Advances in Agronomy v.73

Agronomy
DVANCES I N
VOLUME 73
Advisory Board
Martin Alexander Ronald Phillips

Cornell University University of Minnesota
Kenneth J. Frey Larry P. Wilding

Iowa State University Texas A&M University
Prepared in cooperation with the

American Society of Agronomy Monographs Committee
Diane E. Stott, Chairman
John Bartels Linda S. Lee Wayne F. Robarge
Jerry M. Bigham David Miller Dennis E. Rolston
Jerry L. Hatfield Matthew J. Morra Richard Shibles
David M. Kral John E. Rechcigl Jeffrey Volenec
Donald C. Reicosky
Agronomy DVANCES IN
VOLUME 73
Edited by
Donald L. Sparks
Department of Plant and Soil Sciences
University of Delaware
Newark, Delaware
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01 02 03 04 05 06 SB 9 8 7 6 5 4 3 2 1
Contents
CONTRIBUTORS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii
PREFACE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix
INTERACTIONS AMONG ROOT-INHABITING FUNGI AND

THEIR IMPLICATIONS FOR BIOLOGICAL CONTROL
OF ROOT PATHOGENS
David M. Sylvia and Dan O. Chellemi
I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
II. Functional Diversity in the Root Zone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
III. Interactions among Root-Inhabiting Fungi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
IV. Opportunities for Pest Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
V. Research Priorities. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
DWARFING GENES IN PLANT IMPROVEMENT

S. C. K. Milach and L. C. Federizzi
I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
II. The Biochemical Basis of the Dwarf Phenotype . . . . . . . . . . . . . . . . . . . . . . . . 38
III. Dwarfing Genes and Their Use for Breeding . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
IV. Breeding Challenges and Varieties Developed . . . . . . . . . . . . . . . . . . . . . . . . . . 45
V. Pleiotropic Effects of Dwarfing Genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
VI. Molecular Mapping of Dwarfing Genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
VII. Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
A REVIEW OF THE EFFECT OF N FERTILIZER TYPE

ON GASEOUS EMISSIONS
Roland Harrison and J. Webb
I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
II. The Processes Controlling Emissions of Nitrogen Gases
from Fertilizers. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
III. Measurements of Ammonia Emission Following Nitrogen
Fertilizer Application . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78
v
vi CONTENTS
IV. Ammonia Emission Factors for Nitrogen Fertilizers . . . . . . . . . . . . . . . . . . . 88

V. Measurements of Nitrous Oxide Emissions Following Nitrogen
Fertilizer Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90
VI. Nitrous Oxide Emission Factors for Nitrogen Fertilizers. . . . . . . . . . . . . . 97
VII. Nitric Oxide Emissions from Nitrogen Fertilizers . . . . . . . . . . . . . . . . . . . . . 99
VIII. Summary and Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103
RHIZOBIA IN THE FIELD

N. Amarger
I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110
II. Diversity in Rhizobia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112
III. Rhizobium Systematics. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123
IV. Natural Populations of Rhizobia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129
V. Introduction of Rhizobia into Soil . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143
VI. Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148
INDEX . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169
Contributors
Numbers in parentheses indicate the pages on which the authors’ contributions begin.
N. AMARGER (109), Laboratoire de Microbiologie des Sols, Institut National de

la Recherche Agronomique, 21065 Dijon, France
DAN O. CHELLEMI (1), USDA, ARS, Horticultural Research Laboratory, Ft.
Pierce, Florida 34945
L. C. FEDERIZZI (35), Universidade Federal do Rio Grande do Sul, Faculdade
de Agronomia, Departamento de Plantas de Lavoura, Porto Alegre, Brazil
ROLAND HARRISON (65), ADAS Consulting Ltd., ADAS Boxworth, Box-
worth, Cambridge CB3 8NN, United Kingdom
S. C. K. MILACH (35), Universidade Federal do Rio Grande do Sul, Faculdade
de Agronomia, Departamento de Plantas de Lavoura, Porto Alegre, Brazil
DAVID M. SYLVIA (1), Soil and Water Science Department, University of Florida,
Gainesville, Florida 32611
J. WEBB (65), ADAS Consulting Ltd., ADAS Wolverhampton, Wolverhampton
WV6 8TQ, United Kingdom
vii
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Preface
Volume 73 contains four excellent chapters on contemporary and important top-
ics in the agronomic sciences. Chapter 1 is a thoughtful review of interactions
among root-inhabiting fungi and their implications for biological control of root
pathogens. The fungi are defined, their distribution and abundance are discussed,
and their role in agroecosystems is presented. Chapter 2 discusses advances in the
role of dwarfing genes in plant improvement. Emphasis is placed on breeding and
genetics aspects. Chapter 3 covers a topic that is of great environmental interest—
the effect of nitrogen fertilizers on gaseous emissions. Processes controlling and
measurements of emissions of nitrogen gases are fully discussed. Chapter 4 is a
comprehensive review of Rhizobia, including diversity, systematics, natural popu-
lations, and field introduction of Rhizobia.
I thank the authors for their first-rate reviews.
DONALD L. SPARKS
ix
INTERACTIONS AMONG ROOT-INHABITING
FUNGI AND THEIR IMPLICATIONS
FOR BIOLOGICAL CONTROL
OF ROOT PATHOGENS
David M. Sylvia1 and Dan O. Chellemi2

1
Soil and Water Science Department
University of Florida
Gainesville, Florida 32611
2
UDSA, ARS
Horticultural Research Laboratory
Ft. Pierce, Florida 34945
I. Introduction
II. Functional Diversity in the Root Zone
A. Classification Schemes for Functional Groups
B. Clinical Pathogens
C. Subclinical Pathogens
D. Arbuscular Mycorrhizal Fungi
E. Additional Nonpathogenic Fungi
III. Interactions among Root-Inhabiting Fungi
A. Interactions among Pathogens
B. Interactions of AM Fungi with Pathogenic and Nonpathogenic Fungi
C. Interactions between Pathogenic and Nonpathogenic Fungi
D. Application of Island Biogeography Theory to Root–Fungal Interactions
IV. Opportunities for Pest Control
A. Current and Future Control Strategies
B. Role of Biological Control
C. Obstacles to Implementing Biological Control
V. Research Priorities
References
Soil fungi impact plant health because they grow in, on, and around roots, in-
fecting healthy tissues and colonizing senescent materials. We review the literature
concerning these fungi and discuss the various interactions that occur among the
root-inhabiting fungi and their diversity at the community level. Root-inhabiting
fungi are classified as clinical and subclinical pathogens, mycorrhizal fungi, and
additional nonpathogenic fungi. We define each group, present data on abundance
and distribution, and describe their roles in agroecosystems.We also discuss the
1
Advances in Agronomy, Volume 73
Copyright
C 2001 by Academic Press. All rights of reproduction in any form reserved.
0065-2113/01 $35.00
2 SYLVIA AND CHELLEMI
application of island biogeography theory to the understanding of fungal species

diversity in the root zone. Our goal is to contribute to a better understanding of the
complex ecology of root-inhabiting fungi so researchers can formulate reasonable
and testable hypotheses concerning the roles these fungi play in maintaining the
delicate balance between plant health and disease. We describe the implications of
fungal interactions for biological control strategies of root pathogens using three
diverse approaches: single tactic, integrated pest management, and proactive pest
management. We conclude that it is the very complex nature of the rhizosphere
that makes it imperative that we invest resources into fundamental research of
rhizosphere ecology. C 2001 Academic Press.

I. INTRODUCTION
Fungi contribute significant biomass to soils where they have important func-
tions in nutrient cycling (Harley, 1971) and microaggregate formation (Tisdall
et al., 1997). Soil fungi also encounter plant roots; they grow in, on, and around
roots and infect healthy tissues and colonize senescent materials (Parke, 1991).
In his classic tomes, Garrett (1960, 1970) characterized the edaphic fungal flora
as either soil or root inhabiting. He further characterized the root-inhabiting fungi as
either unspecialized or specialized parasites. The unspecialized parasites, such as
species of Pythium and Rhizoctonia, generally grow on juvenile root tissue. In
contrast, the specialized parasites may grow on more mature tissues and result in
vascular wilts as well as root rots.
The parasitic nature of these associations does not imply that all root-inhabiting
fungi are pathogens. In fact, many fungi growing with roots are beneficial, as
exemplified by mycorrhizal symbionts (Smith and Read, 1997) and nonpathogenic
parasites associated with roots (Deacon, 1987). Here we use “parasite” to describe
an organism that infects roots in order to obtain food for energy and growth, while
the term “pathogen” is used of an organism that specifically incites plant disease—
“the injurious alteration of one or more ordered processes of energy utilization in
a living system” (Bateman, 1978).
Our objectives for this chapter are to review the extant literature concerning fungi
growing on and in roots and to discuss the various interactions that occur among
these fungi. We begin by describing functional classifications of fungi that occur
with roots and then discuss the ecological roles of each major group (clinical and
subclinical pathogens, mycorrhizal fungi, and additional nonpathogenic fungi).
Next we discuss interactions that occur among these root-inhabiting fungi and their
diversity at the community level. Our goal is to contribute to a better understanding
ROOT-INHABITING FUNGI 3
of the complex ecology of root-inhabiting fungi so that researchers will be in a

better position to formulate reasonable and testable hypotheses concerning the
roles these fungi play in maintaining the delicate balance between plant health and
disease. Thus, we conclude this chapter by describing the implications of these
fungal interactions for biological control strategies of root pathogens and propose
further research priorities to achieve this end.
II. FUNCTIONAL DIVERSITY IN THE ROOT ZONE
A. CLASSIFICATION SCHEMES FOR FUNCTIONAL GROUPS
Winogradsky (1924) attempted to classify soil microorganisms on the basis of

their growth habit and modes of nutrition. Those that grow rapidly when nutrients
are readily available were described as zymogenous and those that grow slowly
on recalcitrant material as autochthonous. This is similar to the r-K life strategies
proposed by MacArthur and Wilson (1967) for animal systems. Pugh (1980),
following the reasoning of Grime (1979), expanded on and applied these concepts
to fungi, separating them into four broad life strategies:
1. ruderals, which have high sporulation and fast growth rates on simple, exoge-
nous substrates;
2. competitors, which maintain growth over a longer time period by maximizing
capture of available resources;
3. stress-tolerants, which have low sporulation and slow growth rates as nutrients
are depleted resulting in a stable population; and
4. survivors-escapes, which occupy unique habitats such as the phylloplane or
rhizosphere of roots in waterlogged soils.
The rhizosphere has been defined as the soil adjacent to roots with altered physi-
cal, chemical, and biological characteristics compared to the bulk soil (Bowen and
Rovira, 1999). The input of inorganic and organic nutrients from actively growing
roots stimulates microbial growth, resulting in rapid increases in populations of
bacteria, fungi, and protozoa (i.e., the ruderals). Theoretically, establishment of
competitors should follow the ruderals and, as nutrients are depleted, stress-tolerant
organisms should predominate.
Some have divided the rhizosphere into the ectorhizosphere (zone outside the
root), rhizoplane (the root surface), and endorhizosphere (zone inside the root)
(Balandreau and Knowles, 1978). Though semantically incorrect (Kloepper et al.,
1992), an understanding of the physical, chemical, and biological properties of
these adjacent, but dissimilar, locations should help one understand the growth
habitat and life strategies of root-associated fungi. Unlike most bacteria, the
majority of fungi are filamentous organisms and their expanding vegetative struc-
tures may easily span, and influence, life processes across these zones.
Biodiversity is an important issue and is gaining scientific, as well as politi-
cal, attention. Biodiversity may be viewed as comprising taxonomic, genetic, and
functional components (Solbrig, 1991). Most research has focused on taxonomic
diversity and, with the advent of the new molecular tools, increasing emphasis is
being placed on genetic diversity. However, there are few studies that focus on the
manner by which genetic or taxonomic diversity affects ecosystem function (Zak
et al., 1994). The challenge for soil ecologists is to understand the impact of these
fungi on root function and plant health.
A classification scheme for root-inhabiting fungi may include clinical and sub-
clinical pathogens, mycorrhizal fungi, and additional nonpathogenic fungi (sug-
gesting our lack of knowledge of many fungi that occur in the root). In the remainder
of this section we summarize the natural histories and agroecosystem functions of
these groups.
B. CLINICAL PATHOGENS
1. Definition
Clinical pathogens can be defined as root-inhabiting fungi that cause visual

symptoms of disease. Typically these include mortality or elimination of the re-
productive potential of the host plant, where reproductive potential is inclusive of
both sexual (seed) and asexual (vegetative) propagation. Thus, clinical pathogens
have the potential to dramatically impact the survivorship of plant populations.
While this definition addresses the functional role of the fungus in the ecosys-
tem, it does not differentiate the parasitic nature or host specificity of the fun-
gus. This functional group contains fungi which require living tissue of a specific
plant host to grow and reproduce (obligate parasites), as well as fungi which can
survive for extended periods of time in the soil on organic matter (facultative
parasites).
2. Abundance and Distribution
Clinical pathogens are found throughout the ecological range of terrestrial

plants, and epidemics of plant disease occur in a wide array of ecosystems rang-
ing from the subarctic to the equatorial tropics. Their population dynamics within
crop production systems have been studied extensively. Typically, populations of
clinical pathogens are present at low levels, bordering on the lower detectable
range, until presence of the host coupled with favorable environmental conditions
create an explosion of the pathogen population resulting in an epidemic of plant
disease (Flowers and Hendrix, 1972; Kannwischer and Mitchell, 1981; Mitchell,
1978; Smith and Snyder, 1971). Under most conditions, they probably constitute
a small proportion of the community of root-inhabiting fungi and contribute little
to the total fungal biomass in the soil. Considerably less information exists on the
abundance of clinical pathogens in natural ecosystems (Alexander, 1992; Burdon,
1987).
3. Role in Agroecosystems
While comprising a small percentage of the total fungal biomass in soils, clinical
pathogens perform a major functional role in the ecosystem because they are pri-
mary regulators of plant density and diversity. This is most evident in agroecosys-
tems where large-scale monoculture is practiced (Burdon and Chilvers, 1982).
Epidemics of plant diseases in natural systems have also been observed (Dinoor
and Eshed, 1984; Newhook and Podger, 1972; Schmidt, 1978; Weste and Ashton,
1994). In a study conducted over 4 years in permanent plots, root infection by
Rhizoctonia solani or Pythium irrgulare significantly reduced plant populations of
the annual legume Kummerowia stipulacea (Mihail et al., 1998). The reductions
were more severe at high plant densities. Computer simulation of epidemics caused
by Phytophthora spp. and Fusarium oxysporum have indicated that initial increase
of the pathogen population requires that the host density be above a threshold level
(Thrall et al., 1997).
C. SUBCLINICAL PATHOGENS
1. Definition
Subclinical pathogens invade root tissue and cause localized cell death and
disruption of vascular functions. However, visual symptoms are often difficult to
discern as subclinical pathogens do not cause mortality or eliminate the plants
ability to reproduce. Fungi placed in this classification have been referred to as
“minor pathogens” by Salt (1979). However, unlike Salt’s definition, which limited
this group to fungi that only parasitize root-tips or cortical cells, assignment to the
status of subclinical pathogen does not place any restriction on the type or location
of host tissue colonized within the root. Included in this group are fungal species
belonging to a diverse grouping of genera, including Pythium, Mucor, Fusarium,
and Cylindrocarpon. Subclinical pathogens can negatively impact plant health in
many ways. Through localized necrosis they disrupt vascular function in the root
and alter morphology and limit nutrient uptake or availability in the plant host
(Larkin et al., 1995), which results in a reduction in plant vigor and decline in
plant health. Infection by subclinical pathogens may predispose plants to injury
by other plant pests or environmental stress. Their effects on plant health may be
synergistic when they parasitize root tissue in conjunction with other soil microbes,
such as plant pathogenic nematodes or bacteria. Their effects on the host make the
plant more vulnerable to drought, flooding, or other unfavorable environmental
conditions. Finally, subclinical pathogens can serve as vectors for plant viruses
(Campbell and Fry, 1966; Gerik and Duffus, 1986).
The fact that some fungal species can exist as both clinical and subclinical
pathogens muddies the distinction between these groups. For example, at ambient
temperatures of 28◦ C or less, Pythium aphanidermatum and Pythium myriotylum
function as subclinical pathogens on pepper and tomato (Chellemi et al., unpub-
lished data), parasitizing root cells and causing significant reductions in growth,
but not limiting the plants ability to survive and reproduce. However, at ambient
temperatures near 34◦ C these fungi cause extensive plant mortality in the same
host.
Subclinical root-inhabiting fungi are distributed throughout the range of terres-

trial plants. Their diversity and abundance remains relatively unknown due in part
to the fact that their status as subclinical pathogens remains largely undetermined.
Demonstrable reductions in plant growth or yield in fulfillment of Koch’s postu-
lates are required to confirm their status as plant pathogens. These procedures are
time consuming and labor intensive and, therefore, determination of pathogenic
status has been typically reserved for those fungi suspected of inducing plant
mortality. Thus, investigations to determine the status of subclinical pathogens
are usually undertaken for alternative reasons (i.e., suspicion of vectoring a plant
virus or interaction with other clinical pathogens). In crop production systems, the
abundance of subclinical pathogens has been investigated in replant diseases of
perennial crops (Mazzola, 1999) and citrus declines of unknown etiology (Graham
et al., 1983; Nemec et al., 1980).
Subclinical pathogens also function as regulators of plant density, though to

a lesser extent than the clinical pathogens. They do so by affecting the relative
fitness of plant populations. This is accomplished by reducing the competitive
ability of plants through reductions in vigor or reproduction. There is evidence for
this role in natural plant systems (Augspurger, 1983; van der Putten et al., 1993).
In a study by Holah and Alexander (1999), root-inhabiting fungi unique to soils
associated with Chamaeerista fasciculata (an annual legume) were detrimental
to Andropogon geradii (a native tallgrass and one of the dominant perennials in
the ecosystem). Subclinical pathogens can also initiate processes leading to the
breakdown of plant tissue and recycling of carbon in the soil, as they are present
in root tissue at the time of plant senescence (Waid, 1974).
D. ARBUSCULAR MYCORRHIZAL FUNGI
1. Definition
Mycorrhizae are symbiotic associations of specific fungi with the fine roots
of plants. Several mycorrhizal types have been described, and one or more of
these plant–fungus associations are found in nearly every biome on Earth (Smith
and Read, 1997). The arbuscular mycorrhizal (AM) type is the most widespread
mycorrhiza found on plant roots in agroecosystems. The diagnostic feature of ar-
buscular mycorrhiza is the highly branched arbuscules that develop within root
cortical cells. The fungus initially grows between cortical cells but soon penetrates
the host cell wall and grows within the cell lumen. Neither the fungal cell wall nor
the host cell membrane are breached (Bonfante and Perotto, 1995). As the fungus
grows, the host cell membrane invaginates and envelops the fungus, creating a
new compartment where material of high molecular complexity is deposited. This
apoplastic space prevents direct contact between the plant and fungus cytoplasms
and allows for efficient transfer of nutrients between the symbionts. The arbuscules
are relatively short lived and are often difficult to observe in field-collected samples.
Other structures produced by AM fungi include vesicles, auxiliary cells, extra-
matrical hyphae, and spores. Vesicles are thin-walled, lipid-filled structures that
usually form in intercellular spaces. Their primary function is thought to be for
storage; however, vesicles can also serve as reproductive propagules for the fun-
gus. The term vesicular–arbuscular mycorrhiza or VAM was originally applied to
this group, but because a major suborder lacks the ability to form vesicles in roots,
AM is now the preferred acronym. Auxiliary cells are formed in the soil and can
be coiled or knobby. The function of these structures is not known. Spores can be
formed either in the root or more commonly in the soil. Spores produced by AM
fungi are asexual, formed by the differentiation of vegetative hyphae. For some
fungi (e.g., Glomus intraradices), vesicles in the root undergo secondary thicken-
ing, a septum (cross wall) is laid down across the hyphal attachment, and a spore is
formed, but more often spores develop from hyphal swellings in the soil. The AM
fungi may produce an extensive network of extramatrical hyphae (Sylvia, 1990)
and can significantly increase phosphorus-inflow rates of the plants they colonize
(Jakobsen et al., 1992).
The AM fungi are currently classified in the order Glomales (Morton, 1988).
The order is further divided into suborders based on the presence of (i) vesicles
in the root and formation of chlamydospores borne from subtending hyphae for
the suborder Glomineae or (ii) absence of vesicles in the root and formation of
auxiliary cells and azygospores in the soil in the suborder Gigasporineae. The order
Glomales is further divided into families and genera according to the method of
spore formation. The spores of AM fungi are very distinctive and range in diameter
from 10 to >1000 ␮m. The spores can vary in color from hyaline to black and in
surface texture from smooth to highly ornamented. More than 150 species of AM
fungi have been described; however, taxonomy at the species level is currently
going through extensive revision. The reader may visit the INVAM webpage
(http://invam.caf.wvu.edu/) to obtain current information on AM taxonomy.
Most crop plants are colonized by AM fungi and, in fact, it is much easier
to list the predominately nonmycorrhizal plant families—the Caryophyllaceae,
Chenopodiaceae, Cruciferae, Juncaceae, Polygonaceae, and Proteaceae—than the
mycorrhizal ones. Surveys of field-grown crops reveal wide ranges in the extent
of colonization of roots by AM fungi (Table I). Many edaphic factors, such as soil
type (Frey and Ellis, 1997), soil fertility (Bolgiano et al., 1983), and pH (Clark,
1997), affect the extent of colonization but the conspicuous fact is that the majority
of agronomic crops grown under a wide range of conditions consistently have a
significant portion of their root systems colonized by AM fungi. It is clear that
the critical question for the agronomist is not whether their crops are colonized by
AM fungi but rather what impact these fungi have on crop and soil productivity.
We have incomplete knowledge of the species of AM fungi associated with
agronomic crops because numerous difficulties are encountered when attempting
to characterize diversity of these fungi in the field; spores are difficult to identify,
some species do not sporulate, and there is little relationship between functional
and morphological diversity (Douds and Millner, 1999). The few surveys that
quantify AM fungal spore densities or species richness (Table I) suggest that
there are often less than 10 spores g−1 soil and between 5 to 10 species of AM
fungi present in a given agronomic soil. What these numbers mean relative to soil
productivity is unclear because spores may represent only a small proportion of
the total mycorrhizal propagules in the soil [colonized roots and hyphae may also
initiate new mycorrhizae (Friese and Allen, 1991)]. Furthermore, AM species,
and even isolates, may differ dramatically in their effect on plant growth (Boerner,
1990; Boucher et al., 1999), and with current knowledge it is impossible to predict
which propagules will have the greatest impact on crop response.
Even though AM symbioses are among the best known examples of compatibil-
ity between plants and microbes we have little understanding of the factors that con-
tribute to the specificity of these compatible interactions. The AM symbioses are
often considered nonspecific (Gianinazzi-Pearson, 1984; Sanders, 1993). Nonethe-
less, there is mounting evidence that “host preference” is an important character-
istic of AM symbioses (Dhillion, 1992; Giovannetti and Hepper, 1985). By this
Table I
Examples of AM Fungal Associations of Agronomic Crops
Max. root Max. spore Species

Crop Location colonization (%) densitya richness Reference
Aeschynomene Florida 30b 3 6 Medina et al. (1988)

americana
Allium cepa Israel ca. 50b nac na Krikun et al. (1990)
Apium graveolens Israel ca. 50b na na Krikun et al. (1990)
Capsicum annuum Australia 58b na na Olsen et al. (1999)
Israel ca. 50b na na Krikun et al. (1990)
Cucumis melo Israel >50b na na Krikun et al. (1990)
Eleusine coracana India 30b, d 6 na Harinikumar and
Bagyaraj (1989)
Glycine max Minnesota 25b 67e 14 Johnson et al. (1991)
Pennsylvania 9,b 67d <1,b 1d na Douds et al. (1993)
Gossypium hirsutum Texas 70b na na Zak et al. (1998)
Helianthus annuus India 23b, d 5 na Harinikumar and
Bagyaraj (1989)
Hordeum vulgare Demark 50b 2 na Jakobsen and
Nielsen (1983)
Lycopersicon Florida 52d 4 4 Unpublished data
esculentum
Oryza sativa Japan 55b 3 na Solaiman and
Hirata (1997)
Pisum sativum Denmark 80b 2 na Jakobsen and
Nielsen (1983)
Solanum tuberosum England 53b 3 na Hayman et al. (1975)
Triticum aestivum Pennsylvania 15d 8 na Boswell et al. (1998)
Pennsylvania 35,b 45d <1,b 4d na Douds et al. (1993)
Kansas 27b 3 6 Hetrick and Bloom
(1983)
Denmark 50b 2 na Jakobsen and
Nielsen (1983)
Australia 20,b 70d na na Ryan et al. (1994)
Zea mays Minnesota 34b 71e 14 Johnson et al. (1991)
S. Dakota 10,b 43d na na Vivekanandan and
Fixen (1991)
Pennsylvania 84d na na Boswell et al. (1998)
Quebec 71b, d 21 na Kabir et al. (1998)
India 29b, d 4 na Harinikumar and
Bagyaraj (1989)
a
Data variously presented as spores per gram or per milliliter.
b
Conventional system.
c
na = data not available.
d
Organic/sustainable system.
e
High value due to an abundance of Glomus aggregatum.
we mean that different species, strains, or isolates of AM fungi colonize plant roots
to different degrees and have variable effects on plant growth and development.
Here it is important to distinguish among specificity (innate ability to colonize),
infectiveness (amount of colonization), and effectiveness (plant response to colo-
nization). The AM fungi differ widely in the levels of colonization they produce
in a root system and in their impact on nutrient uptake and plant growth.
Host preference may be under the genetic control of the host, the fungus, or, most
likely, a complex interactive effect of both symbiotic partners with soil edaphic
factors. Much of the extant literature emphasizes the role of the plant in the interac-
tion. Johnson et al. (1991) found that cropping history (maize vs soybean) and soil
type altered the communities of AM fungi in soil, Bever et al. (1996) demonstrated
host-dependent sporulation among common lawn plants, and Zhao et al. (1997)
reported differential development of AM fungi with two legume species. Host
genotype variation in root colonization and plant response also has been demon-
◦
strated for citrus (Graham and Eissenstat, 1994), pea (Martensson and Rydberg,
1995), wheat (Hetrick et al., 1996), barley (Baon et al., 1993), and tomato (Barker
et al., 1998).
Less is know about the effect of the fungal genotype on root colonization and
plant response. Inoculum density can be a confounding factor when one attempts
to differentiate fungal affects (Clapperton and Reid, 1992; Daft and Nicolson,
1969). However, when inoculum densities are not limiting or have been equalized,
important ecotypic variation among AM fungi has been reported (Boyetchko and
Tewari, 1995; Douds et al., 1998; Graham et al., 1996; Hepper et al., 1988; Monzon
and Azcon, 1996; Stahl et al., 1990; Sylvia et al., 1993b). These studies support
the hypothesis that the fungal ecotype will have an important impact on root
colonization, sporulation, and host plant response
We are becoming increasingly aware of the important multifunctional roles of

