....."or.
b Ptulllophota gt'em 101
Ep!ChIo<I
F usarrum 'Spp
Pneumocystidomycetes
Saccharomycetes Pneumocyst,dates
Saccharomycetales
t Saccharomvces cer~'S1at

Fig. 18.2. Phylogenetic tree of the Ascomycota based on tiomycetes (inoperculate Discomycetes), Dothideomycetes
the ITS sequence comparison. Eriksson (1999) has (Loculoascomycetes), Eurotimycetes (Plectomycetes),
proposed a revised arrangement of higher taxa. The corre- Pezizomycetes (operculate Discomycetes)
spondences with most traditional systems are: Leo-

niomycetes and Lecanoromycetes contain discol-
ichenized species.
1. Necrotrophic Fungi
Examples of necrotrophic fungi able to colonize
grass leaves and ears are Alternaria, Epicoccum,
Botrytis, Cladosporium, etc. There is no evidence
of host specificity in these facultatively pathogenic
species, and they can probably attack any stressed
plants.
2. Hemibiotrophic Fungi
Grass pathogens are presented in the Fig.18.2:
Pseudocercosporella herpotrichioides (teleo-
morph Tapesia yallundae), Phialophora graminis
(teleomorph Geaumannomyces graminis), Pyricu-
laria oryzae (teleomorph Magnaporthe grisea),
Septoria tritici (teleomorph Mycosphaerella
graminis) , Rhynchosporium secalis (mitosporic),
Septoria nodorum (teleomorph Leptosphaeria
nodorum), Drechslera spp. (teleomorph
Pyrenophora) , Exserohilum spp. (teleomorph
Setosphaeria), Curvularia spp. and Bipolaris
spp. (teleomorph Cochliobolus), Colletotrichum
graminicola (anamorphie Glomerella), Phylla-
chora graminis, and Fusarium spp. Olivia et al.
(2000) described sequence analysis of ribosomal
genes and the ITS region for a large number of
graminicolous species of Helminthosporium, as
weIl as for some dothideomycetal relatives.
 Coevolution of Pathogenic Fungi and Grass Rosts
3. Biotrophic Fungi
Powdery mildews are one of the most widespread
plant diseases. They affect virtually all kinds of
plants: cereals and grasses, vegetables, ornamen-
tals, and fruit trees. Many taxonomists have placed
Erysiphales among the deistothecial fungi and
others have considered them to be perithecial
fungi.
Saenz et al. (1994) in a study based on the 18S
rDNA showed that Erysiphe graminis does not
belong in the Sordariomycetes or the Euro-
tiomycetes, but is placed in the Leotiomycetes, the
cup fungi (Fig.18.2).
C. Basidiomycota
The basal divergence in the Basidiomycota is
between the Ustilaginomycetes (the smut and
bunt fungi) induding the Urediniomycetes (the
Copnnaceae(A)-22
fCQCWV'lUt ~
Coolnariaceae (Al - 21
Tricholomalaceae (Al - 20
Tncholomataceae (Al · 17
~Ip!lhorales ·15
12 • Ustilaginaceae (U)
13 . Phylum Ascomycota
Fig. 18.3. Phylogenetic tree of the Basidiomycota based on the ITS sequence data. A Agaricales; B Boletes; G Gas-
teromycetes; P Polypores; TTremellales; U Ustomycetes
363
rust fungi) and the rest of the Basidiomycota
(Hymenomycetes). In Hymenomycetes, the
morphologically defined major taxa are , e.g.,
Agaricales (gilled mushrooms, e.g., Coprinus) ,
Aphyllophorales (non-gilled mushrooms, e.g.,
Rhizoctonia, Typhula) , boletes, puffballs, and
polypores, etc.
Many basidiomycetes genera have been das-
sified using the ITS region, e.g., Urediniomycetes
(Gardes and Bruns 1993; Zambino and Szabo
1993), Ustilaginomycetes (Bakkeren et al. 2000),
and Aphyllophorales (Boidin et al. 1998). Despite
some areas of poor resolution (Auriculariales,
Tremellales), the general structure of Basidiomy-
cota is now dear and well supported (Fig.18.3).
Using this framework, long-standing questions
of diversification of gasteromycetes (puffballs ),
aphyllophorales (without gills) and agaricales
(gilled fungi) can be addressed. For example,
gasteromycetes have apparently arisen multiple
times within the Basidiomycota.
4 • Boletaceae (B)
Gomphidaceae (Bl
Rhizopogonaceae (B)
Paxillaceae (B)
5 • Tilletiaceae (U)
CT-'
6 • Tremeilaceae (T)
7 • Ceratobasldlaceae
~ RtuJ:OC«I"r.l
8 • ~~=inaceae (U)
9 • PlJCCinoaceae (U)
( P~)
10· Fllobasid,aceae (T)
11 • Platygloeaceae (U)
364 1. Mugnier
Boidin et al. (1998) demonstrated that the
aphyllophorales represent different independent
lineages. Polyporaceae rivaled with Corticiaceae
in the numbers of species but it is alm ost certain
that these groups are polyphyletic. In the tree pre-
sented in this study, the Ceratobasidiales are dis-
tantly related to the Aphyllophorales.
The agaricales are in a single lineage (Agari-
eales sensu stricto or euagarics). Hibbett et al.
(1997) showed that seven species of gilled mush-
rooms were placed outside of the euagarics
(Lentinus, Panus, Lentinellus, Russula, and
Cantharellus ).
Major basidiomyctous grass pathogens are
exemplified:
Smuts (Ustilago, Sporisorium) and bunts (Tilletia)
are biotrophic, occur worldwide and are persis-
tent pathogens mainly of the Poaceae.
Rusts (Puccinia, Cronartium, Melampso ra ,
Melampsoridium, Pucciniastrum) are
biotrophic specialized parasites. Puccinia spp.
are parasites of many different grasses inc1ud-
ing important cereal crops such as wheat, barley,
rye, and oats.
Several species of Typhula are pathogens of
grasses at low temperatures. Typhula is assigned
to the c1avaroid Telephorales (Aphyl-
lophorales). Using ITS, Hsiang and Wu (2000)
indicated a c10ser affinity of Typhula to the
agaroid forms compared to the Thelephora. The
genus Typhula seems polyphyletic and more
sequences will be sampled in the future in order
to more fuHy address its taxonomy.
Rhizoctonia spp. belong to the Ceratobasidiaceae
(Ceratobasidium, Thanatephorus, Uthatobasid-
ium). They are aggressive pathogens and can
cause extensive damage on economic important
plants (cereals, rice, potato, beet, etc.).
Agaricales. Some agaricales are pathogenic on
grasses, e.g., Marasmius oreades, Lycoperdon,
Psalliote, Clitocybe, and Coprinus psychromor-
bidus (cotony snow mold), which cause fairy
rings on turfgrass.
IV. Reconstructing Grass
and Pathogen Phylogenies
A. Hemitrophic Fungi and Grasses
1. Helminthosporium
Cochliobolus inc1udes a large number of species
known only from their asexual states: Bipolaris
and Curvularia. The ITS analysis indicates
that Cochliobolus and its anamorphs are
monophyletic, c10sely related to Pyrenophora
(anamorph Drechslera) and Setosphaeria
(anamorph Exserohilum).
Molecular phylogenetic results (Berbee et al.
1999) also support the results of morphological
systematics and asexual states c1ustered with their
predicted sexual relatives. Figure 18.4 shows
the phylogenetic relationships among different
species of Helminthosporium and their plant
hosts.
Cochliobolus group 2 inc1uded species with
curved Curvularia type conidia as well as species
with straight Bipolaris conidia. Cochliobolus
group 1 inc1uded species responsible for plant
disease epidemics. It may be coincidental that
the highly virulent, economically important,
Cochliobolus pathogens are c10sely related in
group 1 rather than group 2. There are no rela-
tionships between grass host and Cochliobolus
phylogenies; many of the Cochliobolus species in
both groups 1 and 2 grow on host grasses for more
than one tribe. For the Cochliobolus and their
hosts, there was little evidence of parallel c1ado-
genesis. High virulence in species of Cochliobolus
(group 1) is linked to synthesis of a host-specific
toxin: T-toxin (polyketol) in C. heterostrophus,
(chlorinated polypeptide) in C. victoriae, HC toxin
(cyc1ic tetrapeptide ) in C. carbonum, HS-toxin
(sesquiterpene) in B. sacchari. Given the lack of
homology of these toxins, their role in virulence
must have arisen independently.
2. Fusarium
An intriguing characteristic of some species of
Fusarium belonging to the section Liseola (Fusar-
ium moniliforme, F. proliferatum, F. subglutinans)
is their propensity for colonizing particular hosts.
Heterokaryon tests between different isolates led
to the c1assification of so-called vegetative com-
patibility groups (Esser and Blaich 1994). Fusar-
ium seetion Liseola belongs to six (A-F) distinct
mating-type populations (Table 18.1). The A pop-
ulation is usually found on maize, the B popula-
tion is more frequently found on sugarcane and
millet (Poaceae, Stipoideae), the C population is
commonly found on rice (Poaceae, Bambu-
soideae), and the D population is found on both
maize and sorghum. Only isolates of A and D
populations (G. jujikuroi) are highly virulent on
asparagus. The Band E populations (F. subgluti-
nans) were slightly virulent on asparagus.
 Coevolution of Pathogenic Fungi and Grass Rosts
Table 18.1. Mating population of Fusanum seetion Liseola and their hosts
Species Synonym
R subglutinans R moniliforme
(R oxysporum lineage) var. subglutinans
R proliferatum F. moniliforme
(G. jujikuroi lineage) var. proliferatum
It is interesting that the mating populations C
and F are preferentially associated with a host,
namely rice and sorghum, respectively. The A and
D mating populations have been recovered from
a variety of hosts.
Molecular evidence based on phylogenetic
analyses of sequences obtained from multiple
unlike genes (ITS, tubulin) strongly indicates that
G. jujikuroi and F. subglutinans are nonmono-
phyletic (Fig.18.5). F. subglutinans is phylogeneti-
cally nested with section Elegans (F. oxysporum).
The molecular analysis identified a monophyletic
lineage, the G. jujikuroi complex that comprises
at least 30 phylogenetically distinct species
(Niremberg and O'Donnell 1998). Another char-
acteristic for the G. jujikuroi complex includes
fumonisin production, suggesting that toxin pro-
dnction may have been a key factor in the evolu-
tionary success of this phytopathogenic lineage. G.
jujikuroi also produces metabolites including the
well-known gibberellin phytohormone-induced
bakanae disease of rice.
3. Rhizoctonia
Rhizoctonia solani is a complex of genetically
isolated species with cornrnon rnorphology,
associated with the Thanatephorus teleomorphic
state. Hyphal anastomosis involved in self-nonself
recognition has been used to establish anastomo-
sis groups (AGs). R. solani is composed of at least
13 groups based on the occurrence of hyphal
fusion and other characteristics.
The phylogenies defined by analysis of ITS
variation correlate with the AGs (Boidin et al.
1998). AG1 is divided into subgroups AG1-IB and
AG1-IA. Isolates of R. solani from AG1-IB cause
web blight on rice and seedling damping-off on
various grasses; isolates from AG1-IB cause
sheath blight on rice. AG2 is divided into sub-
groups AG2-1 and AG2-2 based on frequency of
anastomosis and thiamine auxotrophy (AG2-2).
Isolates of AG2-1 cause stern rot in Brassicaceae;
isolates AG2-2 are mainly pathogenic to
365
Mating population Rost(s)
B Sugarcane, millet (tropical)
E Maize (but less frequently than A)
A Maize, asparagus
D Maize, sorghum, asparagus
C Rice (bakanae disease)
F Sorghum
Chenopodiaceae. AG 2-2 is further divided into
subgroups IIIB and IV based on growth at 35 oe.
Isolates AG2-2IIIB primarily cause root rot and
stalk rot in mature maize.
Rhizoctonia cerealis associated with the Cera-
tobasidium teleomorphic state have also been
divided into Ceratobasidium anastomosis groups
(CAG) which are mainly parasites on the roots of
grasses.
B. Biotrophic Fungi and Grasses
Increased parasitism reduced pathogenicity (by
restricted growth and colonization of the host)
and the saprophytic habit has been lost in the
biotrophic fungi. They are correspondingly highly
host -specific. Gene-for-gene interactions suggest
that plant pathogens ought to have similar phylo-
genetic histories to their hosts. Because of their
agriculturally important hosts, the obligately path-
ogenic rusts and powdery mildews have been the
subject of much interest.
1. Clavicipitaceous Fungi
The Clavicipitaceae consists of biotrophic para-
sites of fungi, insects, and plants. The evolutionary
his tory of this family is only vaguely known,
since most of the phylogenetic work has con-
centrated on a few genera, e.g., Claviceps,
Atkinsonella, Balansia, Balansiopsis, Epichloe
and Myriogenospora (Kuldau et al. 1997). These
endophytes, largely restricted to grasses, produce
a variety of ergot alkaloids in their hosts. Chemi-
cal defense against herbivory is the basis for
coevolutionary changes in the host grasses and
endophytic fungi.
Atkinsonella, Epichloe and Acremonium
infect C3, or cool season grasses, while Balansia,
Balansiopsis, and Myriogenospora infect C4, or
warm season grasses. Many grasses are asympto-
matically infected with endophytes, referred to the
an amorph genus Acremonium.
 GENUS TRI8ES