AM fungi in ecosystems (Newsham et al., 1995b). Besides improving uptake of
poorly mobile nutrients (George et al., 1992), AM symbioses may impact drought
tolerance (Schellenbaum et al., 1998) and pathogen interactions (Azcón-Aguilar
and Barea, 1997) and contribute to soil quality by channeling carbon to the soil
and thereby improve soil aggregation (Jastrow et al., 1998). Furthermore, there is
mounting evidence that AM fungi are important determinants of plant community
structure and plant succession (Allen et al., 1995; Gange et al., 1993). van der
Heijden et al. (1998) concluded that below-ground diversity of AM fungi is a
major factor contributing to the maintenance of plant biodiversity and ecosystem
function.
The function of AM fungi in highly managed agroecosystems is less certain
(Hayman, 1987). Under nutrient (Bagyaraj and Sreeramulu, 1982; Beyene et al.,
1996; Osonubi et al., 1995) or moisture stress (Sylvia et al., 1993a), they can
significantly increase crops yields. However, in highly managed, high-input sys-
tems, it is possible to demonstrate growth reductions due to AM colonization
(Graham and Eissenstat, 1998; McGonigle and Miller, 1996). Furthermore,
Hetrick et al. (1993) found that modern breeding practices have reduced my-
corrhizal dependency of wheat. Indeed, in those cases where colonization oc-
curs in the absence of a demonstratable growth enhancement of the plant, the
net cost of the symbiosis may exceed the net benefit (Johnson et al., 1997).
Nonetheless, when one takes a more holistic view of the plant–soil continuum,
the “cost” of the symbiotic association may turn out to be an important benefit
(Schreiner and Bethlenfalvay, 1995). For example, Wright and Upadgyaya (1997)
have described a high-molecular-weight glycoprotein (termed glomalin) that is
produced in abundance by AM fungi. This material accumulates in soil and is
positively correlated with aggregate stability (Fig. 1) (Wright and Upadhyaya,
1998).
Conventional agronomic practices may adversely affect the diversity and abun-
dance of AM fungi in agroecosystems (Johnson and Pfleger, 1992; Thompson,
1994). Large applications of phosphatic fertilizer generally reduce AM root colo-
nization (Harinikumar and Bagyaraj, 1989; Olsen et al., 1996; Vivekanandan and
Figure 1 The relationship between stability of 1- to 2-mm-size aggregates and immunoreactive

easily extractable glomalin (IREEG) from soils in four regions of the United States with ≤80% aggre-
gate stability. From Wright and Upadhyaya (1998); used with permission.
Fixen, 1991); however, addition of phosphorus fertilizer to very low phosphorus

soils may increase root colonization (Bolan et al., 1984). Tillage has been shown
to delay AM colonization of maize roots and result in reduced early season phos-
phorus uptake (McGonigle et al., 1999; Vivekanandan and Fixen, 1991) as well
as reduced hyphal and spore densities near the soil surface (Kabir et al., 1998).
Crop rotations that include nonhost plants (Harinikumar and Bagyaraj, 1988) or
long-term fallow (Thompson, 1987) may also reduce AM fungal populations.
Pesticides, especially fumigants, substituted aromatic hydrocarbons, and benzimi-
dazoles (Johnson and Pfleger, 1992), may adversely affect the activity of AM
fungi in soil. As an overall generalization one may conclude that conventional
management practices reduce AM fungal populations while sustainable, organic,
low-input systems tend to increase their activity (Table I) (Douds et al., 1993;
Kabir et al., 1998; Ryan et al., 1994).
E. ADDITIONAL NONPATHOGENIC FUNGI
1. Definition
This group comprises a diverse collection of fungi capable of invading and

occupying inter- and intracellular spaces within the root tissue without disrupt-
ing the cellular functions of root organelles. As a group, these fungi survive and
function as saprophytes within the soil microbial community and include mem-
bers from many common genera, such as Fusarium, Gliocladium, Microdochium,
Penicillium, Phialophora, Trichoderma, and various poorly defined dark-septate
endophytes (Jumpponen and Trappe, 1998; Skipp and Christensen, 1989). They
differ from mycorrhizal fungi in that they form no specialized organelles or struc-
tures within the root. Also, they are generally thought to have no species-specific
relationships with the plants; however, some host specificity has been suggested
(Skipp and Christensen, 1989).
Fungi included in this group are ubiquitous and occur in large numbers wher-
ever terrestrial plants are found. Many saprophytic fungal species are present in
the rhizosphere shortly after introduction of the plant host into the soil (English
and Mitchell, 1988). Furthermore, epidermal and outer cortical cells of roots are
ephemeral and begin to senesce within a few weeks in the zone of cortical lysis
(Foster, 1986). Deacon et al. (1987) describe this process as early root cortex death
(RCD). These tissues initially appear healthy, but nuclear staining reveals that nu-
clei have disappeared from the cells. A wide range of nonpathogenic fungi readily
colonize these senescing root tissues (Bowen and Rovira, 1976; Huisman, 1988).
Information on the abundance of nonpathogenic root-inhabiting fungi in natu-

ral systems is incomplete, partially because they are not normally investigated
for their contribution to ecosystem health. In crop production systems, docu-
mentation of their abundance usually occurs during investigations related to a
plant pathological disorder. For example, the composition of presumably non-
pathogenic root-inhabiting fungi in association with replant diseases of peren-
nial fruit trees was studied by Mazzola (1999). Difficulties in the identification
of fungal species also limit quantitative studies on their abundance and distri-
bution as few molecular markers have been identified for nonpathogenic soil
fungi.
The role of nonpathogenic root-inhabiting fungi in ecosystems is complex. As

saprophytes present in large numbers they occupy a vital link in the trophic food
web in soils. They participate in carbon recycling through the decomposition of
plant tissue (Waid, 1974). Their relationships to plant health have been docu-
mented in several systems. They may be involved as elicitors of induced systemic
resistance in the plant host (Benhamou et al., 1997; Larkin and Fravel, 1999).
They can also protect plants from infections by pathogens through direct compe-
tition for infection sites on the root or interference with saprophytic colonization
of soil organic matter (Couteaudier, 1992; Martin and Hancock, 1986; Schneider,
1984).
III. INTERACTIONS AMONG

ROOT-INHABITING FUNGI
A. INTERACTIONS AMONG PATHOGENS
Co-infection of root systems by more than one pathogenic fungus is most likely
the rule rather than the exception. Synergism between co-infecting pathogenic
fungi may or may not occur. For example, P. myriotylum interacted synergistically
with Fusarium solani to cause damping-off of peanut seedlings, but no synergistic
effect was observed when P. myriotylum was combined with R. solani (Garcia and
Mitchell, 1975). In the same study, P. myriotylum could not be reisolated from roots
co-infected with R. solani. Thus, although multiple infections may take place in
the root system among pathogens, the resultant level of disease will vary depend-
ing upon the specific species interactions. Elucidation of the interactions among
pathogens may be further complicated by soil edaphic factors and environmental
conditions.
B. INTERACTIONS OF AM FUNGI WITH PATHOGENIC

AND NONPATHOGENIC FUNGI
Mycorrhizal fungi colonize feeder roots and thereby interact with root pathogens
that parasitize this same tissue. A large body of literature exists on the interactions
among pathogens and AM fungi, and several reviews have been written on the
subject (Dehne, 1982; Linderman, 1994; Paulitz and Linderman, 1991; Schenck,
1981). In a natural ecosystem, a major role of mycorrhizal fungi may be protection
of the root system from endemic pathogens (Newsham et al., 1995a). In cropping
systems the role of AM fungi in disease protection is less clear; however, much of
the literature suggests that AM fungi reduce soilborne disease or at least amelio-
rate the effects of disease. Mechanisms put forward to explain disease protection
include (Azcón-Aguilar and Barea, 1997):
r improved nutrient status of the host plant
r competition for host photosynthates
r competition for infection sites
r anatomical and morphological changes in the root system
r microbial changes in the mycorrhizosphere
r activation of plant defense mechanisms
Often inoculation with AM fungi prior to challenging with a pathogen is neces-

sary to achieve disease reduction. This is because many root pathogens germinate
and grow more rapidly than AM fungi and, if co-inoculated, will attack the root be-
fore the mycorrhizae become established. An intriguing finding is that AM fungi
may actually stimulate spore germination of some pathogens (St-Arnaud et al.,
1995). A protective effect may result if germination of pathogen spores close to
the mycorrhizal mycelium, but far from the roots, results in reduced inoculum
potential of the pathogen.
Here we present several recent examples of AM fungal interactions with
pathogens that have been published since the previously cited reviews. Trotta
et al. (1996) reported that precolonization of tomato with the AM fungus,
Glomus mosseae decreased both weight reduction and root necrosis caused by
Phytophthora nicotianae. They concluded that the AM fungus activates a disease-
suppression mechanism to reduce root damage because improved phosphorus nu-
trition could not account for increased disease resistance. Cordier et al. (1996)
found a similar response, reporting that the number of P. nicotianae hyphae grow-
ing in the root cortex of tomato was reduced in mycorrhizal root systems and that
the pathogen hyphae never invaded arbuscule-containing cells. Both localized and
systemic resistance mechanisms have been demonstrated in this system, including
induction of plant wall defense responses (Cordier et al., 1998) and unique chitinase
isoforms (Pozo et al., 1998; Pozo et al., 1997). In contrast, Kjøller and Rosendahl
(1997) found no evidence of a localized defense response when G. intraradices

protected roots of pea from Aphanomyces euteiches, but rather they reported a
general effect on root physiology. Additional biological control interactions with
AM fungi that have been reported include protection of onion from Sclerotium
cepivorum (Torres-Barragan et al., 1996), alfalfa from Verticillium albro-atrum
and Fusarium oxysporum, Java citronella from P. aphanidermatum (Ratti et al.,
1998), and tomato from F. oxysporum (Datnoff et al., 1995). An important criticism
of most of these recent studies is that they do not provide an adequate P-fertilized
control. Future studies should compare mycorrhizal and nonmycorrhizal plants of
similar P status and size in order to separate possible direct effects of mycorrhizal
formation from nutritional responses (Graham, 1988).
In addition to fungal pathogens, mycorrhizae exert a strong influence on bacte-
ria, actinomycetes, other fungi, mycoparasites, and invertebrates that occur in the
mycorrhizosphere (Andrade et al., 1998; Linderman, 1992; Paulitz and Linderman,
1991). Ames et al. (1987) reported that the type of microorganisms found with
AM fungi was influenced by the type of inoculum used, suggesting the existence
of specific microbial associations with AM fungi. The interactions among AM
fungi and closely associated nonpathogenic fungi, such as species of Aspergillus,
Gliocladium, Paecilomyces, Trichoderma, and Wardomyces, are complex and can
vary from antagonistic to neutral to synergistic (Dhillion, 1994; Fracchia et al.,
1998; Garcia-Romera et al., 1998; Mcallister et al., 1996). Rousseau et al. (1996)
demonstrated that a strain of a Trichoderma sp. selected for its superior biologi-
cal control potential actively parasitized G. intraradices, at least in their in vitro
system, but additional research is needed to evaluate these complex interactions
in the field.
C. INTERACTIONS BETWEEN PATHOGENIC AND

NONPATHOGENIC FUNGI
The root and soil surrounding it have high biological activity, including mi-
croorganisms (predominately bacteria and fungi) that promote plant growth and
yield or are deleterious to plant growth (Bowen and Rovira, 1999; Sturz et al.,
1997). The reader should note that in this review we restrict our comments to the
fungal component. Recently, Sneh (1998) reviewed the interactions among plant
pathogens and closely related nonpathogenic (avirulent) and low virulent (hypovir-
ulent) fungi. Competition for infection sites or for nutrients has been demonstrated
for several root-associated fungi, including species of Rhizoctonia, Fusarium, and
Pythium, while mycoparasitism was shown for others, such as Trichoderma and
some Pythium spp.
An interesting case involves Fusarium oxysporum-suppressive soils where dis-
ease remains low despite the presence of high levels of the pathogen (Sneh, 1998).
Competition for nutrients by nonpathogenic strains of F. oxysporum can regulate

the activity of pathogenic strains (Alabouvette, 1990; Couteaudier, 1992; Mandeel
and Baker, 1999). Nonpathogenic stains of F. oxysporum can penetrate the epider-
mis and colonize the root cortex (Olivain and Alabouvette, 1999) and can suppress
disease through parasitic competition for infection sites on the root (Schneider,
1984). Finally, parasitism of root systems by nonpathogenic strains can elicit an
induced systemic resistance response in the host (Larkin and Fravel, 1999). Non-
pathogenic isolates of F. oxysporum from suppressive soil were identified as the
major group of antagonists responsible for disease suppressiveness (Larkin et al.,
1996). In this case the rhizosphere of resistant varieties were less favorable for
the pathogenic F. oxysporum isolates, but more favorable for nonpathogenic iso-
lates. Steinberg (1999) concluded that F. oxysporum is fundamentally a rhizosphere
species and is strongly stimulated in the root vicinity, probably due to organic nitro-
gen present in root exudates. Interestingly, the population structure of F. oxysporum
associated with different plants can vary (Edel et al., 1999), suggesting that plants
have a selective effect on the fungi colonizing their roots. The mechanisms by
which these nonpathogenic strains control disease are not fully understood, but
the evidence to date suggest a combination of effects, including competition for
nutrients and infection sites, induced local and systematic resistance, and myco-
parasitism (Sneh, 1998).
D. APPLICATION OF ISLAND BIOGEOGRAPHY THEORY

TO ROOT–FUNGAL INTERACTIONS
Understanding the interactions within the community of root-inhabiting fungi

and between communities of other soil microflora is paramount to predicting the
outcomes of biological control activities. However, the microcosm comprised of
plant root systems and their rhizosphere is complex, dynamic, and therefore very
difficult to understand.
One way to describe and elucidate this microcosm is to use the dynamic theory
of island biogeography. This theory, as proposed by MacArthur and Wilson (1967),
states that for a given time a habitat island contains a number of species present
(S), as well as an immigration rate (I) of new species onto the island, and an
extinction rate (E) of species presently residing on the island. Application of the
theory to study the population dynamics of fungal communities has been discussed
(Wildman, 1992) and used to study soil fungi colonizing cellophane squares of
various dimensions placed into the soil (Wildman, 1987).
Consideration of roots as habitat islands is based upon the premise that they
are insulated to some extent by the soil medium from exogenous populations
located some distance away. Thus, the primary populations of root inhabiting
fungi arise from reproduction of species present in the rhizosphere. As summarized
by Simberloff (1986), the theory states that immigration and extinction rates are
not constants, but rather monotonic functions of the numbers of species present.
Immigration rate is highest when the island is empty and declines to zero when all
species in the regional species pool already inhabit the island. The extinction rate is
zero when no species are present on the island and rises to a maximum value when
all species are present. The two monotonic curves must cross and this intersection
constitutes an equilibrium number of species (S), about which the island’s number
of species should vary. The equilibrium is dynamic because it is accompanied by
a turnover of species at rate X, as species are locally extinguished on the island
and are replaced by immigrants from the pool. Thus, it is the number of species,
rather that their identity, that is expected to remain constant.
While Simberloff (1986) pointed out that island biogeography should be con-
sidered a hypothesis that is difficult to test, it may facilitate better understanding
of the ecology of root-inhabiting fungi as they pertain to biological control. Plant
roots placed into soil that has been disinfested through practices such as fumiga-
tion with methyl bromide or steam sterilization represent a resource island with
a low number of species present and are rapidly colonized by indigenous fungi.
This phenomenon has been observed in crop production systems (Marois and
Mitchell, 1981; Welvaert, 1974). As initial colonization rates will be high, sys-
tems employing root-inhabiting fungi as biological control agents should consider
the competence of the fungi to compete with the aggressive indigenous fungi
for colonization sites on the root surface. Most applications of biological control
agents are made under the assumption that colonization rates are similar in all
pathosystems. Until this assumption has been verified on a large scale, coloniza-
tion rates by microorganisms specific to individual sites should be considered in
the determination of the competitive fitness of biological control candidates. The
economics of such an approach is not feasible under current systems used for prod-
uct development and sales of root-inhabiting fungi intended for biological control
of root pathogens.
IV. OPPORTUNITIES FOR PEST CONTROL
A. CURRENT AND FUTURE CONTROL STRATEGIES
The strategy of pest management selected by the producer will greatly influ-
ence the opportunities for biological control of root pathogens. Familiarity with
current and future control strategies as they pertain to soilborne pests is necessary
to better understand the application potential of biological control. Three diver-
gent approaches have been used to develop strategies for the control of soilborne
pests.
1. Single-Tactic Approach
The single-tactic approach relies upon the routine application of a broad spec-
trum biocide to eradicate all potential pests. Key to this approach is that applications
are made on a regular basis using materials with a range of efficacy broad enough
to remove the threat of all potential pests. This approach has gained popularity
over the years among large-scale producers of high-cash-value crops. In many
instances the approach has been incorporated into the design of the production
system (Geraldson et al., 1965; Maynard and Hochmuth, 1995).
The single-tactic approach remains popular for several reasons. It eliminates the
need to obtain and manage information regarding pest biology and their population
parameters in the field, thus simplifying the decision-making process. Determina-
tion of cropping sequences and application of materials is based upon marketing
constraints or a calendar date. Because the risks associated with damage from
potential pests is eliminated, growing seasons can be extended and the need for
fallow periods or cultivation of a rotation crop is greatly reduced.
Since the mid 1900s several soil fumigants or fumigant combinations, includ-
ing methyl bromide, chloropicrin, and methylisothiocyanate-generating products,
have been used to control soilborne plant pathogens. Methyl bromide has been
the principal fumigant since the 1970s due to its relatively low cost, ease of han-
dling, performance under a wide range of soil and environmental conditions, low
phytotoxicity, and broad range of activity. In many areas of the world where inten-
sive agriculture is practiced, the use of methyl bromide has become indispensable
(Vanachter, 1975).
Use of a single-tactic approach does have several important disadvantages. Ap-
plications based upon calendar dates or other nonbiological criteria can result in the
over application of pesticides and increase both production costs and the potential
for environmental disruption. Reliance upon a single chemical to control all soil-
borne pests can leave the producer vulnerable to changes in pesticide availability or
regulatory policies. Indeed, methyl bromide has been implicated as a major ozone-
depleting compound, and the United States is required by international treaty to
phase out production and sale by the year 2005.
2. Integrated Pest Management
Integrated Pest Management (IPM) can be defined as the use of multiple tactics
to maintain damage from pests below an economic threshold while conserving
beneficial organisms. Traditionally, IPM has been used as a strategy to manage
foliar insect pests. Far less frequently has it been employed to manage soilborne
pests. Use of multiple tactics ensures that growers do not become dependent upon
a single chemical. Because treatments are applied “as needed,” the threat of envi-
ronmental damage is greatly reduced.
Several fundamental obstacles exist which hinder the widespread adaptation of

IPM for control of root pathogens. Determination of economic thresholds remains
problematic. Sampling methods for soil-inhabiting microbes are technically chal-
lenging, expensive, and labor intensive. Residual populations of the pathogens are
often below the level of detection for most methods. This, coupled with the ex-
plosive growth potential of many root pathogens, can lead to the use of presence/
absence designations rather that quantitative counts. Traditional deployment of
an IPM strategy relies heavily upon intervention after sampling thresholds have
been reached. The soil medium makes uniform delivery of a tactic to mature root
systems difficult to achieve. There are a limited number of chemical and biolog-
ically treatments which act systemically within the root system and even fewer
with therapeutic affects.
3. Proactive Pest Management
Proactive pest management is a strategy which seeks to minimize intervention

through the avoidance of pest outbreaks. When incorporated into the design of the
crop production system this strategy can be very effective and can broaden the
availability of pest-management tactics not routinely considered by conventional
growers. The most common example is the integration of soil-less media into
greenhouse production systems. Through the use of a sterile medium growers are
able to avoid problems associated with soil infested with root pathogens. Crop
rotation can become an effective tactic when incorporated into the design of the
production system. A 3-year rotation with bahiagrass (Paspalum notatum) pasture
has been shown to significantly reduce major soilborne pests of tomato, including
diseases caused by Sclerotium rolfsii and F. oxysporum f.sp. lycopersici as well as
damage from root-knot nematodes (Meloidogyne spp.). This rotation is impractical
for tomato producers in Florida because they cannot afford to lose revenue from
fields which have been modified to utilize their raised bed–plastic mulch production
system. An alternative, low-input production system was designed to utilize min-
imum tillage practices in existing bahiagrass pasture as a means to avoid damage
from the aforementioned pests (Chellemi et al., 1999). Designed to be compatible
with existing bahiagrass pastures, the alternative production system makes avail-
able the 2.5 million acres of improved bahiagrass pasture in Florida for tomato
growers.
To be effective and practical, a proactive strategy should be considered when
designing the production system. This limits its application to existing produc-
tion systems. While a proactive pest-management strategy may be desirable in
theory, avoidance of all potential pests may not be practical. Thus, some flexi-
bility in the strategy that permits intervention when needed is a more realistic
approach.
B. ROLE OF BIOLOGICAL CONTROL
Biological control of root pathogens can provide substantial benefits to growers

utilizing all three pest-management strategies. However, its roles and potential
contributions will vary from strategy to strategy.
While the single-tactic approach has sustained a high level of productivity
and contributed directly to the success of many high-input production systems, it
does not necessarily provide complete control of all potential root pathogens. For
example, fumigation with methyl bromide or chloropicrin did not provide season-
long control of bacterial wilt and Fusarium crown rot of tomato, Fusarium wilt
of tomato and cucumber, and Phytophthora root rot of azalea and rhododendron
(De Ceuster and Pauwels, 1995; Enfinger et al., 1979; Hoitink, 1980; Sonoda,
1976). In most cases, plant disease epidemics were attributed to the reinfestation
of fumigated soil by a pathogen. While fumigation can eradicate or significantly
reduce inoculum in the soil, it also dramatically impacts populations of beneficial
microorganisms leading to a biological vacuum. Fumigated soil is rapidly
recolonized by a diverse group of fungi, including documented biocontrol agents,
although the composition of species and rate of colonization is largely driven by
edaphic and environmental conditions (Bollen, 1974; Marois and Mitchell, 1981).
Despite this information, augmentation of fumigated soil with biocontrol agents
has not been widely practiced. Reinoculation of fumigated soils, using a mixture
of several isolates of Trichoderma harzianum, has been used in commercial
production systems in Belgium (De Ceuster and Hoitink, 1999).
Biological control of root pathogens is ideally suited within the context of an
IPM approach. The narrow spectrum of biological activity, lack of deleterious
effects on the environment, and compatibility with other pest-management tactics
associated with biological control all fit within the goals of IPM. In some cases a
synergistic effect has been observed when biological control agents are combined
with other pest-management tactics. The following examples using soil solar-
ization demonstrated this point: sublethal heating and Talaromyces flavus acted
additively to suppress Verticillium wilt of eggplant (Tjamos and Fravel, 1995);
integration of soil solarization for 6 weeks, the AM fungus Glomus fasciculatum,
and seed treatment with carosulfan were highly effective in reducing damage from
root knot nematode and Fusarium wilt of chickpea (Krishna Rao and Krishnappa,
1996); and combining soil solarization with the fungal antagonist Gliocladium
virens improved the control of southern blight of pepper and tomato caused by the
root pathogen S. rolfsii (Ristaino et al., 1991).
Biological control can provide a supporting role in proactive pest-management
strategies. It can provide short-term control of root pathogens when outbreaks oc-
cur. They offer growers a way of reducing the immediate impact of a root disease
while at the same time minimizing the disruption of the biologically suppressive
or exclusive system which the grower has established. Biological control also can
be incorporated into the design of the system. For example, designing a production
system to encourage and sustain a community of disease-suppressive microorgan-
isms would be in effect designing the system to provide continuous biological
control.
C. OBSTACLES TO IMPLEMENTING BIOLOGICAL CONTROL
Many obstacles remain which impede the widespread adoption of biological

control as a component of root disease control programs. These include a lack of
commercially available products, inconsistency of results from season to season or
region to region, cost, failure by the grower to correctly identify the disease com-
plex, and delivery systems which are compatible with standard grower operations.
Some of these obstacles will be overcome through continual progress in prod-
uct development and technology transfer. However, the most difficult challenges
may lie in addressing the ecological issues underlying some of the obstacles. For
example, financial support by private companies for product development and tech-
nology transfer is dependent upon the assumption that the biological control agent
will have broad market appeal, a requirement to reap a return on their investment.
However, the fundamental question remains as to whether a root-inhabiting fungus
applied outside of the ecological range from which it evolved should be expected
to function in the same manner as in its native habitat. In practical terms, is it
realistic to expect a fungus isolated from wheat in the midwestern United States to
colonize and compete with other rhizosphere organisms on a tomato plant in the
southeastern United States?
V. RESEARCH PRIORITIES
The biology of the rhizosphere is extremely complex due to the interplay of

soil, plant, and microbial factors. Bowen and Rovira (1999) state that a major
impetus for rhizosphere research has been the development of biological controls
for root diseases. Unfortunately, this short-term goal has overshadowed research
aimed at better understanding the basic principles of rhizosphere ecology. It is the
very complex nature of the rhizosphere that makes it imperative that we invest
resources into fundamental research of rhizosphere ecology before we can expect
to predictably manage that environment. Here we suggest research priorities for
advancing our understanding of the diversity and behavior of root-inhabiting fungi
in the root zone.
We are still astonishingly ignorant of the fungi that colonize the roots of crop
plants. While comprehensive guides are available to describe fungi growing in vitro
(von Arx, 1981), there is little information to aid one in identifying the fungi as
they occur in and on the root. There has been some attempt at morphological
characterization of fungi in roots (Abbott and Robson, 1979; Rillig et al., 1998)
and, perhaps, an atlas of fungi growing in root systems would provide a very
practical tool for researchers to better characterize fungi in their system. An obvious
limitation of this approach is that morphology varies with host and soil type.
The popularization of molecular tools offers new opportunities for more pre-
cise characterization of fungal diversity in the root zone (Kohn, 1992; Kowalchuk,
1999). In this regard, AM fungi present a special case because they contain hun-
dreds or thousands of nuclei (Giovannetti and Gianinazzi-Pearson, 1994), each
with multiple copies of ribosomal genes. Glomales-specific primers for the small
subunit ribosomal gene (Simon et al., 1993) and the internal transcribed spacer
region (Hijri et al., 1999; Sanders et al., 1996) have been developed, but variations
have proven either too narrow or too broad, respectively, to allow species or iso-
late characterization. More recently, researchers have found that variation in the
large subunit ribosomal gene may be suitable for separating closely related AM
fungi within roots (Kjøller and Rosendahl, in press). Further refinement of these
approaches should lead to rapid advances in our understanding of fungal diversity
in roots, not only for the most obvious pathogens and symbionts, but also for the
wide array of nonpathogenic fungi for which we presently have little knowledge.
Additionally, the specificity of these molecular approaches should allow us to move
experiments from the laboratory and greenhouse to the field, which is where we
need to evaluate these complex interactions in a real-world context.
As stated above, the challenge for the soil ecologist is not only to character-
ize the fungi that interact with plant roots, but to understand their impact on root
function and plant health. Some progress has been made in this arena. We know
that pathogenic and symbiotic root fungi differentially affect the morphological
patterns of roots (Fusconi et al., 1999). Furthermore, root tissues can react very
differently to the presence of pathogens, symbionts, and saprophytes, with re-
sponses ranging from rapid necrosis to nonrecognition (Asiegbu et al., 1999). We
need to gain increased understanding of the molecular basis of these reactions by
utilizing tools of molecular cytology (Hardham, 1998), molecular genetics (Lange
et al., 1999), and immunology (Slezack et al., 1999).
The role of the host plant on root colonization also needs further clarification,
especially at the level of molecular interactions (Smith and Goodman, 1999). One
example of an important host affect is the variable production of border cells
(originally called “sloughed root cap” cells) by various plant species. It is known
that border cells can attract and stimulate growth of some microorganisms and
repel and inhibit the growth of others (Hawes, 1998). Furthermore, Niemira et al.
(1996) recently hypothesized a connection between a host’s propensity to form
mycorrhizae and its capacity to produce border cells. Future understanding of the
basis for this activity may provide new ways to manage microbial populations in
the root zone.
Understanding the consequences of disturbance events on rhizosphere ecology
will be essential to the development of effective tactics to manipulate populations
of disease-suppressive organisms in the root zone. Pest control activities and cul-
tural practices can have lasting effects on the dynamics and diversity of fungal
communities in and on the root, and the development of effective tactics must be-
gin with the avoidance of activities which are detrimental to the establishment of
those organisms. Commonly used insecticides, such as choropyrifos or paraquat,
may persist in soil for up to 1 year after application (Buyuksonmez et al., 1999;
EXTONET, 1999), even though their application was not intended for the soil.
While the effects of nontarget pesticides on mycorrhizal (see previous section)
and fungal root pathogens (Levesque and Rahe, 1992) have been investigated,
pesticide effects on subclinical or nonpathogenic root-inhabiting fungi and the
interactions among functional groups have received little attention.
The phenomena of disease-suppressive soils offer an opportunity to study natu-
rally derived communities of disease-suppressive organisms. Some well-known
examples include soils suppressive to disease caused by the root pathogens
F. oxysporum, Gaeumannomyces graminis f.sp. tritici, Phytophthora cinnamomi,
and R. solani (Hornby, 1983; Schneider, 1982). Traditionally, studies have focused
on the correlation of specific factors with the attainment of disease suppression in
soil. While strong associations between biotic or abiotic factors and disease sup-
pression have been identified, successful induction of disease suppressiveness in
commercial production systems has been limited, suggesting a more complicated
interaction among factors. Deciphering the mechanisms by which suppressive soils
are obtained will provide valuable insight into the development of effective tac-
tics to manage the populations of disease-suppressive organisms. More attention
should be given to novel approaches for studying disease-suppressive soils. For
example, van Bruggen and Semenov (1999) proposed measuring the amplitude of
fluctuations in microbial populations and resilience to a disturbance or stress as an
approach to the search for indicators of soil health. While many studies concen-
trate on the attainment of disease-suppressive soils, perhaps studies designed to
inactivate the suppressive nature of soils will provide additional insights into the
mechanisms involved.
Efficacy studies of biological control agents intended for use against root
pathogens should be accompanied by basic studies on ecological parameters per-
taining to their competitive fitness at the tested sites. Important considerations
include colonization rates of root systems, persistence in the rhizosphere, and
reproduction and mortality subsequent to the conclusion of the cropping cycle.
Inoculant releases (augmentation) are favored as a biological control strategy by
developers and marketers of biological control agents as a means of recuperating
costs incurred during the formulation process. Because these costs are ultimately
passed on to the grower, they favor application of biological control agents to
high-value crops such as fresh market vegetables and glasshouse crops, where a
large budget for production costs is acceptable (Pickett and Bugg, 1998; Trumble
and Morse, 1993). Further research on the persistence and competitiveness of bio-
logical control agents in the soil will ensure that microbes that contribute to the
development of sustainable disease suppression, but that do not achieve levels of
disease suppression high enough for commercial release, would receive consider-
ation in low-input production systems.
ACKNOWLEDGMENTS
We thank James H. Graham, David J. Mitchell, and Abid Alagely for their reviews of the chapter.
Published as Florida Agricultural Experiment Station Journal Series no. R-07408.
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DWARFING GENES IN PLANT
IMPROVEMENT
S. C. K. Milach and L. C. Federizzi
Universidade Federal do Rio Grande do Sul
Faculdade de Agronomia, Departamento de Plantas de Lavoura
Porto Alegre, Brazil
I. Introduction
A. Historical Perspective
II. The Biochemical Basis of the Dwarf Phenotype
A. GA-Sensitive Mutants
B. GA-Insensitive Mutants
C. Phytochrome Mutants
III. Dwarfing Genes and Their Use for Breeding
IV. Breeding Challenges and Varieties Developed
A. Breeding Challenges
B. Varieties Developed
V. Pleiotropic Effects of Dwarfing Genes
A. Morphological and Physiological Traits
B. Yield and Yield Components
VI. Molecular Mapping of Dwarfing Genes
A. Association to Quantitative Trait Loci for Plant Height and Lodging
Resistance
B. Comparative Mapping to Identify Orthologous Dwarfing Genes
C. Mapping as a Basis for Cloning
VII. Concluding Remarks
References
This chapter attempts to summarize the main findings about the dwarfing genes
in different plant species with emphasis on their breeding and genetics aspects.
Most of the examples presented are on the use of dwarfing genes in cereal breeding
because of their importance for agriculture. The biochemical basis of the dwarf
phenotype is discussed and examples of GA-sensitive, GA-insensitive, and phy-
tochrome mutants presented. Although several dwarfing genes have been iden-
tified and studied in different species, only a few have had wide application for
plant improvement. The most universally accepted dwarfing genes have been the
Rht1 and Rht2 of wheat and the sd1 of rice. The positive pleiotropic effect of these
genes on other plant traits, especially on yield and yield components, is one of
the reasons for the success of these genes. Breeding challenges to transfer these
and other dwarfing genes to different genetic backgrounds are discussed and their
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36 MILACH AND FEDERIZZI
impact in variety development presented. Several dwarfing genes have been asso-
ciated to molecular markers and their orthologous relationships identified through
mapping. Cloning of some of the GA-insensitive genes has been recently achieved
and opened the possibility of transferring these genes to a range of other crop
species. C 2001 Academic Press.