~ ,

I~
e ..
e 0 I
~ m
~ g.. STIPOIDEAE

~ s:::
~
t ]9
:,:ai:;'i"aa:e: .': ..
Cl
it: I 1II',r.,
I
~ I-----. I IIi"I:' POIODEAE

'.WJI_I \ \ ~
_ --'-- ___ _ --'-"-----L~_

.,llliiiJil1MZ! iMt'l!16 i I------.. \\ I .P.P!II . .

:-<
Q,) ~
Cl... W1~Bl ~
:::l [Jq
~ :::I
.~
0 , STIPOIDEAE
..... (S .
5 ~ I ....
"0 ......
.e-
CQ
-=:
0
I~
~
~
'-'
0
I...J
I~ / U-

OJr.l=::h , 1
~:~Jt'Ul! $ IR. 1
'.':~ . t-- \ \ \ \ ,\ N01

.ltill;\~1 1 \ \ \1 ...'

I i PhJl ~ J
Fig. 18.4. Comparison of the helminthosporium ITS phylogeny (ZeJt, derived mainly from Berbee et al. 1999) and the grass host ITS phylogeny
(right). Lines connect parasites with their natural hosts
 Co evolution of Pathogenic Fungi and Grass Hosts 367

...< ...<
... ...
CI
CI
ö

1~1 I~

-
.. I -.
. .- . .
- -
- - -
-- -- -
~

.- - - -

... ... • - -
..:
; ;

U ..... -c.-
T
11 adnoJ~ m/oqO!/qJO) D!J~Dqdsol~S

S!JDIOd!8 pUD D!JDlnAln) wnl!qOJ~SXl

368
F. sambuclnum
F. subglu/inans
F. prol/fet1llum
Fig. 18.5. Simplified phylogenetic tree of the genus Fusar-
ium based on ITS sequence data. The species graminearum,
sambucinum, sporotrichoides, poae (section Discolor) are
pathogenic mainly on cereals and corno F. oxysporum
(section Elegans) is the causal agent of wilts on dicots. The
hosts of F. subglutinans and F. proliferatum (both in section
Liseola) are presented in Table 18.2
About 45 ergot species of Claviceps have
been described, mainly from the tropics and sub-
tropics. The distribution of ergot species through-
out the world has several interesting features
(Pazoutova 1999). First, there is a striking differ-
ence in the number of ergot species colonizing
the chloridoid, pooid, and panicoid subfamilies:
non-grass Cyperaceae (3), Arundinoideae (1),
Pooideae (3), Bambusoideae (1), Centothecoideae
(1), Chloridoideae (4), and Panicoideae (30). Most
species have been found on panicoid hosts,
whereas only few were collected on grasses of
other subfamilies. The most popular species, Clav-
iceps purpurea (rye ergot), is widespread on pooid
grasses with occasional occurrence on arundi-
noids, chloridoids, and panicoids. The host speci-
ficity and grass phylogenetic tree also do not show
direct relationship to the Claviceps phylogeny, as
pooid/arundinoid and panicoid hosts are found in
both main branches.
The c1avicipitaceous endophytes produce
fruiting bodies and the infected plants are
sterilized by hormonal inhibition of flowering
("parasitic castration"). The sterile plants are veg-
etatively vigorous and resistant to herbivory. In
contrast, the Acremonium endophytes do not fruit
on their hosts and the hosts are perfectly fertile;
they are transmitted into ovules and seeds.
In Claviceps, the ergot alkaloids are concen-
trated in the sc1erotium. Ergot poisonings result
from accidental ingestion of sc1erotia in grain or
J. Mugnier
forage. In Balansia and Epichloe, the alkaloids are
systemic, and, therefore, the entire plant is poten-
tially toxic to herbivores. Evolution from a local-
ized infection, as seen with Claviceps , to a systemic
infection, as with Balansia and Epichloe, results in
sexual sterilization of the host plants. Many fungal
endophytes, as with Acremonium, have responded
to the restoration of host fertility by losing the
ability to reproduce sexually themselves.
For Epichloe and their grass hosts (based on
ITS sequences), there was evidence of parallel
c1adogenesis (Schardl et al. 1997; Moon et al.
2000). The monophyly of the "core" Pooideae-
Stipoideae (inc1uding tribes such as Aveneae,
Poeae, Triticeae, and Bromeae) has been substan-
tiated by the Epichloe parasitism (Fig.18.6).
2. Rust Fungi
Puccinia spp. have life cyc1es between their grass
hosts and eudicotyledonous hosts (Table 18.2).
Stages II (uredinial) and III (telial) are produced
on the grass host and stages 0 and I on eudi-
cotyledons (spermogonial and aecial). Some Puc-
cinia appear to lack an alternate host (e.g., P.
striiformis). Zambino and Szabo (1993) and
Gardes and Bruns (1993) have studied the
phylogeny of the rust fungi.
Based on ITS sequence analysis, Szabo et al.
(1996) demonstrated: (1) P. graminis and P. coro-
nata are monophylogenetic groups, while P. recon-
dita is a species complex rather than a single
species; (2) the microcyclic rust P. mesnieriana is
very c10sely related to the correlated species P.
coronata; and (3) two leaf rusts (leaf rust of barley,
P. hordei and the microcyc1ic rust Uromyces
scillarum ) are very c10sely related, indicating
that the taxonomic division between Puccinia
and Uromyces is artificial. Zambino and Szabo
(1993) showed that P. striiformis, which has no
known alternate host, is most c10sely related to the
native grass rust, P. montanensis, which produces
its sexual stage on native barberry (Berberis)
species.
Puccinia seymouriana is a grass rust that pro-
duces both uredia and telia on Spartinia pectinata
in North America. Aecial stages are produced on
Amsonia, Apocynum, Asclepias (Apocynaceae)
and Cephalanthus (Rubiaceae). These families are
grouped in the order Gentianales (Angiosperm
Phylogeny Group 1998). These plants are remote
from their centers of origin and Puccinia sey-
mouriana and P. andropogonis "discovered" the
 Co evolution of Pathogenic Fungi and Grass Hosts
EPICHLO~ SPECIES
I
...-----Vll
"'--- v
L---- IV
__- -VII 1- ___
- - 111
" ' - - - - - IX
Fig. 18.6. Comparative ITS trees from phylogenetic analy-
sis of grass tribes and Epichloe species. There are nine
distinct mating populations (MPs) based on sexual com-
genes for compatibility and in this case act as tax-
onomists. There has been no evolution in con-
junction with the evolving host and this suggests
that there are many plants belonging to the Gen-
tianales that could be susceptible to aecial stages
of P. andropogonis in other parts of the world if
exposed to these pathogens.
Puccinia andropogonis has ure dia and telia
on Andropogon (Poaceae) and aecia on Oxi-
ladaceae, Polygalaceae, Fabaceae, Santalaceae and
Rutaceae. It is interesting that the first three fam-
ilies are closely related in the eurosid; no doubt
these plants also have genes for compatibility that
had a common origin. However, there has been
no coevolution with the evolving host. Again the
rust "discovered" the genes for compatibility.
The other varieties of P. andropogonis occur on
369
GENUS TRIBES
I
STIPOIDEAE
I
~O'AI
STIPOIDEAE
patibility among strains and intersterility between other
MPs established by mating tests
Santalaceae (Santalaies ), Scrophulariaceae
(Lamiales) and Rutaceae (Sapindales), these fam-
ilies being very distant in the APG classification.
3. Erysiphales
The fungi belonging to Erysiphales are obligate
biotrophs commonly known as powdery milde ws.
Over 40,000 plant species representing weH over
40 orders of flowering plants (mainly eudicots) are
attacked by powdery mildews. Phylogenetic analy-
ses revealed that the powdery mildews formed
six evolutionary lineages (Takamatsu et al. 1998;
Saenz and Taylor 1999). The morphological char-
acteristics were analyzed and found not to conflict
with the molecular data, and the morphological
and molecular data generally may be combined.
Table 18.2. Grass rusts and their hosts
I~
Host Rust Aecidial host Host family Host order

Triticum aestivum Puccinia graminis f. sp. tritici" Berberis vulgaris Berberidaceae RANUNCULALES
P. recondita f. sp. tritici" Thalictrum, Isopyrum, Clematis Ran unculaceae RANUNCULALES
P. tritici-durib Anchusa italica Boraginaceae ASTERIDS I
P. striiformis f. sp. tritici Unknown
P. striiformis f. sp. hordei Unknown
Triticum turgidum P. graminis f. sp. tritici Berberis vulgaris Berberidaceae RANUNCULALES
P. recondita f. sp. tritici" Thalictrum, Isopyrum, Clematis Ranunculaceae RANUNCULALES
P. tritici-durib Anchusa italica Boraginaceae ASTERIDS I
P. striiformis f. sp. tritici Unknown
Avena sativa P. graminis f. sp. avenae Berberis vulgaris Berberidaceae RANUNCULALES
P. coronata f. sp. avenae Rhamnus cathartica Rhamnaceae ROSALES
Hordeum vulgare P. graminis f. sp. tritici Berberis vulgaris Berberidaceae RANUNCULALES
P. graminis f. sp. secalis Berberis vulgaris Berberidaceae RANUNCULALES
P. hordei Ornithogalum spp. Hyacinthaceae ASPARAGALES
P. striiformis f. sp. hordei Unknown
P. striiformis f. sp. tritici Unknown
P. coronata f. sp. hordei Rhamnus cathartica Rhamnaceae ROSALES
Secale cereale P. graminis f. sp. secalis Berberis vulgaris Berberidaceae RANUNCULALES :-
P. recondita f. sp. secalis Lycopsis arvensis Boraginaceae ASTERIDS ~
P. coronata f. sp. hordei Rhamnus cathartica Rhamnaceae ROSALES c
CIQ
:;:l
Zea mays P. sorghi Oxalis spp. Oxalidaceae OXALIDALES (ii'
P. polysora Unknown ...,
Physopella pallescens Unknown
Pennisetum glaucum P. substriata var. indica Solanum melongena Solanaceae SOLANALES
Sorghum bicolor P. purpurea Unknown
Eragrostidis tef Uromyces eragrostidis Anthericum torreyi Anthericaceae ASPARAGALES
Andropogon P. andropogonis (North America) Oxalidaceae OXALIDALES
Polygalaceae FABALES
Fabaceae FABALES.
Santalaceae SANTALALES
Scrophulariaceae LAMIALES
Rutaceae SAPINDALES
Spartina pectina P. seymouriana Apocynum Apocynaceae GENTIANALES
Asclepias Apocynaceae GENTIANALES
Cephalanthus Rubiaceae GENTIANALES

'Puccinia recondita f. sp. tritici=Puccinia triticina (http://www.cdl.umn.edu/tritname.html).