I. INTRODUCTION
Advances in grain yield due to the introduction and efficient manipulation of

major genes are common in the literature. For most crop plants, modifications in a
few loci with large phenotypic effects may represent drastic changes in adaptation
and final fitness to the environment. In species of agronomic importance, reduction
of plant height has resulted in large increases in yield, due to lodging resistance,
improved harvest index, and more efficient utilization of the environment.
For the past 4 decades of the 20th century, agriculture has benefited from the use
of semidwarf phenotypes in many different crops. The use of these genes was one
of the main driven forces of the so called “Green Revolution” that resulted from the
adoption of new semidwarf varieties and cultivation methods. The major impact
was in cereal crops, first in wheat and then in rice. The semidwarf wheat varieties
had improved harvest index from the increasing of grain yield at the expense of
straw biomass and were more lodging resistant, thus supporting higher fertilizing
rates.
Because of their importance, a significant amount of research efforts was carried
out to understand the biochemical basis, genetics, physiology, and pleiotropic
effects of the dwarf phenotype in different plant species. Extensive research was
done in cereal crops, particularly with wheat and rice, and one of the few reviews on
this topic with emphasis on all these aspects is that of Gale and Youssefian (1985)
in wheat. Besides that, because of the involvement of the dwarfing genes with
the gibberellin biosynthesis, fundamental research has been conducted with dwarf
mutants involved in the GA biosynthetic and signal transduction pathway. Both of
these aspects have been reviewed recently by Hedden and Kamiya (1997), Ross
et al. (1997), and Swain and Olszewski (1996). Although these represent important
contributions, a review integrating different aspects of the dwarf phenotype with
emphasis in breeding is now appropriate.
This chapter attempts to summarize the main findings about the dwarfing genes
in different plant species with emphasis on their breeding and genetics aspects.
Because a significant amount of work has been done in cereal crops, most of the
examples reported here is on them.
DWARFING GENES IN PLANT IMPROVEMENT 37
A. HISTORICAL PERSPECTIVE
Drastic modifications in plant height due to genetic factors have been reported
since early 1900s, especially in small grain cereals. However, most of these modifi-
cations were described as aberrations or curiosities that were genetically heritable,
but had no practical applications in agriculture (Waldron, 1924). In Japan, never-
theless, since 1873 farmers were using wheat varieties 50 to 60 cm short.
In 1917, the dwarf wheat genotype ’Daruma’ was crossed to ‘Fultz,’ a land
race imported from the United States to Japan. Then, in 1925, Japanese breed-
ers crossed ‘Daruma’—‘Fultz’ with ‘Turkey,’ a Russian wheat land race. From
this combination was selected ‘Norin10,’ which was released in Japan in 1935
(Hanson et al., 1982). It was only after the second world war, in 1946, that S. C.
Salmon, an agricultural research scientist working in Japan, observed that farmers
were growing a number of stiff, short-stemmed wheat varieties. He introduced
16 varieties of this plant type to the United States, and they were available to
breeders in 1947–1948. Voguel from Washington State intensively used one of
them, ‘Norin10,’ in crosses. The crosses between ‘Norin10’ and the American
varieties presented several problems, which required many years of intense selec-
tion until the first variety ‘Gaines’ was released in 1962 (Morrison and Voguel,
1962). That intense selection, especially in progenies of the cross ‘Norin10’ ×
‘Brevor14,’ was of primary importance because it set the basis for a transformation
in the wheat plant that represented a new plant pattern with high number of tillers,
high lodging and shattering resistance, medium size spikes, medium plant height
(semidwarf), medium culm diameter, and medium to small length and width of
leaf (Reitz, 1968). Voguel sent lines of this cross in 1953 to Norman Borlaug,
who was at CIMMYT in Mexico. Borlaug used them intensively to develop new
types of wheat that became responsible for the “Green Revolution.” Since 1911
the Italian wheat breeders were developing stiff and short stature varieties, when
N. Strampelli used the Japanese variety ‘Akakomugi’ in crosses with Italian tall
varieties. Several semidwarf Italian wheat varieties were obtained with the Rht8
and Rht9 genes from ‘Akakomugi’ and these genes have been extensively used in
Europe (Gale and Youssefian, 1985).
Rice varieties on that time were tall and lodged early and did not respond to the
application of nitrogen. In order to increase yield potential, the harvest index had
to be improved and lodging resistance and responsiveness to N increased. This
was accomplished by the reduction of plant height through the incorporation of a
recessive gene for short stature from a Chinese variety called ‘Dee-geo-woo-gen’
(DGWG). The first variety developed was ‘IR 8’ in 1966 that also inherited from
the other parents important traits as sturdy stems, heavy tillering, and dark green
and erect leaves. Due to the advantage of this new type of rice plant, breeders
throughout the world initiated crosses in order to develop short varieties. Short
stature varieties are used now in most of the area cultivated with rice (Khush,
1993).
The new plant type was so revolutionary that all major breeding programs in
the world immediately started to develop short-stature varieties and more than a
thousand varieties of different species are today of short stature. Several major
genes affecting plant height have been reported for wheat, oats, barley, millet,
sorghum, peaches, and so on.
From all the dwarf mutants identified, the most important ones for breeding
purposes have been those from ‘Norin10’ in wheat and ‘IR8’ in rice because of
their positive effects over other agronomic important traits. The intense selection
pressure for fertility and agronomic type in the first crosses was of fundamental
importance for the success of these genes.
The complete sequencing and cloning of these genes will impose new possibil-
ities of using them in improving this trait in other plant species.
II. THE BIOCHEMICAL BASIS OF

THE DWARF PHENOTYPE
Dwarfing genes have been instrumental in dissecting the gibberellins (GAs) bio-
chemical pathway in some species and in elucidating the plant elongation process.
In maize (Zea mays L.), rice (Oryza sativa L.), and pea (Pisum sativum L.), there
are dwarf mutants defective for different steps in the GA pathway and that respond
to the exogenous application of gibberellic acid (Phinney, 1984). In wheat (Gale
and Youssefian, 1985), rye (Börner, 1991), maize (Harberd and Freeling, 1989),
and rice (Mitsunaga et al., 1994), dwarf mutants have been identified that are in-
sensitive to the application of gibberellic acid and are dwarfed due to causes not
directly related to GA biosynthesis. The early 13-hydroxylation GA biosynthetic
pathway leading to active GA1 occurs in maize, rice, and pea (Phinney, 1984). In-
termediates of this pathway have been identified in oat with gas-chromatographic
spectrometry, indicating that the pathway also occurs in this species (Kaufman
et al., 1976).
In wheat, gibberellic acid response assays have been used as a breeding tool
to identify the presence of insensitive dwarfing genes at the seedling stage
(Yamada, 1990; Federizzi et al., 1992). The genetics of the Rht1 and Rht2 re-
cessive dwarfing wheat genes were not clearly resolved until the GA-insensitive
response assay was used in the genetic analysis, probably due to the moderate
effects of these alleles in the wheat hexaploid background (Gale and Youssefian,
1985).
Because the dwarf phenotype may result from mutations related to the gib-
berellin biosynthetic or signal transduction pathway, the dwarfing genes have
been classified as GA-sensitive or -insensitive, respectively. There have also been

reported cases of dwarf transgenic plants resulting from the modified expression
of phytochrome genes (Boylan and Quail, 1989, 1991). The occurrence of these
mutants and their application to breeding differs from one species to another and
is discussed below.
A. GA-SENSITIVE MUTANTS
Shoot extension growth is one of the most studied physiological roles of GAs
(Swain and Olszewski, 1996). Early experiments using exogenous applications of
GA have identified several dwarf mutants that were defective in the production
of at least one enzyme of the GA biosynthesis (Reid, 1990, 1993). These mutants
respond to the application of exogenous GA with stem elongation and are called
GA-sensitive mutants. A list of the better characterized GA-deficient or -sensitive
mutants indicating which is the defective enzyme involved is given by Hedden and
Kamiya (1997).
In maize, the an, d1, d2, d3, and d5 dwarfing genes have been characterized as
recessive mutations that correspond to defective enzymes for the GA biosynthetic
pathway (Fujioka et al., 1988; Hedden and Kamiya, 1997). These together with the
D8 gene are called the “andromonoecious dwarfs” by Coe et al. (1988). Another
class of mutants in maize, which includes the brachytic (br) mutations, is that
of semidwarf or compact plants that show relatively little other morphological
alterations and are conditioned by br1, br2, br3, bv1, cr, ct1, ct2, mi, na1, rd1, rd2,
and td (Coe et al., 1988). We are not aware that the response of all these mutants to
GA has been characterized. Although there are reports that br2, ctc1, and rd may
have yield advantage, especially in high-density plantings (Nelson and Ohlrogge,
1957, 1961), the use of these mutants in corn breeding has been limited (Sprague
and Dudley, 1988).
Of the 20 mutants described in wheat, 16 are sensitive to GA, representing 80%
of the total (Gale and Youssefian, 1985; Konzak, 1987). Rht4, Rht6, Rht7, Rht8,
Rht9, Rht11, and Rht17 are recessive genes; Rht12 is a strong dominant gene and
all the others are partial or semidominant genes. Rht8 and Rht9 have been used
in the European wheat varieties; Rht11 and Rht14 have had some use for durum
or bread wheat varieties; and Rht15, Rht16, Rht18, Rht19, and Rht20 have only
shown good potential but have not been extensively used (Gale and Youssefian,
1985; Konzak, 1987).
A list of the rye dwarfing genes has been described by Börner et al. (1996) and
comprises 11 mutants. Of these, Ddw1, Ddw2 (dominant genes), and d2 (recessive
gene) are responsive to GA. According to the authors, the best known short straw
mutant in rye, which has been included in many Eastern European rye breeding
programs, is the “EM1” mutant that carries the Ddw1 gene.
There are eight dwarfing genes officially described and classified in oat (Avena
sativa L.), but only Dw6, Dw7, and Dw8 are still readily available (Milach et al.,
1998). It is likely that the first five described dwarfing genes have lacked utility
due to the extreme dwarfness or meiotic irregularities of the lines that carry them
(Marshall and Murphy, 1981). Genetic studies have shown that Dw6 (Brown et al.,
1980) and Dw8 (Milach et al., 1998) are dominant genes and Dw7 is a partially
dominant (Marshall and Murphy, 1981). An allele of Dw7 was identified in the
short-stature cultivar ‘Curt’ (Federizzi and Qualset, 1989). All the three dwarfing
loci available in oat are responsive to GA and likely involved in the GA metabolism
(Milach, 1995). These genes have distinct effects on plant height components and
likely different mechanisms of action (Milach, 1995). A double dwarf effect has
been observed in crosses between lines with independent dwarfing genes (Milach
et al., 1998) and likely resulted from the combination of these different mechanisms
to reduce plant height.
A feature of GA-sensitive mutants is that most of them are recessive and deficient
for GA due to a block in the biosynthetic pathway. Recessive genes involve the
loss of wild-type function (Herskowitz, 1987). Interestingly, there are dominant or
semidominant genes in different species that are responsive to applied GA. This
is the case of Rht5, Rht12, Rht13, Rht14, Rht15, Rht16, Rht18, Rht19, and Rht20
of wheat; Dw6, Dw7, and Dw8 of oat; and Ddw1 and Ddw2 of rye, among others.
Herskowitz (1987) suggested that dominant negative mutations can also result in
loss of function and would include mutant polypeptides that when overexpressed
disrupt the activity of the wild-type gene or polypeptides with inhibitory effect. It is
possible that these genes are dominant mutations that regulate the GA metabolism.
While GA-sensitive mutants have played a fundamental role in understanding
the GA biosynthetic pathway, they have not had the same impact in terms of
breeding applications. The likely reason for that is because their phenotype is
usually more extreme than what is acceptable for plant height of a variety. In
cereals, varieties that are too short in height may have problems during harvest
and become even shorter in stress conditions. This is our experience in growing
dwarf oats in Southern Brazil where genes that reduce plant height to 50 to 70 cm
cannot be used because they usually end up producing genotypes that range from
35 to 50 cm across different environments.
B. GA-INSENSITIVE MUTANTS
Another class of GA mutants is of those that do not respond to the exogenous

application of GA. A fewer number of mutants have been identified that belong
to this class compared to the higher number for the GA-sensitive mutants. On
the other hand, some of the GA-insensitive mutants have been the widest used in
plant breeding, as is the case of the Rht1 and Rht2 genes from ‘Norin10’ in wheat
and sd1 from ‘Dee-geo-woo-gen’ in rice. These three genes have been the most
extensively used in developing the wheat and rice semidwarf varieties throughout
the world.
Other GA-insensitive mutants that have been extensively study but have not
had the same use for breeding are those that carry the d8 and d9 genes in maize
(Harberd and Freeling, 1989; Winkler and Freeling, 1994) and the gai gene in
Arabidopsis (Koornneeff et al., 1985).
In barley, GA-insensitive dominant mutants have been recently identified and
studied (Falk, 1994; Ivandic et al., 1999, Börner et al., 1999a). The Dwf 2 dominant
mutant allele has a strong effect on the phenotype and plants that carry it may be
too short for breeding purposes.
Recently, Börner and associates (1996) have proposed a new nomenclature for
the GA-insensitive Rht wheat alleles which takes into account the chromosome
location with the homoeologous set number and assigns a lowercase letter to the
allele present. According to this nomenclature the Rht1 = Rht-B1b, the Rht2 =
Rht-D1b, the Rht3 = Rht-B1c, and the Rht10 = Rht-D1c.
In wheat, there are two GA-insensitive loci that can carry different alleles, one
on chromosome 4B and the other on 4D (Börner et al., 1996). The alleles identified
for chromosome 4B are Rht-B1a (rht, tall allele), Rht-B1 (Rht1), Rht-B1c (Rht3),
Rht-B1d (Rht1S), Rht-B1e (RhtKrasnodari 1), and Rht-B1f (RhtT. aetiopicum).
The alleles identified for chromosome 4D are Rht-D1a (rht, tall allele), Rht-D1b
(Rht2), Rht-D1c (Rht10), and Rht-D1d (RhtAi-bian 1a). Because the names Rht1,
Rht2, and Rht3 are easier to be recognized by breeders, we use that nomenclature
in this Chapter.
Despite their importance, the role of these genes in decreasing plant height
has not been fully understood. It is well known that the wheat mutants do not
respond to the exogenous application of GA because they accumulate several
times more GA than their tall counterparts (Radley, 1970), which indicates that they
have a block in the utilization of GA (King, 1991). Swain and Olszewski (1996)
suggested that since GAs are required for normal internode elongation, all mutants
affected in stem growth by mechanisms other than altered GA metabolism can be
considered as potential GA signal-transduction mutants. They define GA signal
transduction as “the series of biochemical events leading from the perception of the
active GA molecule to the final response.” In a recent study, Peng et al. (1999a)
reported that Rht1, Rht2, and D8 are orthologs of the Arabidopsis gibberellin-
insensitive (GAI ) gene and encode proteins that resemble nuclear transcription
factors and contain an SH2-like domain, which indicates that they may participate
in gibberellin signaling.
C. PHYTOCHROME MUTANTS
There are studies in pea (Weller et al., 1994) and cucumber (Lopes-Juez et al.,
1995) which indicate that type-B phytochromes modify the response to GA. Other
evidences of the involvement of GAs and phytochromes come from studies with
transgenic plants. Tomato transgenic plants transformed with the oat phytochrome
A (phyA) gene are dwarf and dark green and accumulate anthocyanin (Boylan
and Quail, 1989). Transgenic tobacco plants with the rice phyA are dwarf and
dark green (Nagatani et al, 1991) and Arabidopsis plants with the oat phyA gene
have short-hypocotyl elongation (Boylan and Quail, 1991). Arabidopsis transgenic
plants have been produced that overexpress either the Arabidopsis or the rice
phytochrome B ( phyB) genes; they also have a dwarf phenotype (Wagner et al.,
1991). It appears that the overexpression of either phyA or phyB genes can cause
dwarfness in different species, suggesting that these genes may play an important
role in plant elongation.
In oat, we have observed in growth chamber and greenhouse environments
that seedlings of the ‘NC2469-3’ dwarf line with the Dw7 gene are dark green
and accumulate anthocyanin, similarly to what happens to some of the transgenic
phytochrome mutants. Because of the semidominant nature of the Dw7 gene, it
is possible that this mutation results from the overexpression of a gene that can
influence plant height, for instance one of the phytochrome genes. To investigate
this hypothesis, we mapped the oat phyA and the rice phyB structural genes on
the oat hexaploid RFLP map (O’Donoughue et al., 1995) and compared their
map locations to that of the Dw7 gene. In a previous study (Milach et al., 1997),
the Dw7 gene was mapped on linkage group 22 of the oat RFLP hexaploid map.
We mapped the oat phyA gene on linkage group 28 and the rice phyB gene on
the linkage group 30 of the hexaploid oat RFLP map (unpublished data). Because
Dw7 is independent from the mapped phytochrome loci, as are also Dw6 and
Dw8, it cannot be a mutation of these genes. In oat, three phytochromes have been
identified: two in green oat leaves, which differ from the phytochrome that is most
abundant in etiolated oat tissue (Wang et al., 1991). It is possible, however, that
there is homeology between Dw7 and phytochrome genes. Because the hexaploid
oat genetic map has more linkage groups than chromosomes and a chromosome
homeologous series in oat is not completely established, it is difficult to determine
these relationships at the moment. It is also possible that Dw7 is a mutation in a
phytochrome structural gene not investigated in this study or in a gene regulating
phytochrome production. A barley mutant, BMR-1, has been recently identified
which has a dwarf phenotype and contains higher levels of phytochromes A and B
in the dark (Hanumappa et al., 1999). Further study is needed to determine if Dw7
is a photomorphogenic mutant. On the other hand, it is interesting to note that the
wheat Rht1 and Rht2 and the maize D8 genes have been mapped closely to a PhyA
gene (Peng et al., 1999a). Swain and Olszewski (1996) pointed out that very little
has been done to investigate the interaction between phytochromes and GAs using
a genetic approach and that mutants involved in both GA and phytochrome signal
transduction pathways will help to elucidate these questions.
The potential of phytochrome mutants for breeding short-stature plant varieties
remains to be investigated.
III. DWARFING GENES AND THEIR USE

FOR BREEDING
Although several dwarfing genes have been identified and studied in different
species, only a few have had wide application for plant improvement (Table I).
The most universally accepted dwarfing genes have been the Rht1 and Rht2 of
wheat, probably because they have been spread throughout the world in the wheat
germplasm developed by CIMMYT and are present in around 90% of the world
acreage of semidwarf wheats (Dalrymple, 1986).
A similar situation occurred with the sd1 locus of rice that has been used by
IRRI in developing semidwarf germplasm that was sent to several countries of the
world. There have been at least 60 dwarfing genes identified in rice and a list of
them can be found in Futshura and Kikuchi (1997). Even though a large number of
semidwarf high-yielding rice varieties have been developed, semidwarfing genes
of practical importance have been limited only to the sd-1 locus (Futshura and
Kikuchi, 1997). Different alleles of this locus have been used by the American and
Asian countries (Rutger, 1983).
The reasons for the success of the Rht1, Rht2, and sd1 genes are probably due
to the great efforts that were made to transfer them to good genetic backgrounds
early when they were first identified and because they allowed the production
of varieties with semidwarf desirable plant height, combining height reduction
of about 20% with significant increases in spikelet fertility and yield (Gale and
Youssefian, 1985). Their positive pleiotropic effects on different traits made these
genes very attractive for breeding purposes.
Other Rht genes have been most used in Europe, such as Rht8 and Rht9 (Gale
and Youseffian, 1985). The same is the case of the Ddw1 gene of rye (Börner et al.,
1996).
Because the dwarf phenotype in oat is often accompanied by decreases in yield
and changes in internode and panicle length, the use of dwarfing genes has been
limited in oat breeding programs. Dw6 has been used for cultivar development in
Australia (Anderson and McLean, 1989) and in the United States (Marshall et al.,
1987; Meyers et al., 1985; and D. D. Stuthman, University of Minnesota, unpub-
lished observations), and Dw7a has been used to develop the short-stature cultivar
‘Curt.’ Although the Dw6 gene causes a failure of the panicle to fully emerge from
the leaf sheath, panicle exertion genes have been used to help correcting this prob-
lem for cultivar development (Farnham et al., 1990a). However, transferring both
Dw6 and panicle exertion genes to new oat lines is a difficult task, and seed size
often is reduced in the derived lines. The Dw8 gene causes an extreme phenotype
in a series of different genetic backgrounds, which is not desirable for oat breeding
(Milach et al., 1998). There is, therefore, an urgent need to identify and use new
sources of dwarfism in oat, which would produce a phenotype similar to that of
the Rht1 and Rht2 genes in wheat.
Table I
Most Important Dwarfing Genes Used in Cereal Improvement, Their Inheritance, Chromosome Location, and Marker Association
Response Chromosome
Species Gene Source to GAa Inheritance location Marker Linkage Key references
Avena sativa L. Dw6 ‘OT207’ S dominant 18 Xumn 145B (3.3 cM) Brown et al. (1980)
Milach et al. (1997)
Dw7 ‘NC2469-3’ S semidominant 19 Xcdo 1437B (4.3 cM) Marshall and Murphy (1981)
Milach et al. (1997)
Hordeum vulgare L. denso ‘Triumph’ S recessive 3HL WG110 (12.8 cM) Barua et al. (1993) and
OPH7-H800 (31.7 cM) Laurie et al. (1993)
GPert ‘Golden S recessive 5H — Thomas et al. (1984)
Promise’
Oryza sativa L. sd-1 ‘Dee-geo- I recessive 1 Xrg220 (0.3 cM) Cho et al. (1994)
woon-gen’
Secale cereale L. Ddw1 EM-1 S dominant 5RL Xwg199 and Hp1 (6.1 cM) Korzun et al. (1996)
␤-amy-R1 (12.7 cM) Borner et al. (1999)
Triticum aestivum L. Rht1 ‘Norin10’ I part.dominant 4BS Xprs144 (11.0 cM) Konzak (1987)
Xmwg634 (30 cM) Börner et al. (1997)
Rht2 ‘Norin10’ I part.dominant 4DS Xprs921 (0.8 cM) Konzak (1987)
Xmwg634 (1.5 cM) Börner et al. (1997)
Rht8 ‘Mara’, Sava’ S recessive 2DS — Konzak (1987)
Rht9 ‘Mara’ S recessive 7BS — Konzak (1987)
Rht12 Karcag522 S dominant 5AL ␤-amy-A1 (2.5 cM) Konzak (1987)
Xprs1201 (15 cM) Korzun et al. (1997)
Rht14 ‘Castelporziano’ S semidominant ? — Konzak (1987)
a
S, GA-sensitive; I, GA-insensitive.
In the environment of the South Cone of South America, the Dw6 or Dw7
genes produce oat plants that are approximately 77 cm in normal environmental
conditions, but that can be of 50 cm or less and have reduced biomass and yield
under stress conditions.
The use of one or two recessive genes from the Brazilian oat varieties ‘UFRGS 7’
and ‘UFRGS 15’ give an adequate plant height for preventing lodging in excellent
conditions (near 110 cm) and a plant height near 90 cm that will be enough for
producing acceptable grain yield in stress environments (Federizzi et al., 1996).
These recessive genes have been intensively used for developing Brazilian oat
varieties and are GA-sensitive (unpublished data).
A short-statured barley mutant stock containing the sdw gene was obtained
from Norway in 1957 and transferred to American germplasm (Rasmusson et al.,
1973). This gene has been widely used in breeding programs in the United States
and Canada (Rasmusson, 1991). The denso and Gpert (from ‘Gold Promisse’
cultivar) dwarfing genes are present in the European barley germplasm and have
been used for variety development (Ivandic et al., 1999).
Quinby and Karper (1954) found four recessive dwarfing genes, dw1, dw2,
dw3, and dw4, present in different combinations in sorghum varieties grown in
the United States. Pleiotropic effects of these genes (except for dw4) on yield and
yield components have been found (Casady, 1965; Graham and Lessman, 1966;
Blum et al., 1997), probably hindering their potential for breeding.
In foxtail millet (Setaria italica Beauv.), three new sources of dwarf germplasm
have been identified recently and characterized as GA-insensitive (Dineshkumar
et al., 1992). All three sources have a major recessive gene, but it is not mentioned
if these alleles are from different genes or belong to a common locus. The authors
suggested that the high-yielding nature and superior morphological frame of these
dwarfs make them ideal for breeding purposes.
IV. BREEDING CHALLENGES AND