bOnly in the Mediterranean area.
 Co evolution of Pathogenic Fungi and Grass Rosts
L---c!:!5=55=3!~l11l1l1 lpOLYASCUS
Fig. 18.7. Phylogenetic relationships among the
Erysiphales. On the right, the orders of the plant hosts are
indicated. Cleistotheces and myceloid appendages are illus-
Mori et al. (2000) divided the Erysiphales into six
clades, using the ITS sequences (Fig.18.7).
- Clade 1 consisted of Erysiphe, Brassillomyces,
Uncinuliela, and Uncinula, which have a
Pseudoidium mitosporic state.
- Clade 2 consisted of Erysiphe, Microsphaera,
and Uncinula, all of which have a Pseudoidium
mitosporic state.
- Clade 3 consisted of Leveillula and Phyllactinia,
which have Oidiopsis and Ovulariopsis mito-
sporic states and are characterized by the pres-
ence of endophytic or partly endophytic
mycelia. These genera have evolved with plants
classified within the Asterids (except Phyllac-
tinia moricola).
- Clade 4 consisted of Sphaerotheca, Sawadaea,
Podosphaera, and Cystotheca, which have a
Fibroidium mitosporic state. These genera have
evolved with plants classified within the Rosids.
371
POLYASCUS
MONOASCUS
ROSIDS
ASTERIDS
MONOCOTS
trated. Erysiphe graminis is distantly related to the other
Erysiphe species
- Clade 5 consisted of Erysiphe graminis, which
has an Oidium mitosporic state. Erysiphe
diverged from the eudicot-parasitic powdery
mildews at an early stage of evolntion.
- Clade 6 consisted of Uncinula septata that was
placed at the base of the other powdery mildew
taxa.
Traditional taxonomists regarded the
myceloid appendage of the cleistothecia as an
important characteristic for taxonomy of powdery
mildews. The phylogenetic results showed that this
characteristic occurred multiple times due to
convergence (e.g., Erysiphe cichoracearum and
Sphaerotheca pannosa, see Fig.18.7).
4. Smut and Bunt Fungi
Over 900 species of Ustilaginomycetes (300
species of Ustilago) have been described, able to
372 1. Mugnier
infect ca. 4000 plant species belonging to 75
families, most of which belong to the Graminae.
The Ustilaginales were split into three major
lineages: (1) the lineage containing smut species
(U nuda, U nigra, U hordei and U aegilopsidis)
pathogenic on sm all grain cereals and wild rela-
tives within the "core" Pooideae, (2) the lineage
containing Ustilago maydis, U sciataminea and
Sporisorium relianum which share the same host,
Zea mays + teosinte (Panicoidae), and (3) the
lineage of the Tilletia species (bunts).
Species within lineage 1 are all infertile,
which, according to Boidin (1986), would make
them nonspecific. The Avenae species have a nar-
rower host range (tribe Avenae originating in the
Mediterranean basin). The Triticeae species origi-
nated in the Eurasian basin (although U hordei is
also found on Avenae spp.). Bakkeren et al. (2000)
suggested reclassification of the small grain-
infecting smuts, in regards to the limited genetic
variability, in combination with other molecular
and mating data. The new classification might
include varietas designations to define host range.
U maydis and S. relianum appear to be weIl
separated from the other smuts. In addition, they
are the only smuts with a tetrapolar mating
system; the majority of smuts have bipolar
systems.
V. Conclusions
Coevolutionary interactions have received abun-
dant scientific attention, both epidemiologic,
phylogenetic, and otherwise (Charleston 1998;
Burdon and ThraIl1999). A crucial component of
co evolution is phylogenetic analysis based on the
DNA sequence comparison. A really interesting
case of coevolution is that of the relationship
between grasses and fungi. The prominence of the
grasses as an object of molecular research refiects
their persuasive economic importance.
There are two aspects in the host-parasite
associations. First, if the cladograms of the host
and the cladograms of the parasite are congruent,
this certainly suggests coevolutionary phenomena.
Examples of parallel cladogenesis were demon-
strated by Schardl et al. (1997) for Epichloe and
grass hosts, by Holst-Jensen et al. (1997) for
Monilinia and the Rosaceae, and by Bakkeren et
al. (2000) for Ustilago and grass hosts. Second,
there is little phylogenetic congruence between
the pathogens and theirs hosts. Roy (2001) did not
found congruence between the rust fungi and their
crucifer hosts. For the helminthosporium complex
and their grass hosts, there was also little evidence
of parallel cladogenesis (Berbee et al. 1999).
Some time ago, Hijwegen (1988), in a review
on the coevolution of fungi with their hosts,
emphasized the problem of jumps and the practi-
cal implications in crop protection and system at-
ics. The main conclusions of these authors were
that co evolution is relatively rare and that host-
parasite interactions are a mixture of coevolu-
tionary events and jumps to disparate hosts.
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sequence analysis. Mycologia 85:401-414
Biosystematic Index

Abortiporus biennis 20, 23, 26 -fiavus 42,46,49,55,57,58,59,60,61,62,63,64,65,115,

Acacia 346
- mearnsii 146 - fllmigatus 115, 173, 184
- pycnantha 146 - nidulans 44,56,58,59,60,62, 64, 80
Acacia saligna 140,141 - niger 40,41,42,49,79,345,346
Acacia spp. 140 - nominus 55
Acer pseudoplatanus 295 - ochracells 42
Acremonium 365 - oryzae 44
Acremonium alternatllm 96 - parasiticus 55,57,58,59,60,62,63,64,65
Aedes aegypti 117 - penicillioides 42
Aegilops markgrafii 245 - restrictus 42
Aequorea victoria 13 - sydowii 42
Aeschynomene virginica 143 - tamarii 42, 55
Agaricales 359 - terreus 42
Agaricus bisporus 3,22,76 - versicolor 42
Ageratina riparia 140 - wentii 42
Agrobacterium tumefaciens 13,201,292 Atkinsonella 365
Agrocybe aegerita 20, 24, 33 Aureobasidium pullulans 99, 100
Ainus oregona 147
Alternaria 362 Bacillus
- alternantherae 145 - brevis 94
- alternata 40,42,101,315,323,346 - cacticidis 137
- arborescens 42 - cereus 100
- brassicae 206 - megaterillm 94,95,97,98,102
- brassicicola 329 Balansia 365
- citri 42 Balansiopsis 365
- colombiana 42 Beauveria
- cllscuticidae 143 - bassiana 44,113,114,115,116,117,120,121,122,123,
- dumosa 42 133
- eichhorniae 145 - brogniartii 114,116,117,123
- gaisen 42 - nivea 42,43,46, 117
- infectoria 42 Beta vlligaris 324
- interruppta 42 Bipolaris spp. 362
- macrospora 42 Bipolaris sorokiniana 56, 102
- oregonensis 42 Bipolaris sacchari 330
- perangusta 42 Bjerkandera adusta 73,75,76,77
- tangelonis 42 Billmeria graminis 247
- tenuissima 42 Blumeria (syn. Erysiphe) graminis 297
- triticimaculans 42 Boletus sp. 22
- triticina 42 Botrytis spp.
- triticicola 42 - aclada 42
- turkisafria 42,144,200 - cinerea 93,94,95,96,97,98,100,101,102,173,284,
Amanita 22 345
Ampelmyces 101 Brassica
Ampelomyces qllisqualis 96, 100, 101, 102 - alba 206
Arabidopsis thaliana 207,209,248,292,295,301 - arvensis 206
Aristosporum 360 - campestris 294, 329
Armilaria 101 - carinata 329
Armillariella 22 - jllncea 329
Arrhenomanes 360 - napus 124
Aschersonia sp. 112, 113,116 - oleraceae 194,203,329
Aspergillus - oleraceae var. acephala 194
- aculeatus 345 - oleraceae var. botrytis 194
- candidlls 42 - oleraceae var. ita!ica 194
376 Biosystematic Index

- rapa 194 Cryptococcus

Brassillomyces 371 - albidus 99
Byssochlamys fulva 42,43,46,47 - laurentii 96
Cryptostegia grandiflora 141
Cactoblastis 137 Culicinomyces clavisporus 112, 118
Cactoblastis cactorum 136 Curvularia spp. 362, 364
Candida spp. Cuscuta spp. 143
- albicans 276 Cyathus stercoreus 25,26,28,29,30,31,32
- oleophila 42, 96, 100 Cylindrobasidium laeve 146
Cantharellus 22, 364 Cyperus esculentus 144
Cassia obtusifolia 145 Cystotheca 371
Capsella bursa-pastoris 207
Catha edulis 96 Daedalea quercina 26, 28, 29
Ceratocystis 101 Debaryomyces hansenii 42, 43
Ceratobasidium 364 Descurainia sophia 207
Cercospora kikuchü 324 Diabole cubensis 138
Cercospora rodmanü 139 Dichomitus squalens 20,23,25,26,29,30,32,75
Cercospora spp. 315 Dietelia portoricensis 142
Cerco5porella 140 Diplotaxis muralis 209
Ceriporiopsis Discula destructiva 136
- gallica 75 Drechslera spp. 42, 362
- subvermispora 73,75,76,77,79 Drosophila 117
Cerrena unicolor 24
Ceutorhynchus assimilis 124 Echinochloa ssp. 153
Chaetomium Eichhornia crassipes 136,142
- cellulolyticum 22,31,56 Eleusine coracana 153
Chasmanthium 360 Emericella nidulans 42
Chondostereum Enterobacter cloacae 97
- purpureum 146,147, 101,146 Entomophaga
Chromolaena odorata 142 - grylli 112
Chrysosporium - muscae 112
- inops 42 Entomophthorales 111
- farinicola 42 Entyloma ageratinae 139,140
- fastidium 42 Epichloe 365
- xerophilum 42 Eppicoccum nigrum 42
Chylindrosporium concentricum 200, 206 Eremascus
Cinchona succirubra 143 - albus 42
Cladosporium 362 - fertilis 42
- cladosporioides 96 Erwinia herbicola 103
- fulvum 100,291,298,299,300 Erwinia chrysanthemi 341
- herbarum 42, 43, 93 Erisyphe 96
Claviceps 365 - cichoracearum 371
Claviceps purpurea 42,347,348,349 - graminis 297, 354, 371
Cochliobolus 362 - graminis f sp. hordei 284
- carbonum 256,314,315,347 Eupenicillium 47
- heterostrophus 251,315,321 Eupenicillium brefeldianum 46
Colletotrichum 284 Eupenicillium lapidosum 42
- dematium 145 Eurotiales 41
- gloeosporioides 40,139,143,144,145,345,346 Eurotium spp.
- gloeosporioides f. sp. cuscutae 143 - amstelodamii 42, 43
- graminicola 362 - chevalieri 42
- lindemuthianum 346 - herbariorum 42,46,47
- magna 95 - repens 42
- orbiculare 95 - rubrum 42
- truncatum 40,95,144,145 Exserohilum spp. 362
Collybia radicata 22, 24
Conidiobolus spp. 112 Fallopia japonica 143
Coprinus Jimetarius 22,26,29 Fibroidium 371
Coprinus psychromorbidus 364 Flammulina velutipes 3, 20, 22, 24
Coriolus versicolor 20, 31, 73, 76, 79, 80 Fusarium 359,362
Cornus spp. 137 - avenaceum 42
Corolopsis - coeruleum 42
- gallica 73 - culmorum 42
- polyzona 73 - equiseti 42
Cronartium 364 - graminearum 42, 218, 221, 330
Cryphonectria parasitica 94, 175,283,347 - moniliforme 206, 364
 Biosystematic Index 377