VARIETIES DEVELOPED
A. BREEDING CHALLENGES
Although major dwarfing genes are easy to transfer by crosses, breeding semi-
dwarf varieties using these genes has not always been so straightforward. One of
the reasons for this is that not in all crops there are genes available that reduce plant
height and have positive pleiotropic effects on grain yield, as it is the case for some
of the wheat dwarfing genes. In sorghum (Windscheffel et al., 1973; Campbell
et al., 1975), pearl millet (Bidinger and Raju, 1990; Rai and Rao, 1991), and oat
(Meyers et al., 1985) dwarfing genes have generally negative effects on grain yield.
One way of avoiding this problem was first proposed by Law et al. (1978) with
the “tall-dwarf” model for bread wheats carrying dwarfing genes that did not have
positive effects on yield. In this model, the authors proposed to fix the dwarfing
genes in populations at an early stage, followed by positive selection for height in
subsequent generations. Testing this hypothesis in pearl millet, Bidinger and Raju
(1993) were able to produce short-stature hybrids with increased grain yield.
Another situation is that semidwarf varieties may fall below their potential in
terms of farm yields because of poor seedling establishment and low early vigor
that has been associated to the presence of the GA-insensitive genes (Allan, 1980;
Niklas and Paolillo, 1990). In the presence of these genes, cell elongation in ju-
venile leaf and stem tissue is reduced (Hoogendoorn et al., 1990), resulting in a
shorter coleoptile and a plant with decreased initial vigor (Richards, 1992). At-
tempting to minimize this problem, Rebetzke et al. (1999) studied the relationship
of plant height and coleoptile length in order to identify ways to breed shorter
Australian wheat varieties with longer coleoptiles. They found that for GA-
sensitive wheats, height and coleoptile length appeared to be largely under in-
dependent genetic control, which indicates that GA-sensitive Rht genes could be
used to select for short height and longer coleoptile wheats with improved estab-
lishment and seedling vigor. A similar problem identified in rice has also been
overcome with the development of semidwarf germplasm that can produce long
coleoptile (Dilday et al., 1990).
Wheat in Brazil is grown in two major macro environments; in soils with alu-
minum (above parallel 24◦ S) and in soils without aluminum (below parallel 24◦ S).
Since the early 1970s, wheat lines from CIMMYT have been introduced by dif-
ferent Brazilian breeding programs. It is interesting to observe that the varieties
with dwarfing genes have been extensively used only in soils without aluminum.
In the past 3 decade, few varieties carrying Rht genes were released in south-
ern Brazil. While the major gene for aluminum tolerance present in the Brazilian
wheat germplasm is also located on chromosome 4D (Lagos et al., 1991; Riede and
Anderson, 1996), there is genetic evidence that these two traits are independent. So,
the restricted use of the dwarf phenotype is probably due to the variation on plant
height and total biomass produced in soils with high levels of aluminum. This can
be illustrated by observing the plant height obtained for near isogenic lines from
the Brazilian variety ‘Maringá’ carrying Rht1, Rht2, or Rht1 + Rht2 genes grown
in two different environments. In a nonstress environment, without aluminum, the
average plant height is 115 cm for ‘Maringá,’ 93 cm for an isogenic line with Rht1,
and 65 cm for an isogenic line with Rht1 + Rht2 (Fantini et al., 1994). In contrast,
in soils with aluminum, 85 cm is observed for ‘Maringá,’ 74 cm for isogenic lines
with Rht1 or Rht2, and 54 cm for isogenic lines with Rht1 + Rht2 (Zanata and
Oerlecke, 1991). Rosa and Camargo (1991) demonstrated that tall wheat varieties
are more efficient than short varieties in the uptake of phosphorus in soils with
aluminum and low P availability.
In oats, in order to have lines with short plant height that are very competitive
in the yield regional trials, we have developed a screening strategy for segregating
populations with genes for short stature, selecting among F3 and F4 lines for high
vigor and apparent biomass at the six- to seven-leaf stages (Pacheco et al., 1997).
High frequency of tall off-types has been found in some semidwarf varieties, as
in the UK variety ‘Brigand,’ which carries the Rht2 gene. According to King and
collaborators (1996), this frequency was sufficiently high to cause the variety to fail
United Kingdom statutory uniformity tests. The authors prevented the appearance
of tall off-types in lines carrying a translocation involving the 4D chromosome,
where it is located the dwarfing gene.
B. VARIETIES DEVELOPED
The first semidwarf rice cultivar released in California was Calrose 76, in 1976.
In 1981, the seven cultivars with the Calrose 76 semidwarf gene were grown on
an estimated 54% of the 245,000 ha of rice in California (Rutger, 1983). Other
35% of the area was with the semidwarf M9, which carries an allele of sd1 derived
from IR8. In California, as well as in most of other parts of the world, none of
the semidwarf sources nonallelic to sd1 (and to the DGWG source) has become
important in breeding (Rutger, 1983; Futshura and Kikuchi, 1997).
A study conducted in 1985 by Worland (1986) indicated that 25% of the
European winter wheat varieties carried GA-insensitive dwarfing genes and that
their use would be limited to the areas not subjected to temperature stress at a
critical growth stage. By then, only 4 of 18 French varieties carried insensitive
dwarfing genes. In another study conducted 8 years later, the same author pointed
out that there had been a swing toward the acceptance of these genes in French
varieties because they found 10 of the 17 varieties carrying insensitive dwarfing
genes (Worland et al., 1994). A total of 49 GA-insensitive varieties were identified
among the 127 entries tested. Although the authors have not mentioned this, it is
possible that some of the remaining 78 GA-sensitive varieties would carry either
Rht8 or Rht9 dwarfing genes. They also found that all but 1 UK wheat variety car-
ried insensitive genes. On the other hand, only 11 of 49 Germany varieties tested in
their study were GA-insensitive. These data show that the use of dwarfing genes in
wheat varieties does change from one country to the other. The authors explained
that these differences are associated to the fact that in places where the varieties
are subjected to temperature stress during the critical growth stage of flag leaf to
ear emergence, as seems to be the case in Germany during the summer, there is not
much advantage of growing GA-insensitive wheat varieties. They argue that one
way of increasing the use of these genes in German varieties would be to combine
photoperiod-insensitive with dwarfing genes to escape the summer temperature
stress.
Pecetti and Annicchiarico (1998), studying the agronomic value and plant type
of four groups of Italian durum wheats, identified a significant decrease in plant
height from 132 to 83 cm on average in varieties from different eras. Although they
did not indicate which dwarfing genes were present in varieties of groups three and
four, in group 3 they included ‘Castelporziano,’ which according to Konzak (1987)
carries the Rht14 GA-sensitive dwarfing gene. Their group 4 includes varieties
developed from crosses between Italian and CIMMYT materials and it is possible
that some of these have GA-insensitive genes transferred from the CIMMYT bread
wheat program.
In Brazil, of 328 improved wheat varieties released from 1922 to 1992, 32 were
direct introductions from CIMMYT (Lagos, 1983; Sousa, 1994) carrying genes
for short stature grown by farmers in soils without aluminum. In contrast, for soils
with high levels of aluminum, only 4 varieties may have dwarfing genes. Many
crosses were made between Brazilian varieties and different European sources of
Rht8 and Rht9 but no varieties were released with these genes.
Among the semidwarf barley varieties released in North America are ‘Kombar,’
‘Kombyne,’ ‘Samson,’ ‘Duke,’ and ‘Micah’ (Rasmusson, 1991). Several other
released cultivars, tracing to composite populations, are also suspected to have the
sdw dwarfing gene.
V. PLEIOTROPIC EFFECTS OF DWARFING GENES
Dwarfing genes can have positive and negative pleiotropic effects in other plant
traits, probably because of their involvement with the GA biosynthetic or signal
transduction pathways, which is a growth regulator involved in many of the plant
physiological processes.
Gale and Youssefian (1985) pointed out that studies that provide reliable in-
formation about the pleiotropic effects of Rht genes are those that involve ei-
ther comparisons of groups of random lines derived by selfing single hybrids
or comparisons of isogenic lines in which the different alleles have been iso-
lated in a specified genetic background by backcrossing. Care in using the ap-
propriate genetic materials and in minimizing lodging of the tall genotypes is
fundamental in avoiding confounding effects that may cause misinterpretation of
results.
The pleiotropic effects of the GA-insensitive genes Rht1, Rht2, and Rht3
have been the most extensively studied and are reviewed elsewhere (Gale and
Youssefian, 1985; Gent and Kiyomoto, 1998). A summary of the main studies
carried out up to 1985 on the pleiotropic effects of Rht alleles on plant height,
yield, yield components, and grain protein is presented by Gale and Youssefian
(1985). A more recent review by Gent and Kiyomoto (1998) discuss the physio-
logical and agronomic consequences of Rht genes in wheat. Because this topic has
been well reviewed elsewhere, here we attempt to summarize some of the most
important pleiotropic effects on dwarfing genes that have consequences for plant
improvement.
A. MORPHOLOGICAL AND PHYSIOLOGICAL TRAITS
Most of the dwarfing genes appear to be active throughout the plant cycle. So,
the first effects of these genes can be seen at the first stages of plant development.
This is the case with the Rht3 gene, which causes a marked reduction in the rate
of ␣-amylase synthesis during germination (Flintham and Gale, 1982; Gale et al.,
1987). The positive consequence of this is on reducing preharvest sprouting dam-
age to bread-making quality in wheat. Unfortunately, because it causes extreme
dwarfism, this gene has had limited value in improving wheat for reduced pre-
harvest sprouting. An interesting feature of Rht3 is that it also inhibits ␣-amylase
synthesis during the latter part of grain ripening (Mrva and Mares, 1996), which
shows that this gene has constitutive expression in the plant. A less pronounced
effect on reducing the expression of late maturity ␣-amylase is found for Rht1 and
Rht2.
After germination, the effects of Rht genes in wheat and the sd1 dwarfing gene
of rice can be seen on decreasing the length of the coleoptile, which may reduce
stand establishment (Allan, 1980; Dilday et al., 1990; Niklas and Paolillo, 1990).
Subsequently there is a decrease in leaf and stem size (Lenton et al., 1987). Nilson
et al. (1957) were the first to demonstrate that reduced height of some semidwarf
cultivars of wheat was attributable to smaller cells. Alan et al. (1962), investigating
the causes for reduced coleoptile length in two selections with GA-insensitive
genes from ‘Norin10,’ concluded that it was related to fewer parenchyma cells,
while coleoptiles in a Rht3 genotype were shorter because of smaller cells. The
authors suggested that these two dwarfing genes operate in a qualitatively different
manner.
In barley, dwarf mutants are usually associated with fewer cells, but reduction
in cell size has also been noted (Blonstein and Gale, 1984).
The dwarf phenotype in oat has usually been associated with decreases in yield
and internode and panicle length (Brown et al., 1980; Marshall and Murphy, 1981;
Meyers et al., 1985). These drawbacks have probably caused limited use of oat
dwarfing genes in breeding programs. The effect of the Dw6 gene on the plant
height components of internode number and length and panicle length was studied
by comparing OT207 with its tall mother-line OT184 (Brown et al., 1980). The
authors described a significant difference between semidwarf and tall lines in the
length of the three uppermost internodes, particularly the peduncle. This has been
associated with the failure of the panicle to fully emerge from the leaf sheath,
an undesirable trait in oat breeding. Panicle exertion genes correct this problem
in lines carrying the Dw6 gene (Farnham et al., 1990a). In a more recent study
comparing oat dwarf lines carrying the Dw6, Dw7, or the Dw8 with they tall
counterparts, it was observed that the three oat dwarfing loci reduced plant height
in different ways (Milach, 1995). The Dw6 gene reduced primarily the length of
the three uppermost internodes and did not affect internode number. The Dw7 gene
shortened all internodes but particularly the first and the last, and reduced internode
number in the dwarf line. The Dw8 gene significantly shortened all internodes of
the Japanese lines but did not affect internode number. In oat, the lower internodes
are the first to differentiate and elongate. Internodes elongate basipetally from the
intercalary meristem, a typical pattern of elongation in grasses (Kaufman et al.,
1965). Although the Dw6 gene is influencing the elongation of the first internodes
to some extent, the major effect of this gene appeared to be later in development.
This hypothesis is supported by the findings of Farnham et al. (1990b), who were
able to reverse to a great extent the effect of the Dw6 gene on peduncle elongation
by applying GA3 to the dwarf plants at the boot stage. According to Kaufman
and Brook (1992), oat internodes elongate in a sequential fashion: As the previous
internode ceases elongating, the next enters its burst of growth. Because of this
growth pattern, it is possible that the effect of Dw6 is specific to the elongation of
the upper internodes.
B. YIELD AND YIELD COMPONENTS
Many reports indicate that the Rht1 and Rht2 wheat genes have a positive effect
on yield (Gale and Youssefian, 1985; Fischer and Quail, 1990; Börner et al.,
1993). One of the main reasons for this yield advantage appears to be a pleiotropic
positive effect of these genes on spikelet fertility (Gale and Youssefian, 1985), due
to increased kernel number (Fischer and Stockman, 1986). This occurs despite
the fact that dwarf plants have reduced lamina elongation from smaller epidermal
cells (Keyes et al., 1989). There are reports that indicate that the reduction in
leaf cell sizes concentrates the leaf photosynthetic machinery, thereby increasing
photosynthetic capacity per unit leaf area or leaf weight (LeCain et al., 1989;
Morgan et al., 1990). An increase on radiation use efficiency was observed during
the postanthesis in Rht lines and, according to Miralles and Slafer (1997), this
appears to be closely and positively associated with the number of grains per unit
biomass at anthesis. It has been observed that the increase in grain number per
spike is accompanied by a decrease in kernel weight (Brandle and Knott, 1986;
Nizam Uddin and Marshall, 1989), although this is not sufficient to decrease the
yield advantage of the dwarf Rht lines.
VI. MOLECULAR MAPPING OF DWARFING GENES
Classic genetics and cytogenetics were extensively used in the 1970s to place
dwarfing genes on chromosomes and linkage groups. Because these genes have
a major effect on the phenotype and are highly heritable, placing them on chro-
mosomes and linkage groups had a double advantage toward understanding their
genetic relationships as well as using them as morphological markers to map other
traits. So, Rht1 and Rht3 genes (Gale and Marshall, 1976) were placed on former
chromosome 4A, and Rht2 was placed on chromosome 4D (Gale et al., 1975).
Years later, due to a change on the wheat genome nomenclature, 4A became chro-
mosome 4B. Crosses between Rht1 and Rht3 varieties revealed that these were two
alleles of the same locus, confirming their assignment to the same chromosome.
In rice, several dwarfing genes have been placed to chromosomes since the 1960s
(Hsiehh and Yen, 1966; Iwata and Omura, 1973). Barley GPert dwarfing gene was
placed on chromosome 5H through a combination of morphological markers and
translocation stocks (Thomas et al., 1984). Using trisomic analysis, Sturm and
Engel (1980) located rye dwarfing gene Ddw1 on chromosome 2, which corre-
sponds to chromosome 5R.
The advent of molecular markers on the 1980s made possible the construction
of more detailed linkage groups and genetic maps, making easier the association of
important genes to genetic markers. Thus, a number of important dwarfing genes
from different species were associated to molecular markers (Table I) and all this
information today allow us to study the genetic relationships of different mutants
within and across the species.
The sd-1 semidwarfing gene was mapped on rice chromosome 1 (Cho et al.,
1994). Huang et al. (1996) present a list of 13 other rice dwarfing genes, their
linkage to markers, and chromosome position. Except for chromosomes 7, 8 , 9,
and 10, 1 or more dwarfing genes have been found in all other rice chromosomes.
The GA-insensitive Rht-B1 and Rht-D1 wheat dwarfing loci were mapped in
three F2 populations segregating for Rh-B1c (Rht3), Rht-D1b (Rht2), or Rht-D1c
(Rht10) (Börner et al., 1997). Rh-B1c was found linked distally to the RFLP
markers Xprs144 (11.9 cM) and Xprs584 (17.8 cM) and proximal to Xmwg634
(30 cM) on chromosome 4BS in the centromere region. Rht-D1c was closely linked
to the distally located markers Xprs921 (0.8 cM) and Xmwg634 (1.5 cM). Sourdille
and associates (1998), mapping the same loci independently, found similar results.
Korzun et al. (1997) mapped Rht12 on chromosome 5AL with a genetic distance
of 15 cM to marker Xprs1201.
The first dwarfing gene mapped in barley was denso, which was located on
the long arm of chromosome 3HL independently by two research groups (Barua
et al., 1993; Laurie et al., 1993). The Dwf 2 gibberellic acid insensitivity dwarf-
ing gene was mapped recently (Ivandic et al., 1999) on the short arm of barley
chromosome 4H proximal to microsatellite XhvOle (5.7 cM) and distal to RFLP

marker Xmwg2299 (18.3 cM). This gene was found to be at a homoeologous po-
sition to the multiallelic Rht-B1 and Rht-D1 loci in wheat, suggesting that there is
syntheny between these GA-insensitive dwarfing genes within the Triticeae. Other
two genes, gai (GA-insensitive) and gal (GA-less sensitive) were mapped on the
centromere region and on the long arm, respectively, of the barley chromosome
2H (Börner et al., 1999a).
Three dwarfing genes have been associated to molecular markers in rye. These
are ctc2 linked to Xembp (5 cM) and Xprs1194 (13 cM) and located on the chromo-
some 5RL (Plaschke et al., 1993); ctc1 mapped close to the centromere between
markers Xprs163 (1 cM) on the short arm of chromosome 7R and Xa-Amy-2 on
the long arm of 7R (3 cM) (Plaschke et al., 1995); and Ddw1, which is distal to
Hp/Xwg199 (5.6 cM) and proximal to the isozyme marker ␤-amy-A1 (11.5 cM)
on chromosome 5RL (Korzun et al., 1996).
Using bulked segregant analysis (BSA) and F2 RFLP linkage data, three domi-
nant oat dwarfing loci were mapped to different regions of the oat genome. Dw6,
in oat line OT207, was 3.3 ± 1.3 cM from the Xumn145B locus, which has not
been placed on the hexaploid oat map. Dw7, in line NC2469-3, was 4.3 ± 2.3 cM
from Xcdo1437B and 33 ± 4.1 cM from Xcdo708B. This placed Dw7 in linkage
group 22. Dw8, in the Japanese lines AV17/3/10 and AV18/2/4, mapped 4.9 ± 2.2
cM from Xcdo1319A in an AV17/3/10 × Kanota F2 population and 6.6 ± 2.6 cM
from it in an AV18/2/4 × Kanota population. This placed Dw8 in linkage group 3.
Aneuploid analysis of markers linked to the dwarfing genes located Dw6 on the
smallest oat chromosome (chromosome 18) and Dw7 on the longest satellited
chromosome (chromosome 19) (Milach et al., 1997).
A. ASSOCIATION TO QUANTITATIVE TRAIT LOCI FOR PLANT

HEIGHT AND LODGING RESISTANCE
The location of major genes and quantitative trait loci (QTLs) on linkage maps
using DNA technology provides insights about the association between qualitative
and quantitative gene loci. Robertson (1985) suggested that alleles with qualitative
effects may represent the extreme of a spectrum of possible alleles at a locus.
Paterson et al. (1995) noted the existence of both qualitative and quantitative
loci at corresponding map positions among several of the cereals for traits that
have been involved in the domestication of these species for grain production.
The identification and location of qualitative and quantitative trait loci on DNA
linkage maps is important for better understanding the basis of quantitative trait
inheritance and searching for genomic regions of interest in crop breeding.
There are several examples of QTLs for height that have been mapped on close
proximity to major dwarfing genes. Of the three QTLs identified for height in
a double haploid population from a cross between two spring barley varieties
(Blenheim × Kym), that with largest effect was mapped on the region containing
the denso dwarfing gene (Bezant et al., 1996). The other two were in regions
previously identified as carrying the Sh and Sh2 vernalization response genes.
A total of 23 QTLs were located in all 12 rice chromosomes on RFLP maps
of a total of five mapping populations, and 8 of these QTLs were shared by at
least two populations (Huang et al., 1996). The position of the 23 mapped QTLs
were then compared to that of 13 major dwarfing genes previously linked to RFLP
markers. The authors found that the 13 dwarfing genes were in close proxim-
ity to the QTLs, providing evidence to support Robertson’s (1985) hypothesis.
Similar results were obtained for sorghum, where four QTLs detected for plant
height corresponded to map positions of dw1, dw2, dw3, and dw4 dwarfing genes
(Pereira and Lee, 1995). The evidence of a major genes–QTL association became
stronger comparing the sorghum and maize data for plant height because (a) three
QTLs mapped in sorghum were correspondent to QTLs in maize for plant height,
(b) at the QTL positions on chromosome 1 of maize is the br1 dwarfing gene and
at A of sorghum is the dw3 dwarfing gene, (c) the maize chromosome 9 d3–QTL
region (Beavis et al., 1991) corresponded to the sorghum chromosome H dw2–
QTL region, and (d) the maize chromosome 6 Py1–QTL region (Veldboom et al.,
1994) corresponded to the sorghum chromosome E dw4–QTL region (Pereira and
Lee, 1995). Thus, across three regions and two different species QTLs and major
dwarfing genes mapped coincidentally to the same approximate positions.
A QTL for plant height was mapped in rye close to the Ddw1 dwarfing gene
(Börner et al., 1999b).
In oat, the location of Dw7 on linkage group 22 of the hexaploid oat RFLP
map coincides with a major QTL for plant height detected in the Kanota × Ogle
mapping population (Siripoonwiwat et al., 1996).
QTLs for lodging resistance and plant height were identified in a segregating
wheat × spelt population (Keller et al., 1999). The authors indicate that a QTL for
plant height located on close proximity to markers linked to the Rht12 locus is
strong evidence for an allelic relationship between QTL and dwarfing locus.
Thus the locations of major dwarfing loci on DNA linkage maps appear to be a
starting point for the identification of genomic regions which control plant height
in different species.
Another interesting association reported has been that found between dwarfing
and rust resistance genes. Knott (1989) found that the dominant dwarfing gene
present in the wheat cultivar Webster is closely linked to the Sr30 stem rust re-
sistance gene located on chromosome 5D. Resistance to Septoria tritici Blotch in
winter wheat is associated with the presence of the dwarfing gene Rht2 (Baltazar
et al., 1990). In oat, marker Xumn145B, linked to the Dw6 dwarfing gene, appears
to be on an important region of the oat genome that contains several important
and interesting genes, such as Pc 91 (Rooney et al., 1994a), which is a complex
locus of various rust resistance genes (B.-C. Wu, personal communication). The
implication of these results for oat breeding is that the dwarfing gene might be used
as a morphological marker to select lines for rust resistance. However, a 3-point
genetic analysis still needs to be carried out to determine the genetic order and
the distance among Xumn145B, Dw6, and Pc 91. Also, as for Dw6, Dw7 on the
hexaploid oat map is located near a disease resistance gene: Pg13, a stem rust
resistance gene located in linkage group 22 by O’Donoughue et al. (1996).
B. COMPARATIVE MAPPING TO IDENTIFY ORTHOLOGOUS

DWARFING GENES
Molecular linkage maps are available for many agronomically important species.
In the case of cereal crops, a detailed comparison of maps using common markers
shows that there is a extensive collinearity among species within Triticeae. As
several dwarfing genes have been placed on the cereal maps, it is possible to study
their homeology.
Comparative mapping for the known GA-insensitive dwarfing genes of wheat,
rye, and barley has been done recently by Börner and associates (1998). They
concluded that GA-insensitive dwarfing genes are unrelated across species and
belong to four different homeologous groups (group 4 of wheat, groups 5 and 7 of
rye, and group 2 of barley). On the other hand, they found that the GA-sensitive
genes Rh12 of wheat and Ddw1 of rye are members of a homeologous series within
Triticeae. In a subsequent study, however, the Dwf2 barley GA-insensitive gene
was found to be homeologous to Rht-B1c and Rht-D1c of wheat (Ivandic et al.,
1999).
Saghai Maroof et al. (1996) noted the potential orthology of the rice sd-1 and
the barley sdw-b genes, based on their common comparative map positions, and
Van Deynze et al. (1995a) noted putative orthologous loci for dwarfing located on
homeologous chromosomes Triticeae 2L, rice 4, and maize 2 or 10.
There is currently no evidence for homology among the oat chromosome re-
gions carrying the three dominant dwarfing genes nor of these regions with dwarf-
ing genes in wheat and rye (Van Deynze et al., 1995a; Börner et al., 1996), but
homeology is difficult to determine in hexaploid oat, as the species is characterized
by numerous chromosomal rearrangements including translocations, duplications,
and inversions (Rooney et al., 1994b; O’Donoughue et al., 1995; Van Deynze et al.,
1995b; Leggett and Markhand, 1995).
C. MAPPING AS A BASIS FOR CLONING
All the genetic and biochemical information available for various dwarfing genes
make them very good candidates for cloning and sequencing.
One of the first dwarfing genes to be cloned and characterized was gai of
Arabidopsis thaliana (Peng et al., 1997, 1999b). Sequencing this gene has revealed
that the gai allele encodes a mutant protein lacking 17 amino acids from near the
terminus that is thought to confer the altered gibberellin responses characteristic
of the gai mutant (dwarf). The wild-type allele, GAI, encodes a protein containing
features that are characteristic of transcription factors. Based on this information,
Peng and associates (1999a) searched the database and found a rice expressed-
sequence tag (EST; D39460) that encoded a potential polypeptide containing a
sequence nearly identical to the 17 amino acids lacking in the gai mutant allele.
Using this EST they were able to isolate a wheat complementary DNA C15-1 that
ended up being a product of one of the Rht homoalleles. Mappings of C15-1 place
it on wheat chromosome 4B on the region that is homeologous to wheat chromo-
some 4D (where Rht-D1b is located), rice chromosome 3, and maize chromosome
1 (where D8 is located). This is strong evidence that C15-1 is in fact associated
with the GA-insensitive phenotype in all these species, a nice example of how
mapping can be used to support the cloning and characterization of these genes.
C15-1 was then used to isolate genomic DNA clones containing the putative
Rht-B1a and Rht-D1a (the RhtB1 and RhtD1 wild-type alleles) and maize d8 (the
D8 wild-type allele) genes. Comparative sequencing of all these genes revealed an
58% similarity between both Rht-D1a and d8 and GAI and a very similar carboxy-
terminal region in all of them (Peng et al., 1999a). An SH2-like domain was
identified within the C-terminal region, which acts as a transcriptional regulator.
The authors concluded that these dwarfing GA-insensitive genes encode proteins
that resemble nuclear transcription factors and contain an SH2-like domain, in-
dicating that phosphotyrosine may participate in gibberellin signaling. Using the
mutant GAI allele (dwarf) they were able to produce transgenic rice plants with
reduced response to gibberellin and dwarf phenotype. While this has been a great
contribution to our understanding of the GA-insensitive mutants, it remains to be
shown if transferring these genes will increase yield in a range of other crop species.
VII. CONCLUDING REMARKS
In this Chapter we attempted to summarize the main aspects of biochemistry,

genetics, physiology, agronomy, plant breeding, and molecular biology that have
contributed to our understanding of the most important dwarfing genes available
for plant improvement. The research conducted with different mutants of these
genes has had a great contribution toward both the basic and applied areas and it
is a model of how to integrate basic and practical knowledge in agriculture.
Genetics has allowed us to understand the inheritance and biochemistry of
the defective enzymes in some of the dwarf mutants. The consequence was the
elucidation of the GA biosynthetic pathway for different species. Physiology inves-

tigated the many important positive and negative effects of the dwarfing genes for
plant height and other traits. The breeding experience from different crops showed
us that only a few dwarfing genes have had a real impact for plant improvement,
which has not occurred in all plant species. Molecular biology allowed us to clone
and sequence some of these genes and to better understand the possible action of
GA-insensitive genes in the GA signaling transduction pathway.
All this knowledge will be fundamental to our next step in using the dwarfing
genes for plant improvement. This will provide the possibility of transferring them
through the use of transgenic technology to species where valuable mutants for
breeding have not yet being identified.
Although there is a great expectation that these genes will have positive effects
for plant height and other traits in a range of plant species, these will have to be
investigated through the interaction of all the research areas that have been so
fundamental to our current knowledge of the plant dwarf phenotype.
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A REVIEW OF THE EFFECT OF N
FERTILIZER TYPE ON GASEOUS EMISSIONS
Roland Harrison1 and J. Webb2
1
ADAS Consulting Ltd.
ADAS Boxworth
Boxworth, Cambridge CB3 8NN, United Kingdom
2
ADAS Consulting Ltd.
ADAS Wolverhampton
Wolverhampton WV6 8TQ, United Kingdom
I. Introduction
A. The Need for Abatement of Emissions of Nitrogenous Gases from
Agriculture
B. Previous Reviews of the Effect of N Fertilizer Type on Ammonia
Emissions
II. The Processes Controlling Emissions of Nitrogen Gases from Fertilizers
A. Ammonia Volatilization
B. Nitrous Oxide and Nitric Oxide Emissions
III. Measurements of Ammonia Emission Following Nitrogen Fertilizer
Application
A. Field Measurements of Ammonia Emission
B. Agronomic Evaluations: Effects on Fertilizer Efficiency
C. Laboratory Measurements
D. Emissions from Fertilizer Solutions
E. Urease Inhibitors
IV. Ammonia Emission Factors for Nitrogen Fertilizers
V. Measurements of Nitrous Oxide Emissions Following Nitrogen Fertilizer
Applications
A. Comparative Studies of Emissions
B. The Effect of Nitrification Inhibitors on Nitrous Oxide Emissions
VI. Nitrous Oxide Emission Factors for Nitrogen Fertilizers
VII. Nitric Oxide Emissions from Nitrogen Fertilizers
VIII. Summary and Conclusions
A. Ammonia
B. Nitrous Oxide
C. Nitric Oxide
D. Overall
References
65
Copyright
0065-2113/01 $35.00
66 HARRISON AND WEBB
Between 10 and 20% of the N in fertilizers applied as urea is lost to the atmo-
sphere as ammonia (NH3). In contrast only small (<4% of N applied) emissions of
NH3 have been measured following the application of ammonium nitrate (AN) fer-
tilizer. In consequence the replacement of urea fertilizer with AN has been proposed
as a cost-effective measure to reduce NH3 emissions in Europe. However, because
of the greater susceptibility of nitrate- (NO− 3 ) based fertilizers to denitrification,
the replacement of urea by AN may lead to increased emissions of nitrous oxide
(N2O). There was a need therefore to critically review the evidence for substan-
tially greater emissions of NH3 from urea than from other N fertilizers and also to
appraise the effect of fertilizer - N type on emissions of N2O. Ammonia emissions
from N fertilizers are consistent with their known effects on soil chemistry. Those
that increase soil solution pH, for example, by increasing HCO− 3 concentration or
by reducing the concentration of Ca2+ , have the greatest potential for NH3 emis-
sion. In consequence the greatest emissions of NH3 are from urea applied to any
soil and from ammonium sulfate (AS) applied to soils of pHs >7.0. Losses of NH3
from AN were confirmed to be consistently less than from urea. Emissions of NH3
from solutions composed of urea and AN were found to be intermediate between
the two fertilizers. Thus applying urea in solution will not reduce NH3 emissions.
However, NH3 emissions from urea may be reduced by the use of urease inhibitors.
Nitrous oxide emissions are crucially dependent on the interaction between tim-
ing of N fertilizer application and weather. Conditions in spring are more likely
to be wet so that emissions are greater from NO− 3 -based fertilizers than from AS.
In the summer conditions may be dry or wet; under dry conditions emissions are
usually smaller than under wet conditions. For urea the effect of pH appears to
be important. Generally greater emissions can take place from urea, except where
temperature (controlling the rate of urea hydrolysis) and rainfall (controlling the
dispersion of alkalinity) limit this. Thus, the substitution of AN for urea for spring
applications is likely to increase emissions of N2O. For summer applications, the
substitution of AN for urea is likely to decrease N2O emissions providing condi-
tions are relatively dry; when conditions are wet large emissions may occur from
both AN and urea. At this stage it is difficult to say with any certainty whether
a strategy based on urea or AN would result in the smaller N2O emissions. Ni-
tric oxide (NO) may also be released from soils following N fertilizer application.
While soil emissions of NO are small in comparison with other sources of NOx, it
is worth considering the effect of fertilizer type on this gas as well. Insufficient data
is available to predict the effect of urea substitution on NO emissions, but since
these are mainly a consequence of nitrification then replacing urea with AN should
also reduce NO emissions. C 2001 Academic Press.

EFFECT OF N FERTILIZER TYPE ON GASEOUS EMISSIONS 67
I. INTRODUCTION
A. THE NEED FOR ABATEMENT OF EMISSIONS OF NITROGENOUS

GASES FROM AGRICULTURE
Agriculture is the source of a number of gases that pollute the environment.

Ammonia (NH3) causes acidification and eutrophication when deposited to soils
and waters (Roelofs et al., 1992), with indications (e.g., Hettelingh et al., 1992)
that even if maximum feasible reductions in sulfur dioxide (SO2) and oxides
of nitrogen (NOx) are achieved, deposition of ammonium species (NHx) would
exceed critical loads for acidity in some parts of Europe. In consequence reduc-
tions in NH3 emissions are to be included in the forthcoming “multipollutant mul-
tieffects” protocol agreed on by the United Nations Economic Commission for
Europe (UNECE) Convention on Long-Range Transboundary Air Pollution (Bull
and Sutton, 1998). Agriculture is responsible for about 80–90% of NH3 emissions
(Anonymous, 1994), of which about 10–20% is from N fertilizers (Anonymous
1994). While NH3 emissions from N fertilizers such as ammonium nitrate (AN) are
considered to be small (∼1–3%), those from urea are estimated to be much greater
at ∼10–20% of total N application (Anonymous, 1994) and has been estimated to
contribute 50% of NH3 emissions from fertilizers in western Europe (Anonymous,
1994). Reduction of NH3 emissions from fertilizers could therefore be achieved
by using alternative N fertilizers with smaller N emissions. Cost–benefit analy-
sis of means to reduce NH3 emissions by European agriculture has identified the
replacement of urea by AN as one of the most cost-effective measures (Cowell
and ApSimon, 1998). However, the replacement of urea by AN has implications
for the emission of other N gases, since losses of nitrous oxide (N2O) have been
considered greater from AN than from urea.
Nitrous oxide contributes to global warming (Bouwman, 1990) and to depletion
of ozone in the stratosphere (Crutzen, 1981). Mosier et al. (1998) did not consider
there to be sufficient evidence to discriminate between N fertilizer types as sources
of N2O. Following application to soils N fertilizers may also lead to emissions of
nitric oxide (NO) which contributes to acid deposition (Logan, 1983) and to ozone
formation in the troposphere (Crutzen, 1979). Estimates of NO emissions from soil
are very uncertain. Stöhl et al. (1996) estimated soil emissions to be ∼7% of total
European NOx emissions; Simpson et al. (1999) presented a range of estimates
using different methodologies of between 2 and 20% of European NOx emissions.
In all cases soil NO emissions are considered to be largely of agricultural origin.
It was therefore considered necessary to critically review once again the evidence
for substantially greater emissions of NH3 from urea than from other fertilizers
and also of the effect of replacing urea with AN on emissions of N2O and NO.
This chapter focuses on N fertilizers and cropping systems relevant to the UNECE
area, viz. Europe and North America. Losses of NH3 following application of
N fertilizer to flooded rice fields were considered to be potentially greater than
from other cropping systems by Fenn and Hossner (1985), and large losses of
N from denitrification have also been measured. However, rice cultivation is not
extensive in the UNECE area and hence N losses from it are not dealt with in this
chapter. Ammonium bicarbonate fertilizer has also been omitted from the review.
This fertilizer is the main source of mineral N in China and gaseous N losses
following its application may be considerable (e.g., Cai et al., 1998; Hou et al.,
1998). However, this product is little used in the UNECE area.
B. PREVIOUS REVIEWS OF THE EFFECT OF N FERTILIZER TYPE

ON AMMONIA EMISSIONS
The effect of N fertilizer type on emissions of NH3 has been discussed by sev-
eral authors (e.g., Anonymous, 1994; Fenn and Hossner, 1985; Fox et al., 1996;
Sutton et al., 1994). In a review of NH3 volatilization from ammonium (NH+ 4 ) or
NH+ 4 -forming N fertilizers, Fenn and Hossner (1985) focused on possible future
trends for reduction in NH3 losses from surface-applied N. These authors indi-
cated that the influence of fertilizer type on NH3 loss was essentially controlled by
physicochemical volatilization reactions and the N fertilizers which are most sus-
ceptible to NH3 loss are those which produce ammonium carbonate (NH4)2CO3)
when added to the soil system, although some loss occurs from AN (NH4NO3)
in calcareous soils. Both ammonium sulfate ((NH4)2SO4) and diammonium phos-
phate ((NH4)2HPO4) react with calcium carbonate (CaCO3) to produce an increase
in soil solution pH and NH3 loss. They concluded that inorganic fertilizers are not
susceptible to gaseous NH3 loss in acid soils, but that large losses can occur from
urea in all soils.
Byrnes (1990) stated that there had been no in-depth global estimates of NH3
volatilization based on types of N fertilizers or their uses, although a reasonable
database exists to allow this. However, a recent collation of work in western Europe
has been carried out (Anonymous, 1994). ApSimon (1993), in summarizing the
information from a workshop on atmospheric emissions of NH3 and their control,
stated that nearly all the measurements on fertilizer loss apply to conditions in
northwestern Europe and that there was virtually no data for the wide variety of
other climatic and soil conditions and associated agricultural practices in Europe.
As emissions following fertilizer application vary greatly with soil type and pH
as well as the type of fertilizer used, the ECETOC inventory (Anonymous, 1994)
ascribed emission factors for different fertilizers in different countries according
to predominant soil categories.
Fenn and Hossner (1985) also stated that the magnitude of measured NH3
losses depended to some extent on the method of measurement (i.e., laboratory,
greenhouse, and field), with greatest values obtained under optimum conditions
in laboratory experiments. However similar values could be obtained in the field
under favourable circumstances, although this was less likely for urea because of
the requirement that urease activity be optimal. Thus laboratory studies are likely
to overestimate urea losses, both absolutely and in relation to other fertilizers.
Other workers, comparing three field measurement techniques of NH3 volatiliza-
tion from urea granules broadcast to pasture (Black et al., 1985a), found that de-
spite differences in the measured patterns of emission, the total cumulative losses
of NH3 determined by an enclosure method and the micrometeorological mass
balance method (i.e., confined versus unconfined methods) were similar. They
hypothesized that under conditions where the loss is complete within a short time
(i.e., less than 6 days), the total loss depended mainly on the NH+ 4 concentration
developed at the soil surface and the pool of bicarbonate ions (HCO− 3 ) inducing
the pH rise. The loss may be expected to continue until the pH drops sufficiently
to minimize the proportion of NH3 relative to NH+ 4 . Factors such as temperature
and windspeed, which may be expected to vary between the two methods, will
alter the rate of movement of NH3 into the atmosphere, but the total loss will not
change. Ammonia volatilization reactions are elaborated below.
II. THE PROCESSES CONTROLLING EMISSIONS

OF NITROGEN GASES FROM FERTILIZERS
A. AMMONIA VOLATILIZATION
1. Volatilization Reactions
There have been many investigations of the volatilization process (Black et al.,
1985a; Fenn, 1988; Fenn and Kissel, 1973, 1975; Fenn et al., 1981b; Larsen and
Gunary, 1962; Lightner et al., 1990; Meyer and Jarvis, 1989; Roelcke et al., 1996;
Sherlock and Goh, 1985; Sommer and Ersbøll, 1996; Terman, 1979; Touchton
and Hargrove, 1982), which is now well understood. Volatilization is essentially a
physicochemical process which results from the equilibrium (described by Henry’s
law) between gaseous phase NH3 and NH3 in solution [Eq. (1)]; NH3 in solution
is in turn maintained by the NH+4 –NH3 equilibrium [Eq. (2)] as follows:
NH3 (aq) ⇔ NH3 (g) (1)

NH+
4 (aq)
+
⇔ NH3 (aq) + H (aq). (2)
High pH (i.e., low [H+ (aq)]) favors the right-hand side of Eq. (2), resulting in a
greater concentration of NH3 in solution and also, therefore, in the gaseous phase.
Thus, where the soil is buffered at pH values less than ∼7(in water), the dominant
form of ammoniacal-N (NHx) will be NH+ 4 and the potential for volatilization will
be small. In contrast, where the soil is buffered at higher pH values, the dominant
form of NHx will be NH3 and the potential for volatilization will be high, although
other chemical equilibria may serve to increase or decrease this.
The ambient soil pH results in the establishment of a bicarbonate–carbonate
equilibrium with dissolved carbon dioxide (CO2) as follows:
CO2 (aq,g) ⇔ H2 CO3 (aq) ⇔ HCO− 3 (aq)

+ H+ (aq) ⇔ CO2−3 (aq) + 2H +
(aq). (3)
In acid soils, this equilibrium lies to the left so that the concentration of free
carbonate (CO2−3 ) ions is negligible. However, in alkaline (calcareous) soils, the
CaCO3 solubility equilibrium also becomes important:
Ca2+ (aq) + CO2−

3 (aq) ⇔ CaCO3 (s) (4)
It is apparent that the addition of soluble Ca2+ will move this equilibrium [Eq.
(4)] to the right, reducing the concentration of CO2−3 in solution, thus generating
additional H+ ions (i.e., reducing the pH) via equilibrium [Eq. (3)]. Further, the
addition of any other ion which forms sparingly soluble salts with Ca2+ (e.g.,
sulfate) will act in the opposite manner by reducing [Ca2+ ] and hence increasing
[CO2−3 ] [Eq. (4)]. This will move the equilibrium [Eq. (3)] to the left and reduce
[H+ ] (i.e., increase pH). Similar considerations apply to systems buffered by,
say, magnesium carbonate (MgCO3) or barium carbonate (BaCO3). Equations
relating NH3 loss potential to equilibrium constants and the concentrations of
Ca2+ and NH+ 4 were given by Larsen and Gunary (1962). They described laboratory
experiments carried out to investigate NH3 volatilization from NH+ 4 salts applied to
a calcareous soil and an acid soil amended with Mg-,Ca-, or BaCO3. The ranking of
NH3 volatilization potential from the calcareous soil was as follows: ammonium
sulfate (AS) > monoammonium phosphate (MAP) ∼ = diammonium phosphate
(DAP) ∼ = AN > magnesium ammonium phosphate. On the basis of solubility
criteria it was expected that, for calcareous soils, NH3 loss should have followed
the order DAP > AS > AN. It was postulated that the formation of insoluble
calcium ammonium phosphates reduced the potential for NH3 volatilization by
reducing the activity of NH+ 4 in solution. The results for the acid soil were as
follows: MgCO3 amended {AS > AN ≫ DAP}, CaCO3 amended {AS ≫ AN =
DAP}, and BaCO3 amended {AN ≫ AS = DAP}. Small losses were obtained
from DAP in MgCO3-treated soil because of the low solubility of the magnesium
ammonium phosphate product (also observed when this material was added to
the calcareous soil, above). This means that the expected order would be AS >
AN {Ca : = DAP} {Mg : > DAP}. It was suggested that the small losses from AS
from the BaCO3-treated soil were due to precipitation of BaSO4 on the surfaces of
BaCO3 particles, thus reducing the activity of the latter. No reason was given for
the greater N loss from AN compared to DAP in the BaCO3-treated soil.
Fenn and Kissel (1973) measured average NH3 losses after 100 h from NH+ 4
salts applied to Houston Black clay (a calcareous montmorillonitic soil, pH 7.6)
and Wilson clay loam (a neutral to acid montmorillonitic soil). The emissions
(NH3-N as a percentage of NH+ 4 -N applied) from the calcareous soil were as
follows: ammonium fluoride (NH4F), 68%; ammonium chloride (NH4Cl), 18%;
ammonium iodide (NH4I), 16%; AN, 18%; AS, 54%; and DAP, 51%. Thus, larger
emissions were measured where the Ksp (i.e., the solubility product constant) of
the salt formed from Ca2+ and the anion associated with the NH+ 4 compound was
low (i.e., sparingly soluble) and less where the equivalent salt was very soluble.
Volatilization of NH3 from AS added to Wilson clay loam soils saturated with
Mg2+ or Ba2+ (10% by weight of the respective carbonates added) was similar—
21 and 33% respectively. The initial pH of the Mg2+ system was 8.5 compared to
7.4 for the Ba2+ system, which should have favored NH3 volatilization from the
former. However, the solubility of MgSO4 is high while that of BaSO4 is low, which
would favor larger emissions from the Ba2+ system. In fact, the Ba2+ -saturated
soil produced rapid initial NH3 volatilization rates, with the pH rising to 8.9 before
decreasing to 7.4. The pH for the Mg2+ -saturated soil decreased to 8.2 before
returning to the initial value of 8.5. Although not conclusive, these results support
the hypothesis that the formation of sparingly soluble salts between the cation in
carbonate-buffered systems and the anion from added NH+ 4 compounds results in
increased NH3 volatilization.
Fenn et al. (1981b) examined whether the addition of soluble Ca (or Mg) salts
with urea could result in precipitation of CaCO3 and thus reduce the pH which
would otherwise result [see Eqs. (3) and (4)]. Measurements were made from
three soils: calcareous Harkey silty clay loam (15% CaCO3, pH 7.7), Darco fine
sand (pH 5.8), and Beaumont clay (pH 4.8). Measurements of NH3 loss were
made following additions as solutions. Results showed significant reductions from
urea plus Ca(NO3)2, CaCl2, and MgCl2 compared to urea alone (76 and 59% N
loss to 10–15% N loss for the Harkey and Darco soils, respectively; and 46 to
<3% for the Beaumont soil). No reductions were achieved with CaSO4 due (it
was postulated) to its low solubility and hence insignificant effect on removing
carbonate from solution. Addition of CaCl2 with AS or AN also reduced NH3
loss from the Harkey soil—76 to 31% and 33 to 18%, respectively. Other results
(using the calcareous Harkey soil and the acidic Darco soil) showed that increasing
the Ca2+ :N ratio further reduced NH3 volatilization (Fenn et al., 1981a). These
results support the view that NH3 volatilization from NH+ +
4 or NH4 fertilizers can
be explained in terms of the chemical reactions occurring in the soil.
However, further work on this subject (Fenn, 1988) designed to investigate
whether the degree of Ca2+ saturation affected the Ca2+: urea ratio required to
control NH3 volatilization produced somewhat contradictory results. In this latter
study, solutions of urea alone or in combination with CaCl2 (at ratios of 0.25 and
0.50 moles Ca2+ per mole urea) were added to three acid soils and one calcareous
soil. Pretreatments were imposed on the acid soils (nil, Ca2+ saturation, addition of
1% w/w CaCO3, and addition of 5% w/w CaCO3) and the calcareous soil (nil, Ca2+
saturation, 25% Na+ saturation, and 50% Na+ saturation). The results showed that
NH3 loss from urea alone was not affected by soil pretreatment. This suggests that
urea hydrolysis occurs very close to the site of application (i.e., affects only a small
volume of soil) and so the bulk soil pH has little influence on the volatilization
process. However, differences in pretreatment resulted in small but apparently
significant differences in NH3 loss for additions of urea in combination with CaCl2.
Fenn (1988) claimed that these differences reflect the initial soil pH, but this was
not the case, as the lowest pH values were measured for nil soil pretreatment and
NH3 emissions from these treatments were never the least. Even after excluding
these treatments, no consistent pattern emerges between initial soil pH and NH3
volatilized. Fenn (1988) quotes earlier work (Fenn et al., 1981a) which showed
that Ca2+ greatly depressed the rate of urea hydrolysis and states that the Ca–urea
compound must reduce the rate of diffusion into the surrounding soil and (hence)
increase the effect of (bulk) soil pH on NH3 losses. If the effect of Ca2+ is indeed
to extend the existence (“residence time”) of urea, this should serve to increase
the rate of diffusion into the bulk soil which would indeed increase the effect of
soil pH on the volatilization reaction. Nevertheless, although this was not stated
(Fenn, 1988), it is apparent from the figures presented that the effect of increased
Ca:urea ratio in the applied solution was to reduce NH3 volatilization, confirming
the results of the previous study (Fenn et al., 1981b).
Fenn and Kissel (1975) describe results from laboratory experiments which
investigate the effect of CaCO3 on NH3 volatilization from NH+ 4 -containing fer-
tilizers. For AS, NH3 losses increased rapidly with CaCO3 content up to 6.1%,
less rapidly up to 9.7%, and not at all above 9.7%. At low (0.5%) CaCO3 content,
NH3 losses decreased with increasing application rate (110 to 550 kg/ha N). There
was no effect of N application rate with CaCO3 contents of 1.3 and 2.9%. How-
ever, at 6.1% soil CaCO3 and above, NH3 loss increased with N application rate.
These patterns were repeated at three temperatures (12, 22, and 32◦ C), although
for conditions under which less than maximum NH3 volatilization occurred, emis-
sions increased with temperature. At 12◦ C, losses were 20–30% at 2.9% CaCO3
and 30–40% at 14.7% CaCO3 (at 110 to 550 kg/ha N). Differences between N
application rates tended to be less marked at higher temperatures. These results
appear to illustrate both the effect of NH+4 addition in reducing soil pH (i.e., NH3
loss decreases with increasing N application rate, at low CaCO3) and the effect of
CaSO4 precipitation in promoting CaCO3 dissolution and thus increasing soil pH
(i.e., NH3 loss increases with increasing N application rate at high CaCO3). The
pH values 120 h after application tended to be smaller for greater N applications,
and this effect was less marked at greater soil CaCO3 content. Thus, these results
are consistent with the effect of N application in reducing NH3 loss at low soil
CaCO3 content, but do not reflect the effect of rate of N application at high soil
CaCO3 content. Maximum NH3 losses were associated with pH values of 7.6 or
greater. Fewer results were reported for AN. In contrast to AS, NH3 volatiliza-
tion from AN was consistently less from the greater application rate (up to 6.1%
CaCO3, the greatest content for which results were reported). Thus, in the absence
of other reactions, the addition of NH+ 4 serves to reduce pH. Emissions from AN
were less than those from AS.
Other work also confirmed the importance of rate of application of NH+ 4 fer-
tilizers (Fenn and Kissel, 1974) on the extent of NH3 volatilization. For AS and
DAP, both compounds which form sparingly soluble salts with Ca2+ , NH3 loss as a
percentage of N applied was 19–23, 26–45, 38–46 and ∼49% for application rates
(kg/ha N) of 33–66, 100, 275, and 550, respectively. The ranges represent the vari-
ation associated with temperatures 12–32◦ C. In contrast, NH3 volatilization did
not vary with application rate of AN and was 14–26% for the temperature range
12–32◦ C. Although the CaCO3 content of the soil (Houston Black clay) was not
stated, it appeared that this soil was well buffered such that addition of increasing
concentrations of NH+ 4 (as AN) did not result in a reduction in pH and, hence, NH3
volatilization as occurred in the later study of Fenn and Kissel, (1975). In contrast,
for AS and DAP, increasing application rate increased NH3 volatilization because
pH was increased via Eqs. (3) and (4), as described above. Fenn and Kissel (1974)
suggested in their article that increasing temperature had less effect on total NH3
losses from AS and DAP compared to AN. However, the variation in the data
do not allow such a conclusion to be made; it seems more likely that the effect
of temperature is comparable for both compounds which form sparingly soluble
Ca2+ salts and those which do not.
Overall, therefore, it appears that the relative NH3 emissions from different NH+4
(containing or forming) compounds applied to soils under laboratory conditions
can be predicted on the basis of the chemistry involved. Reactions controlling pH
(particularly at, and in the immediate vicinity of, the site of application) are most
important. Urea hydrolysis, in particular, greatly increases pH around the fertilizer
granule leading to a large NH3 potential. Ammonium salts with anions which form
sparingly soluble Ca2+ salts also tend to increase pH, while those with anions which
form soluble Ca2+ salts do not. Addition of Ca2+ reduces pH, while for AN NH3
losses will not increase linearly with increasing N fertilizer addition because of pH
reduction. However, in many cases reactions involving phase changes are involved
(i.e., precipitation of sparingly soluble salts) and these need to be considered with
some care, as the formation of precipitates is not necessarily an instantaneous
process. Nonequilibrium conditions may therefore persist and affect volatilization
reactions.
2. The Role of pH
The key role of pH has been confirmed in a number of studies, although no

consistent method to measure the effective pH at the site of volatilization has been
developed. For example, pH for the fertilizer solution (Al-Kanani et al., 1990),
soil pH after 120 h (Fenn and Kissel, 1975), and soil pH after 24 h (Sommer and
Ersbøll, 1996; Whitehead and Raistrick, 1990, 1993) have all been used to explain
variations in NH3 volatilization. Work by Al-Kanani et al. (1990), investigating
the effect of the urea:AN ratio on NH3 volatilization from solutions applied to
soil and carried out under laboratory conditions, showed that increasing AN at
constant urea induced a logarithmic reduction in NH3 volatilization (postulated
to be due to NH+ +
4 exchange with H on soil colloids, thus reducing pH). The
pH of the fertilizer solution was related to N loss, but this effect was less pro-
nounced for soil with a large clay content. The pH of the soil suspension after
(10 days) incubation was not related to NH3 volatilized. Whitehead and Raistrick
(1990) reported measurements of NH3 volatilization under (laboratory) controlled
conditions over 8 days for surface-applied N fertilizer and from cattle urine at
20◦ C applied to 22 soils (1993), with assessments also made of soil pH. The re-
sults indicate the importance of soil–fertilizer reactions on pH in controlling NH3
volatilization. In general, AN and MAP had either no or a slight acidifying ef-
fect on soils with pH < 7; consequently, NH3 loss was small (<4%). For soils at
pH > 7, NH3 loss increased from AN (up to 10%) and also from MAP where
the soil contained significant CaCO3 (35%) due (it was suggested) to the precip-
itation of octacalcium phosphate (OCP). Emissions from AS increased with pH
(from <2 to 48%). Emissions from urea also increased with pH although not so
markedly (from 24 to 43%, excluding one very acid soil). Thus, comparing urea
with AS, emissions were greater from urea for soils with pH <7 and from AS for
soils with pH >7. The results from DAP were rather anomalous: For soils with pH
<7, NH3 loss was intermediate between AS and urea; for the calcareous soil (pH
>7) NH3 loss was 52% greater than both urea and AS but for the noncalcareous
soil with pH >7, NH3 loss was 8% less than both urea and AS. It was suggested
that this difference could be ascribed to the differing reaction products of DAP in
soils: in those with low CaCO3 it gives calcium ammonium phosphate; in those
with high CaCO3 it gives OCP. This did not seem to be the case for MAP. There
was a strong exponential relationship between NH3 volatilization (as percentage
of NH+ 4 - or urea-N applied) and the pH of the soil–fertilizer mixture after 24 h
[Eq. (5)] which accounted for 85% of the observed variance:
V = −4.61 + 0.0324 × 2.465pH . (5)
The pH of a soil/urine mixture after 24 h was also quite closely correlated with
NH3 loss, whereas two measures of titratable acidity were not (Whitehead and
Raistrick, 1993). Ammonia loss from urine was inversely related to the soil cation
exchange capacity (CEC), and greater losses were measured from grassland soils
relative to arable soils for a given CEC (although “across the fence” comparisons
did not reveal strong differences between grassland and arable soils, except that
volatilization on the first day was greater for the former). It was postulated that
the greater contact between NH+ 4 -N and soil from urine (as opposed to urea)
application meant that the NH+ 4 retention properties of the soil had a greater effect
(compared to pH) on NH3 volatilization.
In another recent study (Sommer and Ersbøll, 1996), NH3 volatilization
from three fertilizers [urea, DAP, and calcium ammonium nitrate (CAN)] was
investigated. A ventilated chamber system was used with a ventilation rate which
had been shown in a separate study to be nonlimiting with respect to NH3 volatiliza-
tion. Two soils (sandy and sandy loam texture), either limed or not limed for 25
years, were used. Addition of CAN always reduced pH [see Eq. (2) above], whereas
urea and DAP generally increased pH. This increase was greater for urea than for
DAP, except for the soil with the lowest initial pH (4.5) which was ascribed to
the low activity of urease under these conditions. For the soil with the highest
initial pH (7.9), DAP and urea addition decreased the pH (possibly through the
“salt effect”) although this rose subsequently following the urea treatment. Soil
pH showed an exponential rise to ammonium from the value measured at 24 h
following urea addition, but there was no change after 24 h following the DAP and
CAN treatments.
3. Models of Ammonia Volatilization
It is apparent that the main factors controlling NH3 volatilization are the rate of
hydrolysis for urea and the changes in soil pH following application for all fertil-
izers, although other factors such as windspeed and CEC also have an influence.
As already stated above, NH3 emissions tend to increase with soil pH, albeit there
is a strong interaction between the fertilizer and the soil solution which may (e.g.,
for urea) override the effects of initial soil pH through hydrolysis and precipita-
tion reactions. Important in this regard is the effect of CEC; large soil CEC (more
specifically, high NH+ 4 retention) tends to reduce NH3 volatilization potential by
reducing the concentration of NH+ +
4 in the soil solution by adsorption of NH4 on
the exchange sites. In the field, however, these factors influence the maximum
potential emission, which may be reduced if significant rainfall occurs during the
main volatilization period, essentially in the 10 days after fertilizer application.
There have been a number of attempts to model NH3 volatilization, both mech-
anistic (Fleisher et al., 1987; Rachhpal-Singh and Nye, 1986; Roelcke et al., 1996;
Sherlock and Goh, 1985) and empirical (Stevens et al., 1989), which have been
more or less successful. The equations developed by Sherlock and Goh (1985)
included the following terms and constants: total NHx concentration, Henry’s Law
constant, volumetric moisture content, pH at the soil surface (site of volatilization),

temperature, the distribution ratio for the partition of NH+
4 between exchange sites
and the soil solution, and the exchange coefficient for transport of gaseous NH3
from the soil to the bulk atmosphere. In a later experiment (Sherlock et al., 1995),
these relationships were incorporated into a linear relationship of the form:
F = k × u z × ␳0, (6)
where, F is the NH3 flux, k is a dimensionless (site-specific) exchange coefficient,
u z is the mean wind speed at reference height z, and ␳ 0 is the NH3 concentration
in equilibrium with the liquid phase and is calculated from the following:
␳ 0 = [NH3 ]soln × 10(1.6937−1477.7/T ) (7)
(0.0918+2729.92/T −pH)
= [NH+