- oxysporum 42,46,95,98,148,173,347 Junghuhnia separabilima 20

- oxysporum f sp. albedinis 185
- oxysporum f sp. batatas 95 Kuehneromyces mutabilis 33,73
- oxysporum f sp. batatas 103
Lactarius 22
- oxysporum f sp. conglutinans 221
Laestisaria 94
- oxysporum f sp. cubense 221
Laetiporus sulphureus 73
- oxysporum f sp. radolens 103
Lagenidium giganteum 113
- poae 102
Lantana
- proliferatum 41,364
- camara 142,143
- sambucinum 62,315,330
Leveillula 371
- scirpi 315
Lentinellus 364
- solani 235
Lentinula edodes 3
- solani fsp. phaeseoli 292
Lentinus 364
- solani fsp .. pisi 292
- crinitus 27
- sporotrichioides 42
- edodes 22, 24, 26, 28
- suglutinans 364, 365
- tigrinus 23, 28, 29
- trivcinctum 40,42,43,93,94,95,96,144
- ulmarium 27,28,29, 101
Fusicoccum amygdali 330
Leptosphaeria
- maculans 194,200,201, 210, 300
Gaeumannomyces graminis 94 - nodorum 362
Galleria mellonella 117 Leveillula sp. 96
Ganoderma Ligneria 359
- lucidum 23, 33, 20 Lycoperdon 364
Geaumannomyces graminis 362
Geotrichum Magnaporthe grisea 154,173,175,251,278,291,300,362
- candidum 40,41,43,79 Malus domestica 317
Gibberella 330 Malva pusilla 145
- jujikuroi 224, 365 Marasmius oreades 364
- pulicaris 221 Maravalia cryptostegiae 138,141
Gleophyllum spp. 76 Massospora spp. 112
Gliocladium Melampsora 364
- virens 94,96,97,100 -lini 300
Gloeophyllum Meligethes aeneus spp. 124
- striatum 73 Menida beryllina 126
- trabeum 73 Metarrhizium
Gloeosporium lunatum 137 - anisopliae 44,113,114,115,116,121,122,123,124,125,
Glomerella cingulata 347 126
Glomus mosseae 292 - anisopliae var. acridum 121,122
Glycine max. 293 - flavoviride 121, 122, 117
Graminicola 360 Microsphaera 371
Grifola frondosa 3 Mikania micrantha 142
Gymnopilus 27 Monascus ruber 42,46
Morrenia odorata 143
Mucor 97
Hamigera reticulata 42
Mycosphaerella graminicola 183
Helminthosporium 359,362
Hemileia vastatrix 244 Mycosphaerella graminis 362
Myriogenospora 365
Hericium
Myrothecium
- clathroides 26, 28
- roridum 330
- erinaceus 3
Heterobasidium 101 Nectria haematocacca 346
Hirsutella spp. Nematoloma frowardii 73,74
- thompsonii 112, 116 Neosartorya
Hordeum spontaneum 245 - fischeri 42,43,46,47,48
Hyalophora 117 - gla-bra 46
Hymenochaete tabacina 22 - spinosa 42
Hyphomycetes 111 - pseudofischeri 42
Neotechina 136
Inonotus Neurospora crassa 62
- andersonii 26, 28 Nezara viridula 113,114,117,118
- dryophilus 26, 28 Nicotiana
- hispidus 23 - paniculata 291
- obliquus 26, 28, 29 - benthamiana 291,295
- rheades 23 - rustica 295
Irpex - tabacum 295
-lacteus 27,73 Nomuraea rileyi 112
378 Biosystematic Index

Oidiopsis 371 - ulaiense 42

Oidium 96 - urticae 42, 123
Ophiostoma - verrucosum 42
- novo-ulmi 94, 101,328 - viridicatum 41,42,43,97
- quercus 329 Peniophora
- ulmi 315,328 - duplex 24
Opuntia spp. 137 - utriculosa 27
Ostrinia nubilalis 120 Periconia circinata 331
Oryza sativa 293 Peronospora parasitica 209
Oudemansiella mucida 76 Petromyces alliaceus 42
Ovulariopsis 371 Petroselinum crispum 293
Paecilomyces Pezicula malicorticis 102
- farinosus 44, 117 Phaedon cochlearidae 124
- fumosoroseus 113, 116, 121, 123 Phaeoramularia eupatorii- odorati 139
- variotii 41,42,46 Phaseolus vulgaris 292
Phanerochaete
Palaemonetes pugio 127 - chrysosporium 20,22,24,25,26,27,28,29,30,31,32,
Panicum ssp. 153 73f
Panus 364 - sordida 73,75,77
Panus tigrinus 20 Phellinus
Parthenium 141 - ignarius var. trivialis 23
- hysterophorus 141,143 - laevigatus 23
Penicillium - pini 31
- albocoremium 42 - robustus 23, 26,28,29, 101
- allii 42 Phialophora graminis 362
- atarmentosum 42 Phlebia
- aurantiocandidum 42 - ochraceofulva 20
- aurantiogriseum 42 - radiata 20, 73, 75, 77
- brasilianum 42 - tremellosa 31,73
- brevicompactum 42 Phloeospora mimosae-pigrae 139
- brevispora 32 Pholiota
- carneum 42 - mutabilis 31
- citrin um 42, 46 - nameko 3
- chrysogenum 42 Phoma
- claviforme 93 - aquilina 193
- commune 42 - betae 193
- corylophilum 42 - chrysanthemicola 193
- crustosum 42 - clematidina 139
- cyclopium 42 - eupyrena 193
- digitatum 40, 42, 256 - exigua 193
- discolor 41,42 -lingam 193, 194, 197
- echinulatum 42 - medicaginis 193
- expansum 41,42,46,102 - napobrassicae 193
- freii 42 - nigrificans 193
- funiculosum 42 - oleraceae 193
- glabrum 42 - siliquastrum 193
- glandicola 42 - tracheiphila 193
- granulatum 42 - wasabiae 193
- hirsutum 40 Phomopsis 144
- hordei 42 Phragmidium violaceum 138
- hysterophorus 141 Phycomyces blakesleeanus 48
- islandicum 42 Phyllachora graminis 362
- italicum 42 Phyllactinia 371
- melanoconidium 42 Phyllosticta concava 137
- miczynskii 42 Physopella pallescens 370
- nalvgiovense 42 Phythium ultimum 97
- neopurpurogenum 42 Phytophora
- nordicum 42 - cinnamomi 294
- olsonii 42 - cryptogea 294
- oxalicum 42 - palmivora 143
- paneum 42 - parasitica 296
- polonicum 42 - infestans 95, 136, 291, 294, 295, 303
- roqueforti 41,42,46 Phytophthora sojae 293,295,300, 303
- sklerotigenum 124 Pichia sp.
- solitum 42 - guilliermondii 97
- spinulosum 42 - pastoris 80, 102
 Biosystematic Index 379