[NH3 ]soln 4 + NH3 ]soln / 1 + 10 (8)
[NH+
4 + NH3 ]soln = [NHx ]total /Mv , (9)
where Mv is the volumetric water content and corrects the amount of NH4-N
extracted in KCl in a specified volume or soil to give a soil solution concentration.
In fact, in this form, k also incorporates a term for a partition of NH+ 4 between
exchange sites and the soil solution, but since this parameter also describes other
site-specific factors such as surface aerodynamic roughness, it is not necessary
to include it explicitly. Thus, NH3 emission was calculated from measurements
of NHx-N in the 0- to 1-mm or 0- to 3-mm soil layers, pH and temperature of
the soil surface, and windspeed at 1.2 m above the soil surface. These results
were compared with those obtained for ∼2-hourly periods over up to 8 days from
the micrometeorological mass balance method (Black et al., 1985a) at two sites.
The correlation coefficients for experiments involving granulated urea fertilizer
and synthetic urine were 0.870 and 0.879, respectively, indicating that a large
proportion of the variation in NH3 emission could be explained on the basis of
variation in windspeed, NHx-N concentration, pH, and temperature.
A mechanistic model of the reaction of urea in the soil has also been developed
by Rachhpal-Singh and Nye (1986). The sensitivity of various parameters was
determined (as was the effect of placement or mixing urea in the surface), and it
was shown that the rate of diffusion of HCO− 3 to the soil surface was an important
rate-limiting process for NH3 volatilization. Roelcke et al. (1996) report the re-
sults of a comparison between NH3 volatilization measured in the laboratory from
vented chambers and these predicted from the Rachhpal-Singh and Nye (1986)
mechanistic model and modifications based thereon. The soil studied had a pH
value of 7.7 and a CaCO3 content of 10%. Because the model (Rachhpal-Singh
and Nye, 1986) was based on a neutral, noncalcareous soil, simulations were car-
ried out using (a) an initial pH of 7.7 but with remaining parameters as in the
original model, (b) bulk density and the parameters relating to gaseous and liquid
diffusion altered to reflect the more porous nature of the soil used in this study (e.g.,
bulk density = 0.95 cf. 1.5 kg/dm3), (c) as (b) but with the pH buffering capacity
increased threefold, and (d) as (b) but with CaCO3 precipitation and dissolution
included in the model equations.
Overall, the best fit was given by (c). In the early stages, simulations (b) and
(d) overestimated NH3 volatilization. Despite the more porous nature of the soil
in this study, the parameters relating to the standard model appeared adequate in
describing NH3 loss in the early stages. In fact simulation (c) also gave a good fit
during this period. It is probable that the good fit for simulation (a) resulted from
the possibly fortuitous canceling of high pH and low gaseous diffusion terms.
Indeed, after about 6 days, simulation (a) began to significantly underestimate
NH3 volatilization, as the effect of the initial high pH became less important
and the lower gaseous diffusion term dominated. It seems likely, therefore, that
the initial overestimation of NH3 volatilization by simulations (b) and (d) was
the result of overestimation of pH during this period. The fact that simulation (d)
overestimated NH3 loss, whereas simulation (c) was reasonably accurate, indicates
the importance of pH; these workers suggest that the description of pH buffering
capacity in Rachhpal-Singh and Nye’s model (1986) may be incomplete, since
increasing this parameter threefold gave a good fit to measured NH3 volatilization,
whereas including the (known) reactions involving CaCO3 did not. In fact, the
pH of this soil was such that it was close to the upper limit of buffering for the
CaCO3–H2O–CO2 system. In addition, there was some evidence that at this pH,
precipitation of CaCO3 was a slow, rather than instantaneous, process. Ultimately,
even simulation (c) was not a good description of the volatilization process over
the full period of the experiment, since after 10 days NH3 loss continued at a rate
greater than predicted.
Another quantitative model of NH3 volatilization from the uppermost 1 cm of
calcareous soils was presented by Fleisher et al. (1987). Factors included were as
follows: monovalent–divalent exchange, diffusion impedance in the gaseous phase,
and nitrification. It was assumed that in practice the addition of NHx fertilizers does
not change soil pH (because of the high buffering capacity of such soils), that most
NH3 losses occur while soil water content in not far from field capacity, and that the
rate of diffusion is the rate-limiting step for volatilization. The model results predict
that volatilization declines with decreasing pH, increasing CEC, and increasing
nitrification rate. The model also predicted a small increase in the proportion of N
volatilized with increasing N application rate.
In contrast to these mechanistic models, Stevens et al. (1989) used results from
ventilated laboratory enclosures and stepwise multiple linear regression to relate
total NH3 loss (Amax ) and time to maximum volatilization rate (Tmax ) and to soil
properties for 36 soils. Values of Amax ranged from 1.6 to 26.1% (mean 16.8%)
and were best explained by variation in titrateable acidity. Values of Tmax ranged
from 0 to 10.6 days and were best explained by nonbuffered urease activity and
loss-on-ignition. These authors also outlined other work where the relationship be-
tween the timing and amount of rainfall (or irrigation) and N volatilization had been
studied. They noted that, in the absence of rainfall, the most important characteris-
tic of the NH3 volatilization process will be Amax ; however, under field conditions,
the dynamics of NH3 volatilization will depend on Tmax and when rainfall occurs.
B. NITROUS OXIDE AND NITRIC OXIDE EMISSIONS
In soil, N2O and NO are predominantly produced biologically, through nitrifi-

cation and denitrification. A number of possible pathways exist for the production
of NO and N2O by chemoautotrophic nitrifying bacteria. Denitrifying bacteria
both produce and consume NO and N2O. In addition, a number of other soil
microorganisms are capable of producing NO and N2O (Robertson and Tiedje,
1987), and abiological reactions involving NO and N2O also occur in soil (Nelson,
1982).
The processes controlling the emission of NO and N2O from soil are illus-
trated by the “hole in the pipe” model (Davidson, 1991; Firestone and Davidson,
1989); namely factors affecting the rates of nitrification and denitrification, fac-
tors affecting the relative proportions of the end products, and factors affecting
gaseous diffusion through the soil to the atmosphere. Early work (ca. pre-1980)
suggested that denitrification was the major source of soil-derived N2O, but work
by Bremner and Blackmer (1978, 1979, 1981) showed that nitrification was also a
significant source. In contrast, in agricultural soils where pH is likely to be main-
tained above 5, nitrification is thought to be the major source of soil-derived NO
(Skiba et al., 1997) since denitrifiers both produce and consume NO (Anderson
and Levine, 1986). The magnitude of N2O and NO emissions from soil is depen-
dent on soil-available NH+ −
4 and NO3 concentrations as well as on factors such as
soil temperature and soil moisture content (soil aeration). Thus N-fertilized soils
are significant contributors to total N2O and NO emissions.
III. MEASUREMENTS OF AMMONIA EMISSION

FOLLOWING NITROGEN FERTILIZER APPLICATION
A. FIELD MEASUREMENTS OF AMMONIA EMISSION
Although there have many measurements of NH3 volatilization from N fertil-

izers, there have been relatively few simultaneous comparisons of losses from
different N fertilizers in the field (Bussink and Oenema, 1997; Fox et al., 1996;
Gasser and Penny, 1967; Gezgin and Bayrakli, 1995; He et al., 1995; Keller and
Mengel, 1986; Lightner et al., 1990; Ryden et al., 1987; Sommer and Jensen,
1994; Stevens and Laughlin, 1988, 1989; Touchton and Hargrove, 1982; van der
Weerden and Jarvis, 1997; Velthof et al., 1990).
Hargrove (1988) stated that N sources influence NH3 losses through cation and
anion effects and indicated measurements from N sources under field conditions
(Hargrove and Kissel, 1979; Hargrove et al., 1977; Keller and Mengel, 1986;
Urban et al., 1987). These studies showed that the NH3 loss potential is greatest
with urea, intermediate with urea-ammonium nitrate (UAN) solution, and least
with NH+ 4 salts on noncalcareous soils, but greatest with AS and much less with
urea or AN on calcareous soils. In general, most fertilizer comparisons in the
field (Table I) have been carried out using either wind tunnels (Ryden et al., 1987;
Sommer and Jensen, 1994; van der Weerden and Jarvis, 1997; Velthof et al., 1990)
or chambers (Gezgin and Bayrakli, 1995; Keller and Mengel, 1986; Lightner
et al., 1990), although a number of agronomic trials involving different fertilizers
expected to differ in their NH3 volatilization potential have also been undertaken
(Gasser and Penny, 1967; Hargrove et al., 1977; He et al., 1995; Stevens and
Laughlin, 1988, 1989; Touchton and Hargrove, 1982; Watson et al., 1998).
1. Micrometeorological Methods
While there have been a number of studies of NH3 emissions from individual
N fertilizers by micrometeorological techniques, the only comparative studies of
NH3 volatilization measured in the field using micrometeorological methods have
been those of McInnes et al. (1986a,b) and Fox et al. (1996) and their results are
reported in the section on fertilizer solutions below.
2. Wind Tunnel Measurements
Ryden et al. (1987) made measurements using wind tunnels following fertilizer
N application to grass swards (no details of soils given). Results for NH3 volatilized
as a proportion of N applied were 5.7, 16.7 and 20.7% from urea at 70 kg N/ha
35.6, 19.9% from urea at 100 kg N/ha, and <2.5 and <1.6% from AN at 70 and 100
kg N/ha, respectively. Velthof et al. (1990) reported the results of measurements
of NH3 volatilization from urea and CAN applied to permanent grassland on a
heavy clay soil, measured using wind tunnels. Their results showed (a) emissions
of 19 and 32% from urea applied at 80 and 120 kg N/ha, respectively, during a
dry period in late March/early April; (b) an emission of 7% from urea applied
at 90 kg N/ha during a wet period in late June/early August; and (c) generally
small or negative emissions [ascribed to deposition of NH3 emitted from adjacent
(1.5 km) slurry application] from CAN. However, on one occasion (the 4th week
of August) an emission of 11% was recorded for CAN, for which no explanation
was given. Over all times and N rates, emissions were calculated as 22.6 ± 15.6%
Table I
Fertilizer Comparisons Carried out in the Field
Emission Measurement
Reference Fertilizersa factors Notes technique
Gasser and Penny (1967) UN; UP; Results of agronomic

UP:U (1:2); AN evaluation
Hargrove et al. (1977) AN Indirect estimates
AS Results over 2 years applied
AS (aq) to calcareous clay
Touchton and Hargrove U; U-AN (aq); AN Results of agronomic
(1982) evaluation
Keller and Mengel (1986) U 0.30, 0.11 For sand loam and silt loam Chambers
Cogranulated U-UP 0.15, 0.06 soils (pH <7), respectively
80
U-AN (aq) 0.09, 0.05

AN 0.04, 0.02
Ryden et al. (1987) U For differing weather Wind tunnel
AN <0.03 conditions and
application rates
Stevens and Laughlin (1988) AS; CN; U Results for utilization
by 15N
Stevens and Laughlin (1989) U; AN Results for utilization
by 15N
Lightner et al. (1990) U Results for spring and Chambers
U (aq) summer applications in
U-KCl (aq) 2 years
Cogranulated U-Up <U
U-CaCl2 (aq) <U(aq)
AN n.s.
Velthof et al. (1990) U For differing weather Wind tunnel
CAN Low conditions and
application rates
Sommer and Jensen (1994) U For sandy soil (pH 6.1) on 5 Wind tunnel
DAP occasions
AS 0.05
CAN 0.02
Gezgin and Bayrakli (1995) AS 0.14 For calcareous clay loam Chambers
AN 0.04 soil
U 0.11
He et al. (1995) U; AS; AC Studied effect of lime-N
agronomic
Fox et al. (1996) U For applications to no-till Micromet
(aq) (sprayed) corn in 3 years
(aq) (dribbled)
81
Bussink and Oenema (1997) U, (C)AN Synthesis of eld data from Agronomic
The Netherlands, southern
England, and Northern
Ireland
van der Weerden and Jarvis U For differing weather Wind tunnel
(1997) AN conditions and application
CN rates on two soil types
(pH 7)
Watson et al. (1998) U
CAN Results of agronomic
evaluation
a
Abbreviations: AC, ammonium chloride; AS, ammonium sulphate; (C) AN, (calcium) ammonium nitrate; CN, calcium
nitrate; DAP, diammonium phosphate; U, urea; UN, urea nitrate; UP, urea phosphate.
and −1.8 ± 6.0% for urea and CAN, respectively; the wide range for urea being
ascribed to differences in N rate, rainfall, and temperature.
Sommer and Jensen (1994) stated that there are few simultaneous studies of
NH3 volatilization from AS, DAP, An and urea applied in the field, despite these
fertilizers being among the most used in Europe. Wind tunnel measurements of
NH3 volatilization from fertilizers (urea, DAP, AS, and CAN) applied to a sandy
soil (winter wheat and grass) were made on five occasions between March and
June. Total losses were calculated from NH3 rate loss measurements over 15–20
days by fitting a sigmoidal (urea) or exponential (DAP, AS, and CAN) func-
tion. Total losses were estimated to be 25% (18–30%) for urea provided there
was sufficient moisture for hydrolysis (otherwise losses were negligible), 14%
(1–17%) for DAP, <5% (not significantly greater than zero) for AS, and <2% (not
significantly greater than zero) for CAN.
Van der Weerden and Jarvis (1997) noted that quoted emission factors for urea
and AN range from 5 to 16.5% and 0 to 10%, respectively. The objectives of their
study were (a) to measure NH3 volatilization from three N fertilizers applied to
grassland on two contrasting soil types, (b) to derive new emission factors based
on this and previous published work, and (c) to estimate NH3 emissions from
fertilizer applied to agricultural land in the UK. Measurements were made using
wind tunnels following application of urea, AN, and calcium nitrate (CN) on three
dates (March: 120 kg N/ha; June: 90 kg N/ha; August: 70 kg N/ha) on two soils
(freely drained sandy clay loam, pH 6.0; poorly drained clay loam, pH 5.6). The
results showed that the greatest NH3 volatilization was from urea. Measured losses
in August were influenced by rainfall, with 17 mm 1 day after fertilizer application
on the poorly drained site and 14 mm 3 days after fertilizer application on the well-
drained site. Losses for March were greater from the poorly drained site compared
to the well-drained site, this being ascribed to the effect of more rapid hydrolysis
outweighing that of lower temperatures; soil moisture contents were 108 and 29%,
respectively. For the June application, emissions were greater from the well-drained
site compared to the poorly drained site. The authors did not comment on this
observation, although they did state that they considered the effect of soil type to
be small in this study as the initial soil pH values were similar. They also noted
that, whereas N loss increased from 12 to 38% as N application increased from 70
to 120 kg N/ha for the poorly drained site, there was no obvious relationship for
the freely drained site. Average emissions for both AN and CN on both soil types
were <1%, and the results indicated that there was little seasonal or soil effect.
3. Chamber Measurements
Lightner et al. (1990) carried out field measurements of NH3 volatilization using
ventilated chambers in spring (May) and summer (August) over 2 years (1982 and
1983). The fertilizers studied were urea, urea solution (Uaq), cogranulated UUP,
urea + KCl in solution (UKCl), urea + CaCl2 in solution (UCaCl2), and AN.
Greatest losses were from urea, Uaq and UKCl, ranging from 27 to 41% of applied
N in the spring and from 12 to 27% in the summer. No significant volatilization
losses were observed with AN. Ammonia losses from UUP (an acidic material)
were significantly less than those from urea in the spring and at least 40% less in the
summer. Relative to Uaq, mixing with CaCl2 reduced volatilization significantly
in both 1983 studies. Smallest losses occurred in summer 1983 when conditions
were particularly dry. The work of Gezgin and Bayrakli (1995) concentrated on
the effect of additives on NH3 volatilization, although useful comparisons for the
unamended fertilizers were included. The results from field (enclosure method)
measurements of NH3 volatilization from fertilizers (AS, AN, and urea) applied
to a calcareous clay loam soil were AS (14.0% of N added), AN (4.4%), and urea
(10.6%).
B. AGRONOMIC EVALUATIONS: EFFECTS ON

FERTILIZER EFFICIENCY
Hargrove et al. (1977) reported some early estimates of NH3 loss on a calcare-
ous (Houston Black clay) soil based on agronomic evaluations. Two methods were
used: indirectly using comparison of N response curves of NH+ 4 -containing fer-
tilizers with CN and directly using enclosure methods. The results over 2 years
(indirect method) showed that N losses (of total N applied) were 3–10% for AN,
36–45% for AS, and 25–55% for AS applied as a solution. Note that all the fer-
tilizers were applied after the soil had first been wetted to field capacity using
irrigation equipment. The results for AS varied somewhat between the 2 years.
Direct measurements were carried out from the AS treatment only and showed
losses of 43–59% and 27–39% in late summer and spring, respectively, due (it
was postulated) to differences in temperature. Application rates were 33–280 kg
N/ha, but there was no evidence of an effect of rate at either time, in contrast to
other reported laboratory results.
Gasser and Penny (1967) investigated urea nitrate (UN), urea phosphate (UP),
and an ∼1:2 urea phosphate: urea mixture (UPU) as N fertilizers to find out whether
the presence of the anion decreased the damage urea causes to germinating seeds
and seedlings and increased the efficiency of urea by preventing loss of NH3.
Touchton and Hargrove (1982) reported the results of field studies which were
conducted for 3 years on a Cecil sandy loam (pH 6.1, CEC = 3.5 meq./100 g) to
compare the efficiency of urea, UAN solution, and AN as affected by method of
application in a direct drilled maize production system.
In the UK, Stevens and Laughlin (1988, 1989) compared AS, CN, and urea and
AN and urea, respectively, using 15N-labeled fertilizers. In the first study, AS, CN,
and urea were applied to ryegrass at three different times in the spring (February,
March, and April) on a lowland and an upland site (in 1983 and 1984, respectively).
The efficiency of fertilizer N use was measured in the following ways: at the first cut
by dry matter (DM) yield, percentage recovery by difference, percentage utilization
of 15N, and for all cuts by percentage utilization of 15N. For the lowland site, only the
percentage utilization for all cuts data showed a significant effect of fertilizer form.
Averages for the three dates of application (note that there was a significant effect of
timing but no interaction with fertilizer form) were AS (48.5%) > urea (46.3%) >
CN (43.1%). It was suggested that more N from AS may have been immobilized
and subsequently mineralized during the growing season. Alternatively, more N
from CN may have been leached or denitrified (see Section V below). For the
upland site, all measures showed significant effects of timing and fertilizer form
with a significant interaction between them. Using the percentage utilization for
all cuts data, the results were as follows: February: AS (41.9%) > urea (28.9%)
> CN (9.6%); March: CN (61.2%) > AS (56.8%) > urea (50.8%); and April: AS
(92.9%) > CN (67.4%) > urea (57.8%). Thus, AS always performed better than
urea, but the effectiveness of CN in relation to the other fertilizers depended on
time of application, being markedly worse for the earliest application.
In the second study, using confined microplots, measurements were made of the
recovery of N in the above-ground herbage from labeled AN and labeled urea and
in soil (0–15 cm) at three sites and two rates of application. In this experiment,
fertilizers were injected as solutions 5 cm below the soil surface. Recovery of urea
was always greater and often statistically significantly greater than that of AN.
Differential labeling also indicated that recovery of the nitrate moiety was less
than that of the ammonium moiety.
Bussink and Oenema (1997) used data from field trials in The Netherlands,
southern England, and Northern Ireland on grassland with urea and CAN to develop
a relationship between the relative yields from these fertilizers and environmental
conditions. They noted that although there are few studies where gaseous and
leaching losses were measured simultaneously after urea and CAN application,
these losses were reflected in dry matter production and N uptake. Their analysis,
combining all trials and grass cuts, generated the following relationship:
AURY = 89.48(±0.781) + 2.188(±0.148) × R3 − 1.091(±0.070) × T 3

(10)
2
Radj = 89.9%, (11)
where AURY is the apparent urea relative yield (i.e., with respect to CAN), R3
is the total rainfall (in millimeters) within 3 days after fertilizer application, and
T 3 is the average temperature (◦ C) within 3 days after fertilizer application. This
relationship suggests that high temperature and low rainfall either promote NH3
volatilization from urea or reduce N losses through denitrification and nitrate
leaching from CAN or both.
C. LABORATORY MEASUREMENTS
There have also been a number of laboratory comparisons of NH3 volatilization

from different fertilizers (e.g., Meyer and Jarvis, 1989; Meyer et al., 1961; Prasad,
1976; Shahandeh et al., 1992; Sommer and Ersbøll, 1996; Whitehead and Raistrick,
1990). Early laboratory experiments (Meyer et al., 1961) with AN; urea; and a
solution made up as 44.3% AN, 35.4% urea, and 20.3% water (SOLN) were carried
out on three soils ranging in pH from 5.6 to 7.8. The results of NH3 volatilization
measurements showed that N loss declined with decreasing pH for all three N
sources. Losses were relatively small from the acid soil and were generally greatest
from the soil with the highest pH; however, for SOLN applied to straw residues
covering the soil surface, emissions from the acid soil were almost as large as those
from the alkaline soils.
Some laboratory studies have focused on the effect of soil or fertilizer amend-
ments on NH3 volatilization. Thus, Prasad (1976) carried out laboratory incubation
experiments to study NH3 volatilization from urea, sulfur-coated urea (SCU), and
AS applied to a calcareous soil at two temperatures (22 and 32◦ C) and three mois-
ture contents (25, 50 and 80% of soil water-holding capacity). Higher temperature
increased the NH3 losses from all sources. Losses usually followed the order
urea > AS > SCU; there were three forms of SCU representing fast (F), medium
(M), and slow (S) dissolution rates, and the order of losses for these was SCU(F) >
SCU(M) > SCU(S). At both temperatures, increasing soil moisture content led
to a reduction in NH3 loss regardless of the N source. It was suggested that this
was because greater moisture content would provide a larger surface area for NH3
absorption and also increase the rate of nitrification. On the other hand, the rate of
urea hydrolysis was only slightly retarded at lower moisture contents, even at the
lower temperature. At 22◦ C, NH3 losses from urea and AS at low, medium, and
high moisture contents were 11.1 and 10.2%, 7.7 and 8.8%, and 4.2 and 3.5% of N
applied. It was noted that the time course of the losses for these fertilizers differed,
with AS showing moderate losses for the whole 21-day experiment, whereas urea
showed high losses for days 3–14 and low or negligible losses for days 0–3 and
14–21. Increased temperature resulted in losses more than double for SCU, about
double for urea, and less than double for AS.
Shahandeh et al. (1992) compared NH3 volatilization from AS, Uaq, and a
sludge produced as a by-product of NutraSweet manufacture (NS). Laboratory
incubations were carried out for 16 days at 25◦ C; a static (i.e., nonvented) system
was used to collect volatilized NH3, which would tend to underestimate the loss
compared to that which occurs in the field. Their results showed average losses of
0.9 and 0.4% from AS solution and 14.2 and 17.9% from Uaq for Tifton [pH 6.8,
CEC = 3.62 cmol(+)/kg] and Dothan [pH 5.5, CEC = 0.54 cmol(+)/kg] soils,
respectively. The difference between the fertilizers was as expected, and that be-
tween the soils for Uaq was ascribed to the effect of the smaller buffering capacity
in the Dothan soil. These authors also discuss the effect of surface residues on NH3
volatilization from Uaq. They state that although other workers have suggested
that emissions are less from soils than from residues (presumably because of high
urease activity and low buffering capacity), in this study NH3 volatilization for the
Dothan soil was reduced by a straw covering (16.9 cf. 18.9%). They suggest that
this may have been due to increased rates of nitrification in the presence of straw.
Other workers have used laboratory studies to investigate the interactions be-
tween fertilizer application and urine from grazing animals by comparing NH3
emissions from urea and AN alone and in the presence of urine from intact cores
and disturbed soils (Meyer and Jarvis, 1989). The results for intact cores show that
emissions from urea (6.2 and 17.8% for applications of 2 × 30 and 2 × 60 kg N/ha,
respectively) are much greater than from AN (<0.1%). However, the addition of
urine resulted in increased emissions from the fertilizers. Emissions were 72.0 and
21.8% for urea and 47.1 and 16.0% for AN from application rates of 2 × 30 and
2 × 60 kg N/ha, respectively. It was suggested that this was the result of localized
increases in pH associated with the hydrolysis of urea (in urine). There was no
consistent effect of disturbance, except that the period of emission was shorter,
presumably due to greater adsorption. These authors suggest that although other
workers consider that changes in fertilizer practice for grazed swards (compared
with cut grass, which receives no urine) are not necessary (since only a small
proportion of the soil surface receives urine each day), there is a need to assess the
longevity of the effects reported in field conditions.
Ammonia volatilization from five nitrogen compounds used as fertilizers fol-
lowing surface application to soils reported by Whitehead and Raistrick (1990) has
already been discussed above. However, these workers considered that their results
(obtained in the laboratory) were probably twice those which could be expected in
the field for spring N fertilizer application. This assessment was made on the basis
of a comparison of the results for urea on one of the soils (pH 7.1, CaCO3 1.8%)
in this study with those from a field study using wind tunnels (Ryden, 1984). A
similar study with urea, DAP, and CAN (Sommer and Ersbøll, 1996) and two soils
(sandy and sandy loam texture), either limed or not limed for 25 years, indicated
that cumulative emission followed a sigmoidal pattern with urea, a negative expo-
nential pattern with DAP, and a linear pattern with CAN, except for the unlimed
soil with the (lowest) pH value of 4.5, where very small emissions were obtained
for urea. Overall losses were urea (23–28%) > DAP (23–28%) > CAN (<5%),
excluding the results from urea for the most acidic soil.
D. EMISSIONS FROM FERTILIZER SOLUTIONS
McInnes et al. (1986a,b) reported 4–17% loss from 120 kg N/ha applied to bare
soil as urea solution and 7–17% from 200 kg N/ha applied to wheat straw and
stubble as UAN solution. Similarly, Keller and Mengel (1986) reported the results
from field experiments carried out on two soil types to measure NH3 volatilization
using ventilated chambers from urea, cogranulated urea-urea phosphate (UUP),
UAN, and AN. Fertilizers were applied at 168 kg N/ha to a maize residue. The soils
were Lyles sand loam ( pH 5.6, CEC = 6.7 cmol(+)/kg) and Bedford silt loam
( pH 5.5, CEC = 12.3 cmol(+)/kg). For the Lyles sand loam, NH3 volatilized was
30, 15, 9, and 4% of the applied N for urea, UUP, UAN, and AN, respectively.
Although the overall losses were less from the Bedford silt loam (11, 6, 5, and 2%),
relative losses from the different N fertilizers were similar. These results show that
emissions from UAN solutions are intermediate between those from urea and AN
granules, but it is difficult to make firm conclusions on the effect of application in
solution per se.
However, Lightner et al. (1990) measured NH3 volatilization from urea, Uaq
and AN. No significant volatilization losses were observed with AN; greatest
losses were from urea and Uaq. Measurements were made in spring (May) and
summer (August) over 2 years (1982 and 1983). There was no significant difference
between urea and Uaq in May 1983 (∼31% volatilized) and August 1982 (∼34%
volatilized). In May 1982 the losses were 41% for urea and 29% for Uaq. This
was ascribed to two rainfall events of 5.0 and 3.3 mm which occurred during
the 2nd and 4th day after application which (it was postulated) were sufficient to
enhance urea granule dissolution, urea hydrolysis, and evaporative water flux from
the soil, but inadequate to move the urea into the soil and terminate volatilization.
Smallest losses occurred in August 1983 when conditions were particularly dry,
and emissions were 10 and 14% from urea and Uaq, respectively. In this case, it
appeared that the additional moisture associated with fertilizer solution treatment
was important in promoting additional volatilization.
Fox et al. (1996) report the results of field (ZINST method) measurement of
NH3 volatilization from urea, sprayed UAN, and dribbled UAN on direct-drilled
maize at an application rate of 134 kg N/ha. Results from measurements in three
(annual) experiments indicated that N losses were as follows: urea, 30% of N
applied; and UAN, 15%. In all 3 years, the 6 days following N application were
relatively rain free. In 1 year, however, 10 mm rain fell 6 days after application,
and this appeared to reduce emissions from urea but not from UAN. The results
were as follows: urea, 39–47% and 33%; sprayed UAN, 19–24%; and dribbled
UAN, 16–19%. Approximately 90% of the total emission took place within the
first 10 days in the drier years, although emissions appeared to continue beyond
day 16 (for urea) for the year when significant rainfall fell 6 days after applications.
E. UREASE INHIBITORS
Watson et al. (1998) investigated the effect of the urease inhibitor nBTPT by
comparing dry-matter production and N uptake by grass fertilized with urea (with
and without inhibitor) and CAN. Results from 15 fertilizer application periods in
Northern Ireland during the period 1994–1996 were reported (and results from six
equivalent fertilizer application periods in the Republic of Ireland were also noted).
On 4 of the 15 occasions in Northern Ireland (and 2 of 6 occasions in the Republic
of Ireland), CAN significantly outperformed urea. Fertilizer applications were
carried out during the summer when conditions for NH3 volatilization are optimal in
Ireland. Addition of the inhibitor significantly improved the performance of urea so
that dry-matter production and N uptake were almost the same as those with CAN.
Laboratory soil enclosure studies showed both that NH3 volatilization was less and
the peak of volatilization occurred later (4.9 cf. 1.6 days after urea application) with
the inhibitor. There was no evidence for a decline in the efficacy of the inhibitor
with repeated application; indeed the inhibitor appeared to be more persistent than
had been indicated in earlier pot studies, due (it was thought) to lower temperatures
in the field. There was still an effect of the inhibitor 10–12 weeks after the third
application, but this had disappeared by the following growing season.
IV. AMMONIA EMISSION FACTORS FOR