Plasmopara viticola 102 - tritici-duri 370

Pleurotus - xanthii 138
- cornucopiae 27,28,33 Pucciniastrum 364
- ryngii 22f, 33, 73, 75, 78, 79 Pulverulentum 23
- gigantea 33 Pycnoporus cinnabarinus 25,28,73,75, 80
- ostreatus 20,23,24,26,27,28,29,33,34,73,76,78 Pyrenophora 362
- ostreatus-mutant 28, 29 Pyrenophora tritici-repentis 256,315,326
- pulmonarius 26,27,28,73 Pyricularia orizae 362
- pulmunoarius 76 Pythium 94,96,97,359,360,361
- sajor-caju 20,23,24,26,27,28,30,31,33,34
- sapidus 23,27,28 Raphanus raphanistrum 206,207,209
- serotinus 22, 23 Raphanus sativus 294
- spodoleucus 27 Reticulitermes flavipes 120
- squarosulus 27 Rhizobium meliloti 292
- tuber-regium 3,20,22,29, 33 Rhizoctonia 359
Podosphaera 96, 371 Rhizoctonia solani 97,98,103,200,206,365
Polymyxa 359 Rhodnius prolixus 122
Polypaecilum pisce 42 Rhynchosporium secalis 291,362
Polyporus Rottboellia cochinchinensis 142
- brumalis 23, 28, 29 Russula 364
- ciliatus 26, 28, 29
- tomentosus 20, 24 Saccharomyces
Poria rivulosa 24 - brevicaulis 42
Prospodium tuberculatum 139 - cerevisiea 43,45,46,48,273,347, 361
Prunus serotin 146 Saccharum spp 330
- dulcis 330 Sawadaea 371
- persica 330 Sclerotinia
Pseudocercosporella herpotrichioides 362 - sclerotiorum 93,94,100,174,182,200,206
Pseudoidium 371 Sclerotium rolfsii 98
Pseudomonas Scopulariopsis
- aeruginosa 95 - brevicaulis 42
- cepacia 97 - candida 42
- chlororaphis 100 - fusca 42
- fluorescens 99, 103, 104 - halophilica 42
- putida 95,103 Scytalidium 101
Psilocybe 27 Scytinostroma galactinum 31
Puccinia 364 Septoria 359
- andropogonis 369,370 - myricae 139
- abrupta 138 - passiflorae 139
- canaliculat 144 Septoria nodorum 362
- cardui 138 Septoria tritici 362
- carduorum 138 Serpula
- chondrillina 138 - lacrimans 22, 45
- chondrillina 140 Setaria spp. 153
- coronata 368 Setosphaeria 362
- coronata jsp. avenae 370 Serratia marcescens 103
- coronata f.sp. hordei 370 Sinapsis
- evadens 138 - alba 209
- graminis 243, 244, 368 - arvensis 206
- graminis jsp. secalis 370 Sorghum bicolor 331
- graminis jsp. tritici 300, 370 Sorosporella 112
- hordei 370 Sphaerotheca 371
- lantanae 143 - pannosa 371
- melampodii 138 Spaerotheca
- montanensis 368 - fuliginea 96, 101
- myrsiphylli 138 - fusca 96, 100
- polysora 370 Sphaerulina mimosae-pigrae 139
- purpurea 370 Sporisorium 364
- recondita fsp. secalis 370 Sporisorium ophiuri 139,142
- seymouriana 368, 370 Sporobolomyces roseus 96
- sorghi 370 Sporothrix
- spegazzinii 142 - flocculosa 97, 100, 101
- striiformis 368 - rugulosa 97
- striiformis fsp. hordei 370 Sporotrichum 23
- striiformis tritici 370 Stachybotrys 330
- substriata vaL indica 370 Stemphylium spp. 42
380 Biosystematic Index

Strenotrophomonas maltophilia 102 - virens 98

Streptomyces griseovirides 100 - viride 40, 93,94,96,97,98,101
Striga hermonthica 148 Triticum aestivum 326
Strongwellsea castrans 112 Typhula 359,364
Stropharia
- rugoso 23 Ulocladium chartarum 42
- rugosoannulata 20, 22, 24, 26, 33 Uncinula 96, 371
Uncinuliela 371
Talaromyces Uromyces
- bacillisporus 42 - eragrostidis 370
- flavus 46,47,48 - galegae 138
- macrosporus 42,43,44,46,47,48 - heliotrophii 138
- trachyspermus 46 - tepperianum 138
Tapesia yallundae 362 - tepperianum 140
Thanatephorus 364 Ustilaginales 137
Theobroma cacao 137 Ustilago 364
Thlaspi arvense 207 Ustilago
Tilletiopsis 97 - aegilopsidis 372
Tilletiopsis minor 99 - hordei 372
Tilletiopsis washingtonensis 101 - maydis 278, 280, 372
Tolypocladium spp. - nigra 372
- inflatum 116, 173 - nuda 372
Trametes - sciataminea 372
- gibbosa 26, 28, 29
- hirsuta 23 Verticillium
- hirsutus 73 - Verticillium dahliae 206
- hispida 73 - Verticillium latericium 206
- versicolor 73,75,76,77,78,80 -lecanii 96,101,102,112,113,114,116,118,122,123
- villosa 79 Volvariella volvacea 20, 24
- zonata 20, 24 Wallemia sebi 42, 43
Trialeurodes vaporariorum 123
Trichoderma Xeromyces bisporus 42, 43
-hamatum 97
- harzianum 13,31,95,96,97, 98, 99, 100, 101, 103 Zea mays 280
- koningii 99 Zygosaccharomyces
- longibrachiatum 103 - bailii 42, 43, 45, 46, 48
- pseudokoningii 97 - chevalieri 46
- reesei 97 - rouxii 42, 43, 44
Subject Index

ß-galactanases 344 aspergillosis

ß-galactosidase 344 - invasive 184
ß-1,3-glucanase 350 Aspergillus
ß-glucans 293 - afiJ 59,63
ß-tubulin gene 13 - aflR 59, 60, 61, 63
3-methyl-1-butanol 65 - fadA 62
- flbA 60
ABC transporter 255 - morphological development 61
- multidrug resistance 256 - nor-l 57
acetyl esters 344 - norA 57
adenylyl cyclase 61,277,283 - ord-l 60
aecidiospore 244 -pkaA 60
AF see 'aflatoxin' - rcoA 62
aflatoxin 55, 56, 326 - stc 60,61
- biosynthesis 57,58,59 - stcQ 59
- control measures 64, 59 - stvV 57,59
- environmental stimuli 62 - ver-l 63
- inhibition 63 Aspergillus fumigatus
AFLP see 'amplified fragment length polymorphism' - Afutl 184
Agaricus bisporus - genetic variability 184
- breeding 10 attenuation 118
- transformation 12, 13 averantin 57,59
agriculture averufin 57
- yield reduction 193 avirulence
- quality reduction 193 - factors 299
Agrobacterium tumefaciens -genes 162,163,201,273,299,300
- media ted transformation 201 - perception 300
agro-business 14 - pro tein 302
allergenicity 125 - species-specific 294
amplified fragment length polymorphism 197,223,
259 bacterial blotch 8
antagonistic 101 basidiomycetes 22, 27
- fungi 94 Basidiomycota
- genetic modification 103 - phylogenetic 363
- microorganisms 103 basidiospores 13
anthracnose disease 40 bassianin 117
antibiosis 97 bassianolide 117
antibiotic BCA see 'biological control agent'
- activity 117 beauvericin 117
- producers 96 benomyl resistance 13,253
antifeedant 116 benzimidazole
aphid 124 biocontrol agents
appressorium 40,113,153,156,279 - microbial 93,207
arabinofuranosidases 344 bioconversion technology 32
- (arabino )xylan backbone 343 biodiversity 135
arthrospores 43 biological control
aryl alcohol oxidase 20,27,76,80 - challenge 100
ascomycota - chestnut blight 94
- phylogenetic tree 360 - dutch elm disease 94
ascospores 43,46,210 -RNA 94
- heat-resistant 47 - take-all disease 93,94,136,142,143,148,230
asexual biological control agent
- development 64 - additives 101
- reproduction 61 - survival 101,102,103,111,127,142,143,228,229
382 Subject Index