NITROGEN FERTILIZERS
Emission factors for different N fertilizers have been published by a number of

authors (see, e.g., Anonymous, 1994). Although NH3 emission from urea has been
extensively studied, information for other fertilizer types is more sparse. In general,
it is considered that emissions from other fertilizers are less than those from urea,
with the possible exception of AS and DAP on calcareous or otherwise alkaline
soils. In a recent report Sutton et al. (1994) examined the different emission factors
for fertilizers used in UK inventories (Table II). These authors noted (with surprise)
the fact that despite the large range in emission of measurements from urea, the
range for emission factors is rather narrow. Nevertheless, they concurred and stated
that a reasonable best estimate for emission from urea (after accounting for some
recapture by vegetation) was 10%. The suggested range for AN is (proportionally)
somewhat larger. This also is surprising. However, Sutton et al. (1994) point out that
the values quoted by Buijsman et al. (1987), and consequently Klaassen (1992) and
Eggleston (1992), were based partly on the work of Fenn and Kissel (1974), which
investigated NH3 volatilization from surface applications of NH+ 4 compounds on
calcareous soils. If these estimates are excluded, and noting that Jarvis and Pain
(1990) excluded emissions from fertilizer other than urea from their calcuations,
the range for emission factors from AN is much narrower, and Sutton et al. (1994)
considered that 1% was a reasonable best estimate.
More recently, van der Weerden and Jarvis (1997) have revised emission factors
for fertilizer N applied under UK conditions. These authors considered that the
emission factor for urea suggested by Buijsman et al. (1987), Klaassen (1992),
Table II
Comparison of Average NH3 Emission Factors from Application of Nitrogen Fertilizers
Loss as % applied N
Author Country Urea NH4NO3 Othera Average
Buijsman et al. (1987) UK 10 10 1 5.8

Kruse (1986) GB — — — 1
Jarvis and Pain (1990) UK 15, 5b 0 0 0.6c
Whitehead and Raistrick (1990) UK 16.5 2.5 1.5–4.9d 3.4
Asman (1992) Europe 15 2 4d 3.8
Klaassen (1992) UK 10 10 1–15d 5.4
Eggleston (1992) UK 10 10 1 —
ECETOC (1994) Europe 15/20 3 8 4.4
Sutton et al. (1994) UK 10 1 2.5 2.4
After Sutton et al., 1994.

a
Mainly combined NPK-N fertilizer in the UK.
b
For grassland and arable and respectively.
c
Calculated using total N fertilizer consumption in the UK of 1460 Gg/year N (Asman, 1992).
d
Several other subcategories provided.
e
Based on Buijsman et al. (1987); see Klaassen (1991).
Eggleston (1992), and Sutton et al. (1994) was too low because it lies near the
bottom of the range of losses from field experiments. They estimated an emission
factor for urea based on their work and other field results using only data where
applications were <200 kg N/ha. Averaging the information from these studies
they calculated an emission factor of 23% for urea applied to grassland. They ac-
knowledged that this represents a value applicable to “average” conditions and note
that it will be influenced substantially by application rate, weather, and soil con-
ditions. This value is somewhat larger than the figure proposed by Whitehead and
Raistrick (1990), who calculated their factor by, first, correcting their laboratory
results for field conditions (×0.5, see above) and, second, by making assumptions
about the representative nature of the soils in their study. Hence, they considered
that soils represented by the Hucklesbrook (pH 6.1, CaCO3 0.6%) and Frilsham
(pH 7.1, CaCO3 1.8%) series were three times as common as represented by the
Batcombe (pH 5.5, CaCO3 0%) and Andover (pH 7.4, CaCO3 75.0%) series. For
AN, an emission factor of 1.6% was estimated (van der Weerden and Jarvis, 1997)
based on this study and other published results (Meyer and Jarvis, 1989; Ryden
et al. 1987; Whitehead and Raistrick, 1990). This factor was also suggested for
other N fertilizers such as CAN and compounded mixtures (e.g., containing CN,
ammonium phosphate, and AS). Whitehead and Raistrick (1990) had proposed
emission factors of 1.5, 4.9, and 9.9% for MAP, DAP, and AS, respectively. Emis-
sion factors for fertilizers applied to arable soils were estimated as half of those for
grassland soils. This was based on work presented by Black et al. (1985b) which
showed emissions from urea applied to winter wheat were 65 and 4% of those from
urea applied to pasture for surface application and incorporation, respectively. In
contrast, Whitehead and Raistrick (1990) had suggested that emission factors for
arable crops should be reduced by 30% to allow for a proportion being applied by
drilling rather than top-dressing.
V. MEASUREMENTS OF NITROUS OXIDE EMISSIONS

FOLLOWING NITROGEN FERTILIZER
APPLICATIONS
A. COMPARATIVE STUDIES OF EMISSIONS
Early reported results from “highly replicated” field experiments (Breitenbeck

et al. 1980) showed that fertilizer-derived emissions of N2O from plots treated
with nitrifiable forms of fertilizer N markedly exceeded those from plots receiving
an equivalent application as nitrate. The fertilizer-induced emissions over 96 days
were 0.11–0.18% of N applied for AS, 0.12–0.14% for urea, and 0.01–0.04 for
CAN. Measurements supported earlier laboratory studies (Bremner and Blackmer,
1978, 1979) which showed that most of the N2O evolved from soils fertilized
with AS or urea is generated within 2 weeks and that N2O emissions from such
soils after about 3 weeks are not significantly greater than N2O emissions from
unfertilized soils. Soil analyses showed that most of the N added to the AS and urea
plots was nitrified within 3 weeks. These workers also noted that despite rainfall
considerably above average and moisture contents frequently near, and at times
above, field capacity, there was no marked increase in N2O emission from CN-
treated plots. In a later study (Breitenbeck and Bremner, 1986), the effect of 180 kg
N/ha as anhydrous NH3, aqueous ammonia, and urea on N2O emission from three
soils (pH 6.9–7.9, CaCO3 0–9.8%) was determined in the field using chambers
with measurements at 3- to 7-day intervals over a 140-day period. The anhydrous
NH3 was applied by injection at a depth of 20 cm, while the other fertilizers were
applied as solutions evenly spayed over the surface; applications occurred on June
3, 1980, and all plots were rototilled to a depth of 20 cm shortly afterward. The
results showed that fertilizer-derived N2O emissions were (as a proportion of N
applied) as follows: anhydrous NH3, 0.86–2.08% (average = 1.33%); aqueous
NH3, 0.04–0.12 (average = 0.07%); and urea, 0.07–0.11% (average = 0.08%).
A laboratory study (utilizing one of the soils) showed that the proportion of N
applied which was emitted as N2O increased markedly with increased application
rate of NH3, rising from 0.18% at 100 ␮g/g soil to 1.15% and 1.19% at 500 and
1000 ␮g/g soil, respectively. It has been shown that the concentration of NH+ 4 -N
frequently exceeds 500 ␮g/g soil where anhydrous NH3 is applied by injection.
Thus, these workers suggest that the large emissions observed with anhydrous
NH3 were due, at least in part, to the highly alkaline conditions which occur due
to the method of application and which were not present where the fertilizers were
broadcast. Thornton et al. (1996) also reported the results of measurements of N2O
emission from anhydrous NH3 and urea solution. Fertilizers were banded 15 and
10 cm below the surface, respectively, at a rate of 168 kg N/ha. Measurements
were made every 3 h from May 6 to September 12, 1994. The results showed that
fertilizer-derived emissions were 7.3 and 3.8% of the applied N for anhydrous
NH3 and urea, respectively. N2O emission from urea solution was much greater
than had previously been reported. The large N2O emission from urea solution in
this study was ascribed to the fact that it was banded below the surface rather than
surface applied. Correlations with soil properties in the zone of fertilizer application
suggested that the main source of N2O was denitrification (correlated with nitrate
concentration and water-filled pore space >60%). Two other points were worthy
of note. First, whereas most studies extrapolate total emissions from, say, weekly
measurements, in this study determinations were based on quasicontinuous data.
It was shown that for these data, N2O loss estimates would have varied by a factor
of up to 2.6 if these had been based on extrapolation from weekly measurements.
Second, while other studies with AN have shown that most of the N2O was lost
in the first 42 days after application, in this study the period of elevated emissions
lasted for about 60 days.
Duxbury and McConnaughey (1986) reported the results of field measurements
(using static chambers) of N2O emission and denitrification (in situ acetylene
block technique). The experiment was carried out over an 85-day period between
fertilizer application (July 5) and harvest (September 28, 1981) of maize on a silt
loam soil. Measured N2O fluxes were greatest following rainfall, especially during
the middle 14 days of August when there was 72 mm of rain. Total emissions during
the experiment period were 0.3, 0.3, and 2.5 kg/ha N2O-N for nil fertilizer, CN,
and urea, respectively. The fact that there was no difference between nil fertilizer
and CN suggested that NO− 3 availability did not limit emission, which may have
resulted from nitrification or denitrification. The additional emission from urea
was ascribed to nitrification.
Keller et al. (1988) reported measured N2O emissions following fertilization
of tropical forest soils. Their results showed that NO− 3 addition resulted in a
loss of ∼0.5% of the applied N, which was more than five times greater than
that for NH+ −
4 addition. The emission factor for NO3 was considered by these
authors to be large in comparison to other reported values, particularly as their
data were integrated over only 2 weeks; they quoted values for 28 separate mea-
surements of N2O from N fertilizer. They concluded that their study revealed a
large potential source of N2O from denitrification, with a much smaller response
associated with nitrification. No heavy rainfall events were recorded during the
experiment period and soil temperature at 5 cm remained in a narrow range be-

tween 24 and 26◦ C.
Recent studies in Scotland (Clayton et al., 1997) and France (Hénault et al.,
1998b) clearly showed that N2O emissions from N fertilizers may vary substan-
tially within and between seasons. Clayton et al. (1997) presented the results of field
measurements over 2 years of N2O emission from different fertilizers applied at
120 kg N/ha to grassland at three times of the year (April, June, and August). Their
results showed that over the 2 years N2O losses followed the order urea ∼ = AN ∼=
CN > AS, with annual fertilizer-derived emissions ranging from 0.2 to 1.4% of the
N applied. However, the temporal pattern of losses was different for different fer-
tilizers and between years. Emissions following the April application followed the
order CN ∼ = AN > urea ∼ = AS. Thus, in both years conditions were more favorable
for denitrification than for nitrification. The observation that emissions from CN
were similar to those of AN in April (and at other times of the year, see below) might
be explained in part by the difference in soil pH between these treatments; higher
pH values in the CN treatment (probably associated with the liming effect of cal-
cium and root anion exchange) would tend to decrease the N2O:N2 denitrification
ratio (Granli and Bøckman, 1994). This might more than offset any increase in den-
itrification rate due to increased pH. However, the denitrification product ratio also
increases with increasing NO− 3 concentration (Granli and Bøckman, 1994). These
workers noted, however, that a large proportion of the total N2O emission from CN
and AN in each period was emitted within 1–3 weeks of fertilizer application. They
suggest that this might indicate a rapid decline in the soil NO−3 concentration due to
plant uptake (and denitrification). Emissions following the June and August appli-
cations varied depending on the prevailing conditions. When these were dry (June
1992 and August 1993) emissions were small; greatest for urea (0.5 and 0.4%)
and least for AS (0.1 and 0.1%). Emissions from AN (0.1 and 0.4%) and CN (0.1
and 0.1%) were more or less intermediate. It appears that for these applications,
conditions were not especially favorable for denitrification, and probably both ni-
trification and denitrification contributed to the observed fluxes, with the effect
of increasing temperature and increasing N2O:NO− 3 nitrification ratio being more
important relative to the situation in April. The marked feature of these measure-
ments was the consistently greater emissions from urea and lesser emission from
AS. This observation was again ascribed to the differing effects of the fertilizers
on soil pH. It is generally thought that the large NH3 concentration associated with
the pH rise resulting from the hydrolysis of urea inhibits the oxidation of nitrite to
nitrate by Nitrobacter (Cochran et al., 1981), leading to nitrite accumulation (Van
Cleemput and Samater, 1996) and hence enhanced N2O production. In contrast,
lower pH values (in this case associated with AS application) have been shown
to inhibit nitrification and the production of N2O. When the prevailing conditions
were wet (June 1993 and August 1992), the order of emissions was urea > AN ∼ =
CN > AS. Under these conditions, denitrification became more important relative
to drier conditions at the same time of year. However, recent data comparing AN
and urea have suggested that the relative emissions from different fertilizers may
be more complicated than originally suggested (K. A. Smith, personal commu-
nication). While emissions in spring tend to be greater from AN than from urea,
this is not always the case: In fact, from 11 series of measurements, emission was
greater from AN on 8 occasions, but N2O emission was greater from urea than AN
on the occasion with the greatest emission. The picture is even more complicated
for the later fertilizer applications. From 21 series of measurements, emission was
greater from urea on 14 occasions, but for the 10 occasions with the greatest N2O
emissions, there were 5 each with the greater emission from AN and urea.
Velthof and Oenema (1994) measured N2O emissions in the spring following
the application of different fertilizer types or urine. Frequent measurements were
carried out over a 23-day period using closed chambers and photoacoustic in-
frared spectroscopy. Fertilizers AS, CAN, CN, and urea were applied at 80 kg
N/ha, and urine was applied at 275 kg N/ha. Emissions from the fertilizers were
not significantly different from each other. Fluxes were generally small (mean 2.5
g N/ha/day), which was ascribed to the cold and dry conditions. In a further series
of experiments (Velthof et al., 1997), carried out to investigate the effect of fertil-
izer type on N2O emission from grassland in The Netherlands, fertilizer-derived
emissions measured over 3–4 weeks under cold and dry conditions (6.0◦ C, 13
mm rainfall) were small (<0.1%) and showed no differences between the differ-
ent fertilizers CAN, CN, AS, and urea. However, in another spring experiment
under slightly warmer and significantly wetter conditions (8.2◦ C, 42 mm rain-
fall) emissions were much greater, especially from the nitrate-based fertilizers.
Fertilizer-derived emissions were 5.2, 5.2, 0.2, and <0.1% from CAN, CN, AS,
and urea, respectively. That these emissions were derived largely from denitrifica-
tion was confirmed by the large total denitrification fluxes and by the fact that the
use of a nitrification inhibitor (DCD) with AS only reduced N2O emissions slightly
(to <0.1%). Similar results were obtained from a summer experiment (16.0◦ C, 68
mm rainfall), with emissions of 8.3, 12.0, 1.0, and 0.7% from CAN, CN, AS, and
urea, respectively. In this experiment, N2O appeared to be the main product of deni-
trification (i.e., N2O fluxes were equal to total denitrification fluxes). DCD reduced
N2O emission from AS to 0.1%. In comparison to other workers (e.g., McTaggart
et al., 1994), N2O emissions from urea and AS were similar. It was postulated
that in the experiments reported above, conditions were either cold enough to slow
down urea hydrolysis and hence limit the pH rise at microsites or wet enough to
disperse the alkalinity such that N2O emission from urea and AS were similar.
In contrast (Velthof and Oenema, 1994), emission from urine was 0.5% of the N
applied. It was postulated that this was due to the high NH+ 4 concentration; this
would support a high NH3 concentration which would lead therefore to enhanced
N2O production. Indeed, Wang and Rees (1996) found (under laboratory condi-
tions which were favorable for nitrification) that ammonium concentration alone
was not sufficient to predict N2O emission; including pH as an additional variable

in the regression equation allowed 92% of the variability to be accounted for. The
experiment (carried out on a range of 10 Scottish soils at a matric potential of 500
mm) consistently showed that emissions over a 14-day period were greater for
urea (average-60.1 mg N2O/kg soil) than for CN (11.6 mg N2O/kg soil; cf. 8.4 mg
N2O/kg soil for no fertilizer control). Emissions from a wider range of fertilizers
with a single soil over 16 days were greatest for AS (215 mg N2O/kg soil) and
least for CN (6.3 mg N2O/kg soil; cf. 4.2 mg N2O/kg soil for no fertilizer control).
Hénault et al. (1998a) carried out a comparison of N2O emissions from different
fertilizers applied to oilseed rape at Longchamp, Burgundy, France. Measurements
were made periodically from November 1996 to July 1997. Four fertilizers were
used: AN, AS, urea, and, potassium nitrate (PN). The results showed that fertilizer
type affected both soil pH and soil mineral N. Soil pH was lower in fertilized plots
(compared to the no fertilizer control) and the decrease was particularly marked
for AS. Nitrate concentrations were greatest in the PN treatment in March and in
the AS treatment at the beginning of May. Measurement of N2O emission showed
that fertilizer type had an effect during two periods: for 1 month following fertilizer
application and from mid-June until harvest. For the earlier period, N2O emissions
were greatest with AN and AS and least with PN. The urea treatment showed
a strongly increasing trend, but with high temporal variability which could not
be accounted for on the basis of soil mineral N (NH+ −
4 or NO3 ) or pH. For the
later period, N2O emissions were greatest for PN and least for AN and urea.
These differences between fertilizer treatments could not be explained in terms
of differences in soil mineral N or pH. Overall, fertilizer-derived emissions were
0.53, 0.55, 0.42, and 0.42% of applied N for AN, AS, urea, and PN, respectively.
However, these values were small compared to measurements obtained 2 years
earlier from a similar soil approximately 2 km from the site used in this study,
where emissions had been ∼2.5% of the applied N (Hénault et al., 1998b). In that
study, the largest fluxes were obtained from AN and urea > nitrate > NH+ 4 . The
smaller losses in the later study were ascribed to a drier spring for that period
compared with the equivalent period in the earlier study. The results indicated that
the effect of N fertilizer type were very limited with respect to time and that in this
study differences were due to differences in nitrification rates.
It is clear that although N fertilizer type certainly affects the extent of N2O
emission, appropriate emission factors for individual fertilizer types have not been
generally agreed. Leaving to one side the magnitude of emissions from anhydrous
NH3 and from injected urea solution, it is apparent that emission factors for broad-
cast urea, AS, AN, and NO− 3 salts depend on the conditions in the period after
fertilizer application. Both nitrification and denitrification contribute to the emis-
sion, and it is possible to rationalize (but more difficult to predict) results for NH+ 4
and nitrate fertilizer types on the basis of the season of application. The position
of urea appears anomalous, however; whereas Clayton et al. (1997) suggested
Table III
Proposed Scheme for Assessing Relative Emissions of N2O from
Differing Fertilizers
Relative emission Relative emission

Moisture for N forms from urea Notes
Dry Low Nitrate ∼

= Urea ≥ ammonium Rate of urea
ammonium hydrolysis limited
Wet High Nitrate > Urea ≫ Rate of urea
ammonium ammonium hydrolysis increases
with temperature
Very wet High Nitrate ≫ Urea ∼
= ammonium High pH associated
ammonium with hydrolysis
dispersed by moisture
that emissions can be relatively large under conditions when emissions from AS
are relatively small, Velthof et al. (1997) obtained similar emissions from both
fertilizer types. Work by Smith and co-workers for a range of Scottish sites indi-
cated that there was a good relationship between rainfall at and around the time
of fertilizer application and N2O emission from AN. Although earlier results had
suggested a similar relationship for urea (albeit with lower emissions), greater
than expected emissions were measured from urea in the extremely wet 1998
season (K. A. Smith, personal communication). The suggestion that temperature
(controlling the rate of urea hydrolysis) and rainfall (controlling the dispersion of
alkalinity) are important determinants obviously needs further investigation.
As a first approximation, we suggest that the relative emissions from N fertilizers
mught be considered as outlined in Table III. Thus, emissions are generally greater
from NO− +
3 -based fertilizers compared to NH4 fertilizers, and this difference
increases with increasing moisture. Under dry conditions, emissions from urea
may be slightly greater than from other nitrifiable fertilizers, but since emissions
under these conditions are small this is not of great significance. Under wet (and
particularly, warm) conditions, emissions from urea are significantly greater than
those from NH+ 4 -based fertilizers and urea emissions may exceed those from
NO− 3 -based fertilizers, particularly at the lower end of this moisture category.
However, the work of Smith and co-workers shows that as moisture increases so
does the relative emission from NO− +
3 compared to NH4 -based fertilizers, and so at
the high end of the “wet” moisture category, there are significant emissions from
both NO− 3 -based fertilizers and urea; the fertilizer from which gives greater emis-
sion is probably extremely sensitive to both moisture and temperature. The results
of Velthof et al. (1997) seem to suggest that there is a further “very wet” category
where emissions from urea become similar to those of NH+ 4 -based fertilizers;
however, it is important to account for the effect of soil type on the “wetness”
category. The results of Velthof et al. (1997) were obtained from a poorly drained
sand soil compared to the heavier textured soils studied by Smith and co-workers.
B. THE EFFECT OF NITRIFICATION INHIBITORS ON NITROUS

OXIDE EMISSIONS
Studies with nitrification inhibitors have demonstrated that there is consider-

able scope for reducing N2O emissions from NH+ +
4 or NH4 -forming fertilizers.
Magalhães et al. (1984) reported results from measurements of N2O emission
following banded application of anhydrous NH3 at a depth of 15 cm. The re-
sults from this study suggest a much smaller N2O emission factor for anhydrous
NH3. In one soil (Mywybilla, pH 6.9) there was no significant effect of fertil-
izer application on N2O emission. For the other two soils (Anchorfield, pH 7.5;
Norillee, pH 8.5, calcareous) there was an effect of fertilizer application; how-
ever, the largest emission factor was only ∼0.05% (for the Norillee soil). This
experiment was carried out under relatively cool and dry conditions, with aver-
age soil temperatures at 17 cm of between 12.5 and 17.5◦ C and the soil water
content at 0–20 cm—well below field capacity. It has been suggested that N2O
losses in soil fertilized with anhydrous NH3 result from the reduction of nitrite,
which tends to accumulate in the soil surrounding the zone of NH3 injection. In
this study, application of a nitrification inhibitor (nitrapyrin) significantly reduced
N2O emission from the Norillee soil; this was associated with a large (72%) de-
crease in nitrite concentration but only a moderate (48%) decrease in the rate of
nitrification of the fertilizer. In contrast, nitrapyrin had no effect on N2O emis-
sions from the Anchorfield soil but slightly reduced nitrite concentration and the
rate of nitrification of the fertilizer. The difference between these two soils was
ascribed to a lower bioactivity of the inhibitor in the higher organic matter content
Anchorfield soil.
Aulakh et al. (1984) carried out a field experiment on a wheat stubble field
to compare N2O emissions from PN and urea. Fertilizers were applied at 50
kg N/ha in autumn and measurement showed that fertilizer-derived emissions
continued for about 4 weeks. There was a significant increase in N2O emission
from the urea treatment over and above the unfertilized control but this did not
occur for the PN treatment. Addition of the nitrification inhibitor N-serve (2-
chloro-6-(trichloromethyl)-pyridine) reduced emissions from urea to background
levels. These results suggest that the mechanism for N2O production in this ex-
periment was nitrification. Soil moisture was 26–37% saturation during the pe-
riod of the experiment. Bronson (1993, 1992) and co-workers determined N2O
emission from soils in the field (using vented chambers) in irrigated corn sys-
tems. In one experiment (Bronson et al., 1992), the treatments were urea alone,
urea plus nitrapyrin (U+np), urea plus 20 kg/ha encapsulated calcium carbide
(U + ECC20), and urea plus 40 kg/ha encapsulated calcium carbide (U + ECC40).