biotrophic 353 - P-450 56

biotrophic pathogens 95 - P-450 monooxygenase 76
breeding program 234
brown-rot 20,71,76
defoliation 141
dehydrogenase 57
Ca2+ 278
demethylsterigmatocystin 57
caffeic acid 32
destruxins 116
calmodulin 278
development
calmodulin-dependent protein kinase 278
- pathogen-related 279
calmodulin kin ase 278
- pseudohyphal 277
cAMP 61,277,279
dichloroisonicotinic acid 208
- signaling 277,282
differential display 257,283,285
CDPK see 'calmodulin-dependent protein kinase'
digestibility of lignocellulosic materials 34
cecropins 117
dihydrobisfuran rings 55
cell wall degrading enzymes 341
disease
- coregulation 345
- control 217,261
- glucan ase 202
- diagnostics 224
- virulence factors 353
- Fusarium ssp. 219,220
cellobiose dehydrogenase 76
- management 165, 193
cellobiohydrolase 349,351
- resistance gene see 'resistance gene'
cellulose 342
- Turkey-X 55
cellulose-hemicellulose 342
disease suppression
cellulose-binding domains 80
- natural 94
chitin 292, 293
dispensable
chitinase 97,98,233,260
- B-chromosomes 198
chlamydospores 46,143
DNA fingerprinting see 'fingerprinting'
chloroplast genome 360
DNA sequences data 359
chromosomal polymorphism 7
dormancy
chromosome length polymorphisms 9
- exogenously 47
chromosome segregation 5
- constitutive 47,48
citrus fruit rots 95
dry matter
Cochliobolus carbonum 313,314
- cellulose 29
-HM1 313
- disappearance 32
- HC toxin 313,314
- hemicellulose 29
coevolution 289,359
-lignin 29
colonisation
-losses 29
- community changes 40, 99
dry matter digestibility
commercialisation of
-improvement 20,22,25,32,34
- fungal products 144,145
commercialized 100
composting technology 4 ecological fitness 119
conidia 44, 46 ecological recource 39
co-regulation 59 ecosystems
crop - agricultural 135
- biological treatment 19 - natural 135
- nutritive value 19 efrapeptins 116
- residue 19 electroporation 13
- weeds 135 elicitor 113, 290
crossing over 4 elicitors
cryotolerance 45 - acidic ß-elicitins 294
Cryphonectria parasitica -AVR4298
- double-stranded(ds)RNA molecule 104 -AVR9298
- G-protein - Avr9 disruption mutants 298
- hypovirulence 104 - chitin fragments 292
- hypovirus 283 - cultivar-specific 298
culture - elicitin-encoding 293
- semicontinuous 31 - elictitins 293,
cuticle degradation - a-elicitins 294
- enzymes 114,115 - ergosterol 291
cutinase 98, 234 - inß 295
CWDE see 'cell wall degrading enzymes' - race-specific 295
cyanide hydratase 202 - 34-kDa glycoprotein 296
cyclic peptides 116 endoarabinanases 344
cyclopenenone ring 56 endoglucanase 103
cytochrome endo-l,3-ß-glucanases 293
- c peroxidase 77 endo-l,3-ß-glucanases inhibitor 293
 Subject Index 383

endopolygalacturonase 97,341,344,345,346,349,353, - postharvest pathogen 221

354 - trans pos on trapping 179
- gene 351 Fusarium oxysporum f sp. dianthi
entomogenous fungi - Fotl transposon 187
- generalists 111,112 - lmpala transposon 187
- mitosporic fungi 111
- specialists 111,112,113,115 galacto(mannan) backbone 343
environment gene expression
- extreme 41 - culture conditions 78
- factors 233 gene transfer
- indoor 41 - horizontal 199
- niches 99 gene-for-gene interaction 273, 290, 298, 365
Environmental Protection Agency 143 - LEMl/alml 201
epicuticle 112 - Rlml/AvrLml 162,201
epidemics 98, 194,243 gene knockout 314,318,320,352
epipolythiodioxopiperazine 198 genetic locus
epizootics 118 - hypervariable 178
ergosta-5,24(28)-dienol 250 - unstable 178
ergosterol genetic markers 5
- inhibitor of biosynthesis 207 genotoxic agents 56
evolution genotypes
- adaptive 202 - recombinant 7
exopolygalacturomnase 97,346 germ tube 112
expression germination spore
- pathogen-inducible 261 - inhibition 95
germplasm 9
fatty acid synthase 57 Giberella fujikuroi
feeding value 32 - mating population 224
ferrocytochrome c 74 - pulsed field gel electrophoresis 225
ferulic acid 21,32 - RAPD 225
field conditions 93, 98 - sexual fertile 225
finger millet Gleophyllum
- blast 153 - culture 76
fingerprinting glucanase 98, 202, 258, 352
- microsatellite 174,180,196 glyceraldehyde-3-phosphate dehydrogenase 13
food glucurono-arabino-xylans 348
- industry 146 glyoxal oxidase 80
- living crop 39 G-protein
- processed food 39 - a subunit 276,279,282
fruit body - ß/y heterodimer 276,277,279,282
- formation 8 - heterotrimeric 276, 277
- primordia 8 green fiuorescent protein 13
- edible 27
fungal growth haemocoel 117
- inhibition 112 haplotypes 9, 11
fungal pathogen 136 heat activation 48
fungal populations heat-resistant
- asexually reproducing 175 - fungi 46, 47
- genetic variability 171 hemicelluloses 342
- molecular markers 171 hemibiotrophic 137,360
fungal spores 41 hepatocarcinomas 56
fungi 29 hepta-ß-glucan 293
fungicide herbicide 144
- high risk 253 herbicides
- organomercury 227 - resistence 135
- resistance 253 heteroallelic 5
- sulfur-based 248,249,250,253,262 heterokaryosis 178
fungistasis 123 heterokaryotic 11
fusaric acid 222 heterotrimeric G-proteins
Fusarium - a subunits 61,297
- compatibility groups 223 hirsutellin 117
- genetic diversity 221 histone deacetylase 313
- Fotl transposon 179 homokaryons 7, 11
- nia gene 179 homothallic 4
- phylogenetic tree 368 host factors 64
- phytohormone 226 host -parasite interaction 359
384 Subject Index

host plant - population studies 157

- genetic background 99 -PWL 296
host range 137 - PWL2 297
hostpathogen - repeated DNA 154
- co-evolution 140 - sexual reproduction 160
humidity 122 male sterile cytoplasm
hydrolytic enzymes 97 - URF13 321, 322
hydroperoxylenic acid 64 management strategy 141,142
hydroperoxylinoleic acid 64 manganese peroxidase 71,72,74
hydrophobins 117,328 manganese-dependent peroxidase (MnP) 20, 27
hydroxyaverantin 57 MAP kin ase see 'mitogen-activated protein kinase'
hygromycin B 13 marker
hyperconidiation 61 - genetically unlinked 174
hyperparasites 96 - mendelian inheritance 174
hypersensitive reaction 204 mating-type locus 6
hypersensitive response 291 - a-locus Ustilago maydis 281
- b-locus Ustilago maydis 281
indole glucusinolate 204 metabolite
insecticidal propertis 117 - antifungal 102
insects - futile cycles 45
- behavior 120 - secondary 198
interactions metapopulation 224
- host-pathogen 114 Metarhizium anisopliae
interfertile 9 - Prl 115
internal transcribed spacer 197,359 methyl benzimidazole carbamate
intramixus 5 methylation 8
introgression 208 microarray 280
inverted repeated 11 microhabitat 120
ITS see 'internal transcribed spacer' micronutrients 146
microorganisms
karyotype 6 - establishment 101
knockout mutant see 'gene knockout' - persistence 101
mitochondrial 9, 11
laccase mitochondrial DNA 321
- differentially gene regulated 79 mitogen-activated protein kinase 277,281,282,284
-gene 27,71,72,75,79 - cascade 278
lactone ring 56 - signaling module 278
leucine-rich repeat 162, 163 Mn(III) oxidize 72
lignification 342 MnP/lipid peroxidation system 75
lignin monocultural practice 13
- decomposition 23, 33 morphogenesis 279
- degradation 22, 27 morpholines 248
- metylated 71,74 MSF transporters
lignin depolymerization 71 - fungicide insensitivity 256
ligninolytic agents 71 multimechanism action 98
lignin-peroxidase (LiP) 20,27,71,72,74 multiple mutagenesis strategy 354
lignocellulose mushroom
- conversation 20 - production 3, 4
lignolytic enzymes 20 mutagen 56
LRR see 'leucin-rich repeat' mutualistic symbioses 95
lycomarasmin 222 mycoherbicides 144,145,147,148
mycoinsecticides 111
Magnaporthe grisea mycoparasite 102
- ACR1 gene 156 Mycosphaerella graminicolla
- AVR-Pita 300,302 - partial TE sequence 184
- avirulance gene 162 - pSTL70 probe 184
- chromosome length polymorphism 160 mycotoxins 311,326
- clonal population 160 - aflatoxin see 'afiatoxin'
- genes of 296 - trichothecenes 49,55,218,223,224,232,235,236
- host range 154, 155
- leaf spot disease 153 necrosis-inducing proteins
- mating type genes 160 - NIP1 299
- MGR fingerprint groups 157, see also 'transposons' - NIP2 299
- neck blast 153 - NIP3 299
- panicle blast 153 necrotroph 95,98,284,350,353,360
- pathotypic variation 154, 156, 178 nonproteinogenetic amino acids 312
 Subject Index 385

nonribosomal peptide synthetase 314,318 - stern canker 193,195,200

non-target organisms 125 - taxonomy 195
norsolorinic acid 57,59 phomalide 198
nuclear localization signal 60 phomaligol 199
nutrients phylogeny 359
- competition for 96 p-hydroxybenzoic acid 32
nutrition phytoalexins
- insect 120 - biosynthesis 291
- kievitone 233
- maackiain 233
O-methylsterigmacystin 60
- medicarpin 233
oosporein 116
- pisa tin 233
opportunistic
- saponin 202,233,260
- fungi 137
phytotoxic
- infections 40
- host-selective 199,200,202
organic matter digestibility 31
phytotoxins 311,312
organopollutant 73
PKA see 'protein kinase A'
organopollutant degradation 71
plant
osmotolerance 44
- defence 289
osmotolerant 43
- pathogenic 39
oxidase 71
plant pathogen
oxidative enzyme 72
- opportunistic 350
plant-resistance genes
pasteurisation 39,48 - species-specific 297
pathogenesis 64, 96 plasmid
- related 291 - linear 12
pathogenicity plasmolysis 97
- climatic factors 119 polycyclic aromatic hydrocarbons 74
- factors 290 polygalacturonase
- test (Phoma) 198,125 - inhibiting proteins 353
pathophobia 141,147 polyketides 320
p-coumaric 21 polyketide synthase 57,62
p-coumaric acid 32 polymerase chain reaction 5
peanut polysaccharidase
- pnloxl 64 - endo-acting enzymes 343
pectate lyase 97,341,345,346 - exo-acting enzymes 343
pectin 342, 343 post-harvest
pectin acetylesterase 344 - blue mold 102
pectin lyase 344,347 - diseases 40
pectin methylesterase 97,344,345,352 - quality 3, 102
pectin network 343 post-meiotic 4
pectinase 234,345,351 post -tranlational
peptide synthetases 312 - modifications 74
peptide synthtase genes 312 - regulation 60
peroxidase post-transcriptional 60, 61
- extracellular 71 powdery mildew
pcroxidase 74 - antagonists 101
pest - hyperparasites 101,246,250
- risk assessment 140,142 powdery mildew 243f
pest control 113 preservation
phenolic acids 21 - technique 39,41
pheromone 124 press ure-resistance
phleomycin resistance 77 - high pressure 48
Phomasp. programmed cell death 292
- blackleg disease 193 promoter
- chromosome length polymorphism 197 - GPD 13
- diagnostic approach 205 proteases 114
- DNA polymorphism 196 protein degradation
- extrachromosomal element 198 - inhibit 34
- fingerprinting 196 protein kinase A 61,280,282
- genetic variability 195 proteolytic activity 98
- host range 199 protoplast fusion 259
- incompatibility 196 PR-proteins 259
- karyotyp 197 pulse field gel electrophoresis 5
-leaf spot 193 Pyricularia grisea
- pathogenicity 195,196,199,205 - phylogenetic tree 177
386 Subjeet Index

quarantine 142 rumen

quinolines 251 - fermentation 31
quinoxyfen 251 - microbial digestion 31
rust
R gene see 'resistanee gene' - on cereals 243
radieal-eooper oxidase 76 - on eoffee 137,243
random amplified polymorphie DNA 5, 177, 196,205,
223,259 Saccharomyces cerevisiae
RAPD see 'random amplified polymorphie DNA' - SNFl gene 347
reeeptor salicylic acid 95
- G-protein-eoupled 247,276 SAR see 'systemie aquired resistance'
- ligand model 162 SCAR see 'sequenced characterized amplified region'
- membrane channels 276 sclerotia 46
- receptor-like kinase 301 Sclerotinia sclerotiorum
- tyrosin kinase 276 - non-LTR-retrotransposon 183
recognition 289 - phylogenetic analysis 183
- nonspecific 290 - repeated element 183
recombination secondary metabolites
- homologous 297 - biosynthesis 49,55,59,74
- parasexual 161 sequenced characterized amplified region
regulatory - markers 6, 179
- domains 60 sexual development 64
- gene 59, 162, 165 signal perception 303
REMI see 'restrietion enzyme mediated integration' signal transduetion
resistance - easeade 162,273
- by genetie manipulation (riee) 165 - genes involved 274,275
- disease 209,290 - modular strueture 273
- endophyte associated 95 - pathway 61,273
-induced 95,258,261 signalling apparatus 114
- interspecific 209 sirodesmin 198
- nonhost 290 soil-borne fungi 46
- race specifie 246, 248, 257 solar radiation 120
- suppressor 290 Sorghum
- systemic 95, 208 - stover 21
resistance gene 273 spawn strains 4
- Cf 299 spoilage
- L6 245 - erops 39
- Lr13 246 - food 39,41,48
- Mla 246, 247 spoilage fungi 40
- Mlg 246, 247 ST see 'sterigmatocystin'
- Mlo 246, 247 sterigmatocystin
- Pi-l 158 - biosynthetie pathway 58, 59
- Pi z-5 158 - carbon sourees 62
- Pi ttl 158 - control 59
-Rph16248 - environmental stimuli 62
- Rrsl 301 - produetion 60, 61
restriction enzyme mediated integration 280,285,322 - repression 64
restriction fragment length polymorphismus 5, 176, 178, sterilisation 48
196,223 stigmast-7-enol 250
reverse genetics 300 storage 102
RFLP see 'restriction fragment length polymorphismus' strain
rhamnogalaeturonan hydrolase 344, 352 - instability 6
rhamnogalacturonan lyase 344, 345 straw
ribosome-inactivating proteins 259 - barley 21
nce - biological treatment 21
- biological treatment of straw 29 - chickpea 21
- production losses 153 -lucerne 21
RIPs see 'ribosome-inactivating proteins' - oat 21,31
risk assessment 121, 125 - wheat 19,22
RNA polymerase 12 strobilurins 251,254
root rot structural polysaccharides 20
- suppression 102 subtilisin-eoding genes 115
r-strategist 244 sugar oxidase 76
RT-PCR sugareane bagasse 21
- competitive 80 survival structures 46
 Subject Index 387

sweet-rot 20 - Afutl 184

syringie acid 32 - Boty 173
systemie acquired resistance 95, 208, 256, 259, 291 - dass I 172
- dass 1I 172
tenellin 117 - Crypt-l 173, 175
tertrabisfuran 56 - diagnostic tool 172, 175, 184, 186
thiabendazole - differential hybridization 176
toxic metabolites 39,49 - fingerprinting 159, 180, 187
toxigenicity 125 - Flipper 173
toxin - Folyt 179
- AAL toxins I 326 - Foret 179
- AB toxins 329 - Fosbury/Maggy 156, 173
- ACR-toxin 346 - Fotl 156, 173, 174, 179, 180, 185
- AK toxin 323 - Foxy 179
-AKTl 323 - genetic variability 157
-AKT2323 - Grasshopper 156, 173, 176
-AKT3-1 323 - Gypsy family 156
-AKTR-l 323 - hAT family 175, 182
- AK toxin biosynthesis 324 - horizontal transmission 175
- AM toxin I 318 - Impala 179, 180, 185
- cerato-ulmin 328 - intraspecific recombination 172
- cercosporin 324 - Joyrider 179
- cercosporin biosynthesis 325 - UNE 156, 176
- doned genes 315 - MGR386 156,158,159,175,176,177,178
- destruxin B 319 - molecular markers 171, 173
- detoxification 233 - Palm 179, 181, 187
- electrolyte leakage 327 - Pot2 156
- enniatins 319 - Pot3 156, 158, 159
- ergopeptines 319 - Restless 173
- HC-toxin 313,314 - selfish DNA 172
- HC toxin biosynthesis 314 - Skippy 179
-HM1313 - Tfol 182
- host-selectivity 203 - trapping 6,172,179,225
- host-specific 311 transversion 56
- HS toxin 330 trap crops 125
- HTSI 314 trehalose 44
- fusieoccin 330 trehalose synthetase 45
- maitotoxins 320 Trichoderma harzianum
- nonribosomally synthesized peptides 312 - chitinase 103
- nonspecific toxins 311 - proteinase 103
- PC toxin 331 Trichodex 101,102
- phytotoxins 311 turgor
- polyketide/fatty acid derivatives 312 - glycerol 154
- production 56, 365
- proteinaceous toxin 329 uniparental inheritance 9
- Ptr ToxA 327 urediospore 244
-TOXA 314 uredium 244
-TOXC 314 Ustilago maydis
-TOXD 314 - dimorphie switch 283
-TOXE 314 - mating type locus 281
-TOXF 314 - transcription factor 281
-TOXG 314
- t toxin 321 vascular wilt disease 179,222
- trichothecenes 330 veratryl alcohol 74
- victorin 315,317 versicolorin A 57
toxin biosynthesis versicolorin B 57
- regulatory genes 103 versieonal 57
Trametes versicolor versiconal hemiacetal acetate 57
-mnp279 viral-like 12
transcription factor virulence factors 347
- zink-finger 60
transgenic plants 303 wasabidienone 199
transformation 8,77,259 water activities 43
transposon weed
- 300-bp mobile repetitive element 8 - invasive 141,142,147
388 Subject Index

- management 136 - growth 22

- pathology 136 - maturation 22
- population dynamics 144 wilt disease
wheat blast 154 - oil palm 181
white-rot 20,71,72,76,77,80 wooddecaying 71
white-rot fungi
- autolysis 22 xylanase 349
- colonization 22 xylan backbone
- fruiting bodies 22 xylogalacturonase 344