Fluxes of N2O were positively correlated with soil NO− 3 , indicating that the ni-
trification inhibitors indirectly controlled N2O emissions by preventing nitrate
from accumulating in the soil. For the first crop (1989) total losses were 3.2, 1.1,
1.0, and 1.0 kg/ha N2O-N from urea, U + np, U + ECC20, and U + ECC40,
respectively. For the second crop (1990) losses were less (1.7 kg/ha N2O-N for
U), probably because there were fewer irrigation events. In a second experiment
(Bronson and Mosier, 1993) the results for cumulative emissions (from time of
fertilization 2 months after planting to harvest 97 days later) were as follows:
1.65, 0.98, 0.48, 0.43, and 0.11 kg/ha N2O-N for urea alone, with nitrapyrin,
with ECC at 20 kg/ha, with ECC at 40 kg/ha, and for the no-fertilizer control,
respectively.
McTaggart et al. (1994, 1997) demonstrated that there was considerable scope
for reducing N2O emissions by applying nitrification inhibitors with NH+ +
4 or NH4 -
forming fertilizers. This is especially so for crops such as intensively managed grass
where there are several applications of fertilizer N over the season, as the effect
of inhibitors applied in April persisted until after a second fertilizer application in
June. Their results showed that for fertilizer applied to grass at 120 kg N/ha in April,
June, and August (i.e., 360 kg N/ha in total), DCD applied in April and August
reduced the annual N2O emission from urea by 58% in 1992–1993 and by 56% in
1993–1994 and reduced the annual N2O emission from AN by 35% in 1993–1994.
The results for nitrapyrin applied in April showed a reduction of 40% in the annual
N2O emission from urea in 1992–1993. There was no effect of DCD or nitrapyrin
on the annual emission from AS in 1992–1993, although there was a reduction
for DCD in 1993–1994. However, emissions from AS were less than from the
other fertilizers. These authors also reported the results of measurements of N2O
emission from fertilizers applied to spring barley. Their results show that total N2O
emissions over 56 days were 0.6, 0.4, and 0.3 kg N/ha from two spring applications
of 60 kg N/ha as urea, from AN, and from a no fertilizer control, respectively.
Because water-filled pore space was <60% throughout the measurement period,
it was suggested that nitrification was the main mechanism for the emissions. The
nitrification inhibitor DCD reduced emissions from urea by 40% but had no effect
on those from AN.
VI. NITROUS OXIDE EMISSION FACTORS FOR

NITROGEN FERTILIZERS
In contrast to NH3 emissions, there have been fewer studies of the effect of N
fertilizer type onN2O emissions. Bolle et al. (1986) stated that “until recently it was
generally assumed that about 10–15% might be lost as N2O [but] new data show
that this figure is much too high and that the loss as N2O is strongly dependent
on the mode of application and type of fertilizer.” These authors noted that the
greatest loss rates have been observed for anhydrous NH3 and NH+ 4 fertilizers,
which supports the view that nitrification is the dominant N2O production process,
and also note results (Breitenbeck et al., 1980; Conrad et al., 1983; Slemr and
Seiler, 1984) which show average N2O loss rates of 0.04% for nitrate, 0.15–0.19%
for ammonium and urea, and 5% for anhydrous NH3.
Eicher (1990) reviewed direct measurements of fertilizer-derived N2O emissions
from 104 field experiments published in the literature and carried out between 1979
and 1987. Of the 104 experiments, 49 did not sample emissions from an unfer-
tilized control site so fertilizer-derived emissions could not be distinguished from
natural sources of N2O and 8 of the controlled studies had fertilizer application
rates >250 kg N/ha and were therefore excluded from subsequent analysis because
such rates are much greater than typically used in agricultural systems. The results
showed fertilizer type to be an important factor influencing emissions. The averages
were as follows: anhydrous NH3 (2.70%, range 0.86–6.84%) > AN (0.44%, range
0.04–1.71%) > ammonium (0.25%, range 0.02–0.90%) > urea (0.11%, range
0.07–0.18%) > nitrate (0.07%, range 0.001–0.50%). It was noted that several im-
portant variables that affect emissions were not summarized or considered in the
experiments surveyed; e.g., temperature and the amount or intensity of rainfall,
the timing of these events with respect to emission, and the presence of resid-
ual plant material. In addition, the studies reviewed suggested that most of the
fertilizer-derived N2O is emitted during the growing season (often shortly after
fertilization), although a significant amount has been found to be released during
spring thaw. In most of the experiments, the duration of the sampling period cap-
tured the large, often episodic flux that occurs shortly after fertilization. Thornton
et al. (1996) briefly reviewed other work dealing with emissions from different
fertilizers. They pointed out that although the OECD/OCDE use different fac-
tors for different fertilizers (based on the work of Eichner, 1990), Mosier (1993)
noted that these were based on very limited data; they cited recent laboratory
studies (Mulvaney and Khan, 1994) of N2O and N2 losses which indicated that
emissions decreased in the order anhydrous NH3 > urea > DAP > AS > AN >
MAP. Median values of N2O loss from urea appear to be in the range 0.1 to 0.6%
of applied N (Granli and Bøckman, 1994). Most recently, Smith et al. (1997)
noted that no allowance has been made for different fertilizer types in IPCC emis-
sion factors for N2O on the basis that soil management and cropping systems
and unpredictable rainfall inputs were more important variables. Mosier (1994)
and Bouwman (1994) concluded that the literature data relating to N2O emissions
from agricultural soils were too limited to calculate the individual emission factors
for different fertilizers. Smith et al. (1997) also noted that it had been suggested
that the large emission factor suggested for anhydrous NH3 may be unrepresen-
tative because in the data set used there was no plant sink to compete for the
applied N (fallow soil) and cropresidues were also present possibly enhancing
denitrification.
VII. NITRIC OXIDE EMISSIONS FROM NITROGEN

FERTILIZERS
There is very little published data concerning nitric oxide (NO) emissions from
different N fertilizer sources. Veldkamp and Keller (1997) have summarized and
evaluated 23 published studies of NO emission following fertilizer addition. Emis-
sion factors were estimated using only studies carried out over at least a full growing
season and where measurements had been made on a field scale. Only 6 studies
met these criteria, all of which were carried out in temperate regions. An emission
factor of 0.5% was determined (although this was considered to represent the lower
limit), and the data were too limited to separate the effect of fertilizer type (or soil
type or crop management). Slemr and Seiler (1984) made measurements of NOx
emission at two sites: Finthen, Germany and Utrera, Spain. Their results showed
that application of mineral fertilizers increased NO and NO2 emission rates. The
largest fertilizer-derived emission rates were obtained from urea (3.3 and 2.2% for
NO and NO2, respectively), followed by NH4Cl, AN, and least from NaNO3 (0.04
and 0.07% respectively). Thornton et al. (1996) measured emissions of 0.2 and
0.3 kg/ha NO-N for anhydrous NH3 and urea banded 15 cm and 10 cm below the
surface, respectively, at a rate of 168 kg N/ha. Measurements were made every 3 h
from May 6 to September 12, 1994.
VIII. SUMMARY AND CONCLUSIONS
A. AMMONIA
There have been many investigations of NH3 volatilization, which is now well
understood. Volatilization is essentially a physicochemical process. Overall, results
from field comparisons of NH3 loss from different fertilizers follow the pattern
expected on the basis of known chemistry. Emissions from urea are the most
variable, ranging from 6 to 47% of applied N, and are very dependent on factors
such as soil type (via CEC rather than soil pH), weather conditions, and application
rates. In contrast, reported emissions from AN (and CAN) were small, never
exceeding 4% of applied N. There are fewer studies involving other fertilizers
such as AS and DAP, but for these, emissions are greater than that for AN and less
than that for urea, although on calcareous or other high pH soils losses from AS
may be greater than those from urea. Variations in emissions result from differences
in soil type and time of application. In general, it is considered that emissions from
other fertilizers are less than those from urea, with the possible exception of AS
and DAP on calcareous or otherwise alkaline soils.
With respect to urea, a greater NH3 loss on calcareous soils does not appear
to be justified. While NH3 losses from AS and AN have been found to increase
markedly with increasing pH (e.g., Whitehead and Raistrick 1990), the hydrolysis
of urea to (NH4)2CO3 increases pH around the fertilizer granule to ∼9 and so
tends to override bulk soil pH. Moreover, reaction with calcium ions reduces
the volatilization potential of (NH4)2CO3 produced by urea hydrolysis (Fenn and
Hossner 1985), and hence, NH3 losses from urea have not been found to be greater
on a calcareous soils (Whitehead and Raistrick, 1990; Gezgin and Bayrakli, 1995).
Results show that emissions from urea–AN solutions are intermediate between
those from urea and AN granules (Fox et al., 1996; Keller and Mengel, 1986;
McInnes et al., 1986a,b). Studies by Lightner et al. (1990) indicate that the effect
of applying urea in solution depends on the moisture status of the soil at and
immediately after application. Where the soil is dry, emissions will be small but
application as a solution may increase NH3 volatilization. Rainfall appears to have
a greater influence in increasing emissions from urea granules than from urea
applied in solution. Addition of urease inhibitor has been shown in field studies to
significantly improve the performance of urea (Watson et al., 1998).
There have been a number of attempts to derive NH3 emission factors for fer-
tilizers in recent years. Without additional data or specific cause, it does not seem
appropriate to increase this number. We, therefore, suggest that, with the exception
of AS, the factors proposed by van der Weerden and Jarvis (1997), who consid-
ered that NH3 emissions are greater from fertilizers applied to grassland than arable
land, should be accepted. Thus, emission factors are as follows: grassland: urea,
23%, and AN (and compound fertilizers), 1.6%; arable land: urea, 11.5%, and AN
(and compound fertilizers), 0.8%. However, there is strong evidence that emissions
from AS are strongly dependent on soil pH, and we therefore suggest factors of
2 and 18% for this fertilizer for soils with pHs <7 and >7, respectively. These
estimates have been made following the methodology of Whitehead and Raistrick
(1990). Applying these emission factors to current N fertilizer use, we estimate
that replacing urea with AN would reduce UK NH3 emissions by ∼6,700 tonnes
NH3-N. It should be noted that on calcareous soils, or those limed to a pH >7
(as may be the case in arable rotations involving sugarbeet), losses of NH3 from
AS may be as great or greater than from urea. Due to very large reductions of
emissions of SO2 and subsequent decreases in sulfur deposition since 1998, sulfur
deficiency has become apparent in some crops in some areas. The use of AS fer-
tilizer is one means of overcoming this deficiency. As yet, use of AS is small, but
in the interests of minimizing NH3 emissions it may be argued that other means
of preventing sulfur deficiency need to be encouraged.
A major criticism of the present estimates is their reliance on simple fixed

(%) emission factors, given in relation to amounts of N applied. More work
needs to be done in the development of mechanistic process-based models for
predicting NH3 emissions from N fertilizers and the foliage of fertilized crops,
which take into account the known physicochemical equilibria as well as inter-
actions with biological processes to predict net fluxes. It is well established that
NH3 may be exchanged with the soil surface and with leaves via stomata and cu-
ticular absorption/desorption as well as with decomposing leaves, and future work
needs to quantify the interactions and exchange cycles between these different
components.
This study confirms there is potential for reducing NH3 emissions by switching
from urea to other N fertilizers. A further possibility is to add urease regula-
tors/inhibitors to urea fertilizer, which are expected to reduce emissions. Costs of
these measures would include the differential price of more expensive fertilizers
or of inhibitors. Emissions may also be reduced by placing the fertilizer granule
into the soil at the same depth as the seed (∼7–8 cm). This will only be applicable
for crops sown in the spring (apart from grass reseeds in autumn).
B. NITROUS OXIDE
In contrast to NH3 emissions, there have been fewer studies of the effect on N
fertilizer type on nitrous oxide (N2O) emissions. Emission factors of 0.04, 0.15–
0.19, and 0.50 for nitrate salts, ammonium salts and urea, and anhydrous NH3,
respectively, have been proposed by Bolle et al. (1986). Recent laboratory studies
(Mulvaney and Khan, 1994) of N2O and N2 losses indicated that emissions de-
creased in the following order: anhydrous NH3 > urea > DAP > AS > AN >
MAP. Median values of N2O loss from urea appear to be in the range 0.1 to 0.6%
of applied N (Granli and Bøckman, 1994).
Studies in Scotland (Clayton et al., 1997) and France (Hénault et al., 1998b)
clearly showed that N2O emissions from N fertilizers may very substantially within
and between seasons. Results from the Scottish study indicated that emissions are
likely to be greater from calcium nitrate and AN than from urea in the spring
if conditions are cool and wet and vice versa in the summer if warm and wet.
Emissions from AS will probably be less than from other fertilizers. However,
recent data comparing AN and urea have suggested that the relative emissions from
different fertilizers may be more complicated than originally suggested. While
emissions in spring tend to be greater from AN than from urea, this is not always
the case.
Overall, the literature appears to support the view that a significant proportion
of N2O emissions occur from nitrification, although this is crucially dependent
on the interaction between timing of fertilzer application and weather. Conditions
in spring are more likely to be wet, and in this situation (and excluding urea for
the moment) emissions are greater from NO− 3 -based fertilizers and least from AS.
In the summer conditions may be dry or wet: under dry conditions emissions are
smaller than under wet conditions. Again excluding urea, greatest emissions in the
summer occur from AN. For urea, the effect of pH appears to be important. Gener-
ally, greater emissions can occur from urea, except where temperature (controlling
the rate of urea hydrolysis) and rainfall (controlling the dispersion of alkalinity)
limit this. Thus, the substitution of AN for urea for spring applications is likely to
increase N2O emission. For summer applications, the substitution of AN for urea
is likely to decrease N2O emissions, providing conditions are relatively dry; when
conditions are wet high emissions may occur from both AN and from urea. At
this stage it is difficult to say with any certainty whether a strategy based on AN
or urea will result in the lowest N2O emissions; further work both on the factors
controlling emission from urea (and AN) combined with assessments of weather
variablity are required. However, it seems likely that the optimum strategy will be
one involving a sophisticated appreciation of the interaction between N fertilizer
form and timing of application.
Studies with nitrification inhibitors (e.g., McTaggart et al., 1997) have demon-
strated that there is considerable scope for reducing N2O emissions from
ammonium (NH+ +
4 ) or NH4 -forming fertilizers. This is especially so for crops
such as intensively managed grass where there are several applications of N fer-
tilizer over the season, as the effect of inhibitors applied in April persisted until
after a second fertilizer application in June.
C. NITRIC OXIDE
On theoretical grounds, since most NO emissions occur during nitrification,

replacing urea with AN should reduce those emissions. The results from Slemr
and Seiler (1984) are consistent with this hypothesis, but these conclusions can
only be tentative as there is still a paucity of data from field experiments.
D. OVERALL
We conclude that replacing urea with AN has the potential to significantly reduce
NH3 emissions without increasing losses of N2O, albeit emissions of this gas are
less predictable and more dependent upon season and weather. The effect on emis-
sions of NO is uncertain, but current data do not suggest these will be increased.
Published studies do not suggest the use of urea in solution will reduce losses of
NH3. Data on the use of urease inhibitors is limited, but those available suggest
the use of urease inhibitors may significatly reduce NH3 emissions from urea.
ACKNOWLEDGMENTS
This work was funded by the UK Ministry of Agriculture, Fisheries and Food. We thank K. A. Smith
for discussion during the preparation of this chapter and E. Lord for helpful comments on the text.
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RHIZOBIA IN THE FIELD
N. Amarger
Laboratoire de Microbiologie des Sols
Institut National de la Recherche Agronomique
21065 Dijon, France
I. Introduction
II. Diversity in Rhizobia
A. Cultural, Physiological, and Biochemical Characteristics
B. Serological Characteristics
C. Chemical Composition
D. Symbiotic Characteristics
E. Deoxyribonucleic Acid
III. Rhizobium Systematics
A. From Cross-Inoculation Grouping to Polyphasic Taxonomy
B. Phylogeny and Taxonomy
C. Identification
IV. Natural Populations of Rhizobia
A. Diversity of Populations
B. Population Structure
V. Introduction of Rhizobia into Soil
A. Inoculation
B. Soil Colonization by Inoculant Rhizobia
C. Interactions with Indigenous Rhizobial Populations
D. Agricultural Implications
VI. Concluding Remarks
References
Most of the legumes have the ability to establish a dinitrogen-fixing association

with bacteria defined as rhizobia. The legume crops will benefit from this symbiosis
only when the plant roots encounter, during their development, compatible and efficient
rhizobia that can induce the formation of fully effective nodules. The rhizobial pop-
ulations present in the field soils therefore play a key role in legume productivity. In
recent years, the development of molecular biology has provided new tools that have
allowed molecular characterization of rhizobia. This has lead to the description of a
new diversity and to the creation of new taxons in order to handle it. Studies on the
distribution of this diversity among rhizobial populations isolated from the nodules of
the most widespread legume crops have shown that these populations are composed of
a great variety of genotypes which can belong to different species or genera. Although
environmental constraints may reduce the diversity, in many instances a great part of
the genetic variation present within each species can be maintained within populations
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and individual plants. The structures of the rhizobial populations suggest that genetic
recombination plays an important role in generating diversity within these populations.
Following introduction of new rhizobial genotypes through seed inoculation, highly
diversified populations can develop in relatively short periods of time. The extensive
diversity that has been revealed has now to be managed in order to optimize nitrogen
fixation by legume crops. C 2001 Academic Press.
I. INTRODUCTION
The contribution of legumes to the maintenance of soil fertility has been rec-
ognized for centuries. However, it was only in the middle of the 19th century
that Boussingault demonstrated that leguminous plants have the ability to ob-
tain their nitrogen from a source other than mineral nitrogen, which thus could
only be from the atmosphere. Later in that century, in 1888, Hellriegel and
Wilfarth established that the small tubers, or nodules, present on the root sys-
tems of legumes are the seat of the assimilation of atmospheric nitrogen. The same
year, from root nodules of several legumes, Beijerinck isolated bacteria that he
demonstrated were the causative agents of the fixation of atmospheric nitrogen.
These bacteria which possess the property of forming nodules on the root sys-
tems of legumes are now collectively referred to as rhizobia. The nodules are
the expression of a symbiotic association between a rhizobium and a legume: the
bacteria reduce dinitrogen to ammonia and supply nitrogenous compounds to the
plant, which in return supplies nutrients to the bacteria. The bacteria can multi-
ply in a protected habitat from which they are released in large numbers upon
senescence of the nodules, and the plant gains independence of the presence of
nitrogenous compounds in the soil environment. Rhizobia are facultative sym-
bionts. In the free-living state, they are common soil inhabitants and are, with
very few exceptions, unable to fix dinitrogen. Legumes are very diverse and dis-
tributed worldwide, and many, but not all, of the 10 to 15% that have been checked
for the presence of nodules are able to nodulate. Among the 16,000 to 19,000
species composing the Leguminosae family (Allen and Allen, 1981), less than 1%
mostly Papilionoideae, are of agricultural importance. Nevertheless, these plants,
in addition to being economically important, with edible and highly nutritional
crops for both human and animal consumption, forage crops, and green manure,
also play a key role in sustaining long-term soil fertility in agricultural systems.
Global annual inputs of biologically fixed nitrogen in agricultural systems through
the activity of rhizobium–legume associations are estimated to be equivalent to
RHIZOBIA IN THE FIELD 111
inputs in the form of nitrogen fertilizer. Some 35 to 44 million tons would come
from legumes growing in arable land and another 40 million tons from permanent
pasture (Peoples et al., 1995).
The symbiotic interactions between rhizobia and legumes are host specific. It
became apparent soon after the isolation of the first rhizobia that a given nodule
isolate has the ability to produce nodules on certain plants and not on others. The
range of hosts a particular strain of rhizobium nodulates can be rather narrow, a few
legume species, or extremely broad, hundreds of species. These differences in host
range between strains of rhizobia served as the first basis of the classification into
“cross inoculation groups” and later into species. Further steps in the development
of the nitrogen fixing symbiosis can also show some degree of host specificity.
As a result, a given bacterial strain can form nitrogen-fixing nodules with certain
hosts (the symbiosis is effective) and nonfixing nodules with others (the symbiosis
is ineffective). Great variations in specificity of interaction with rhizobia are also
observed among legume species. Some legumes form nodules with a restricted
range of strains with similar nodulation and physiological characteristics that are
usually classified in the same taxon. Other legumes, said to be promiscuous, nodu-
late with strains differing in many respects and belonging to different taxa. In this
latter case, various levels of effectiveness of the nitrogen-fixing symbiosis may be
attained depending on the rhizobia that formed the nodules. Legumes cultivated
in temperate regions tend to be less promiscuous than those of tropical regions.
The ability of a legume crop to benefit from the fixation of atmospheric nitrogen
will be dependent on the presence in the field soil of rhizobia able to nodulate
the legume and to form an effective nitrogen-fixing symbiosis with the host plant
under field-growth conditions. When soils are devoid of the specific rhizobia,
inoculation with effective, host-specific rhizobia usually allows the legume crop
to profit from nitrogen fixation. When the crop nodulates but fails to reach its
yield potential, the question is whether the rhizobia which nodulated the plants are
optimally effective in N2 fixation or not. Although other biotic and abiotic environ-
mental factors may exert constraints on N2 fixation by legume crops, inadequacy
of compatibility between the field rhizobia and the legume is often considered as
being the major cause of suboptimal yields. Whether this is the case is difficult to
ascertain, as long as it is not shown that fully effective strains allow expression of
the crop potential in the same environment. This proof is most often missing since
current inoculation practices usually fail to displace the soil rhizobia from the
nodules. Due to the lack of suitable methodologies to characterize and identify
rhizobia, our knowledge of field populations of rhizobia is rather restricted.
As a consequence, the possibilities that we have to better control and exploit them
are very limited. However, since 1985, biochemical and genetic approaches have
allowed important advances to be made in our understanding of the host-specific
relationships of rhizobia with leguminous plants. In parallel, the development
112 N. AMARGER
of molecular biology has provided new tools to characterize rhizobia, tools that
are currently used to study natural rhizobial populations. In this chapter we first
consider the progress made in molecular characterization of rhizobia and the new
diversity this has revealed. We then approach the distribution of this diversity and
its variability in field rhizobial populations and the impact that the introduction of
an alien rhizobium might have on this diversity. Our overall intention is to pool
current knowledge on the symbiotic partners a leguminous crop is confronted with
during its growth.
II. DIVERSITY IN RHIZOBIA
The characteristic that qualifies a bacterium to be named rhizobium, as presently

understood, is its capacity to form a definite nodule on the root or on the stem of
a leguminous plant. This designation has no taxonomic significance; as is shown
below, bacteria belonging to different species and classified in distantly related
genera are capable of nodulating leguminous plants. Rhizobia can show wide
variations in numerous characteristics; only those useful for identification or as
markers in population studies are considered here.
A. CULTURAL, PHYSIOLOGICAL, AND BIOCHEMICAL

CHARACTERISTICS
Differences in rates of growth allowed early separation of the rhizobia into

two basic groups (Fred et al., 1932). Fast growers have generation times of less
than 6 h and generally form visible colonies (2–4 mm in diameter) on agar me-
dia within 2–5 days, whereas slow growers have generation times exceeding 6 h
and give detectable growth after more than 5 days. These differences in rates of
growth between strains can be lessened, but not completely eliminated, by modi-
fication of the carbon or/and nitrogen sources (Allen and Allen, 1950). Recently,
extraslowly growing rhizobia were isolated from nodules of soybeans collected in
the People’s Republic of China (Xu et al., 1995). Their generation times varied
with the strain from 16 to 40 h. Rhizobia isolated from nodules of alfalfa, clover,
pea, and bean are typical fast growers; those isolated from US-grown soybean,
lupine, and Vigna spp. are typical slow growers. Under standardized conditions
most of the slow-growing rhizobia produce alkali while the fast-growing produce
acid (Fred et al., 1932; Norris, 1965). Variations in colony morphology among
isolates from the nodules of a given legume are relatively common. They have
been used as a convenient criterion for differentiation of isolates from nodules of
cowpea (Sinclair and Eaglesham, 1984; Eaglesham et al., 1987; Mpepereki et al.,
1997), common bean (Beynon and Josey, 1980; Amarger et al., 1994), soybean
(Desa et al., 1997; Martins et al., 1997), and diverse legumes grown in Kenya
(Odee et al., 1997).
Rhizobia are chemoorganotrophs that have long been known as being able to
utilize a variety of carbon and nitrogen compounds for their growth (reviewed in
Allen and Allen, 1950; Graham and Parker, 1964; Stowers, 1985). Although vari-
ations are seen within each group, fast growers tend to metabolize a wider variety
of carbohydrates than slow growers. The former can use a broad range of hex-
oses, pentoses, disaccharides, trisaccharides, polyols, and organic acids, whereas
the latter seem less able to use disaccharides, trisaccharides, and organic acids for
growth (Elkan and Kwik, 1968; Chakrabarti et al., 1981; Parke and Ornston, 1984;
Stowers and Eaglesham, 1984). Martinez-Drets et al. (1972) have pointed out that
a key enzyme of the pentose pathway, the NADP-linked 6-phosphogluconate de-
hydrogenase, present in the fast-growing rhizobia, was absent in the slow growers.
This characteristic has proven useful to ascertain the classification of rhizobia into
fast- or slow-growing groups (Kennedy and Greenwood, 1982; Sadowsky et al.,
1983; Anand and Dogra, 1991; Batzli et al., 1992).
Besides sugars, a wide variety of aromatic compounds can be metabolized by
rhizobia through degradative pathways involving inducible or constitutively ex-
pressed enzymes (Glenn and Dilworth, 1981; Muthukumar et al., 1982; Chen
et al., 1984; Parke and Ornston, 1984; Gajendiran and Mahadevan, 1988; Hartwig
et al., 1991; Hopper and Mahadevan, 1997). The spectrum of compounds that can
be metabolized seems strain dependent and may reflect differential adaptation to
legume rhizosphere or soil organic components. Its variation among isolates might
have ecological implications.
With the exception of rhizobia isolated from stem nodules of Sesbania rostrata
(Dreyfus et al., 1988), free-living rhizobia are incapable of utilizing dinitrogen
for growth. They can use nitrate, ammonium, or amino acids as a sole source of
nitrogen. Variability in the use of these different sources has been demonstrated
among isolates of slow- as well as fast-growing rhizobia. Some amino acids, such as
glutamate, can be used as an N source by almost every rhizobium and others, such
as glycine, by only a few strains (Elkan and Kwik, 1968; Chakrabarti et al., 1981).
Differences in substrate utilization are useful not only to differentiate between
slow- and fast-growing rhizobia but also to reveal the diversity among each type.
When the substrates are various and numerous enough, meaningful groupings
can be made using numerical analysis. Recently, miniaturized systems that allow
many substrates to be easily tested have been developed primarily for clinical
applications. They use color reactions to indicate metabolic activities such as the
oxidation of carbon compounds. Two of these systems, API 50 or Biolog, which
allow testing of the ability of 147 and 95 compounds, respectively, to serve as
sole carbon source, were used with rhizobia isolated from tropical woody legumes
(de Lajudie et al., 1994; Dupuy et al., 1994; McInroy et al., 1999) and clover
114 N. AMARGER
(Leung et al., 1994a; Nour et al., 1994a). The results showed that such systems
enable grouping of otherwise genetically related rhizobial strains. Therefore, they
might be useful for identification purposes, provided that many more reference
strains are entered into the databases.
Physiological traits such as tolerance to abiotic or biotic factors are intrinsic traits
of each rhizobium strain. Their variation among strains can be wide enough to allow
distinction between nodule isolates. Such variations have been observed in fast- as
well as slow-growing rhizobia for tolerance to elevated temperature (Graham and
Parker, 1964; Munevar and Wollum, 1981, 1982; Hartel and Alexander, 1984; de
Lajudie et al., 1994; Michiels et al., 1994; Surange et al., 1997), to acidity (Bryan,
1923; Graham and Parker, 1964; Norris, 1965; Graham et al., 1982; Hartel and
Alexander, 1983; Aarons and Graham, 1991; Graham et al., 1994; Elidrissi et al.,
1996; Surange et al., 1997), and to salinity (El Essawi and Abdel-Ghaffer, 1967;
Bhardwaj, 1975; Mendez-Castro and Alexander, 1976; Yelton et al., 1983; Saxena
and Rewari, 1992; Mpepereki et al., 1997).
Large differences in degree of tolerance to antibiotics among fast- and slow-
growing rhizobia have been reported (Graham, 1963; Pattison and Skinner, 1974;
Pinto et al., 1974; Pankhurst, 1977; Cole and Elkan, 1979; Hagedorn, 1979). Since
the first exploitation of these natural strain to strain variations in intrinsic resistance
to antibiotics (IAR) for identification and differentiation of nodule isolates from
common bean (Josey et al., 1979; Beynon and Josey, 1980), IAR has been used
extensively in ecological studies to identify inoculant strains and to determine
heterogeneity in natural populations (Eaglesham, 1987). The method has proven
practical, rapid, and reliable with a discriminating ability dependent on the number
of antibiotics and of concentrations used. IAR has been used either as a primary
criterion or as a complement to other(s) method(s) to describe diversity in nodule
isolates from alfalfa (Jenkins and Bottomley, 1985a; Shishido and Pepper, 1990),
pea (Turco and Bezdicek, 1987; Brockman and Bezdicek, 1989), clover (Hagedorn,
1979; Glynn et al., 1985), Phaseolus sp. (Arredondo-Peter and Escamilla, 1993),
chickpea (Kingsley and Ben Bohlool, 1983; Garg et al., 1985), soybean (Meyer and
Pueppke, 1980; Dowdle and Ben Bohlool, 1986; Mueller et al., 1988; Mpepereki
et al., 1997; Ramirez et al., 1997), cowpea (Sinclair and Eaglesham, 1984; Xavier
et al., 1998), and various tropical legumes (McLaughlin and Ahmad, 1984; Date
and Hurse, 1991; Subramaniam and Babu, 1993).
In recent years the results of tolerance tests to different abiotic factors have
tended to be analyzed as a whole. They form, with the tests involving trophic
capabilities on different carbon and nitrogen sources the core of the phenotypic
characters that are used for numerical taxonomic purposes (Batzli et al., 1992;
Novikova et al., 1994; Madrzak et al., 1995; van Rossum et al., 1995; Elidrissi
et al., 1996; Desa et al., 1997; Gigova et al., 1997; Odee et al., 1997; Struffi et al.,
1998; Vasquez Arroyo et al., 1998).
Root nodule bacteria were found to differ in their susceptibility to differ-
ent phages abundant in soils as early as 1932 (Laird, 1932). Such differences
in susceptibility to various numbers of phages have served as the basis for
phage typing of nodule isolates from alfalfa (Lesley, 1982; Bromfield et al.,
1986), clover (Kankila and Lindström, 1994), Galega spp. (Lindström et al.,
1983), common bean (Dhar et al., 1993), soybean (Hashem et al., 1996; Ali
et al., 1998), sulla (Struffi et al., 1998), and different-temperate legumes (Conn
et al., 1945; Staniewski, 1970; Lindström and Kaijalainen, 1991; Novikova and
Limeshchenko, 1992; Novikova et al., 1993). Although phage typing can be highly
discriminatory, its use is limited because it requires prior isolation and consti-
tution of a battery of phages differing in their ability to lyse the strains under
study.
B. SEROLOGICAL CHARACTERISTICS
Serological methods, which are based on the antigenic nature of the cell surface
and on the specificity of these antigens, provide rapid means for identifying bac-
teria. Several variants of the serological method, agglutination, immunodiffusion,
direct or indirect immunofluorescence (FA), or enzyme-linked immunosorbent
assay (ELISA), have been used in the examination of serological diversity of rhi-
zobial isolates (Schwinghamer and Dudman, 1980; Vincent, 1982; and references
therein). On the basis of reactions with a set of antisera against reference strains
rhizobial isolates can be assigned to a given serogroup or serotype. The antigenic
diversity of the slow-growing nodule isolates from soybean is probably the best
known and most of the populations can be described from the existing serogroups
(Johnson and Means, 1963; Date and Decker, 1965; Caldwell and Weber, 1970;
Ham et al., 1971; Berg and Loynachan, 1985; Ayanaba et al., 1986; Vargas et al.,
1993, 1994; Madrzak et al., 1995; Ramirez et al., 1997). Other rhizobia whose sero-
logical diversity has been examined are those that nodulate clovers (Hagedorn and
Caldwell, 1981; Dughri and Bottomley, 1983; Renwick and Gareth Jones, 1985;
Valdivia et al., 1988; Leung et al., 1994b), peas (Mahler and Bezdicek, 1980;
Turco and Bezdicek, 1987), chickpeas (Kingsley and Ben Bohlool, 1983), alfalfa
(Purchase et al., 1951; Olsen and Rice, 1984; Rajapakse and Macgregor, 1992),
lotus (Irisarri et al., 1996), Hedysarum spp. (Kishinevsky et al., 1996), common
bean (Robert and Schmidt, 1985), and different tropical legumes (Sinclair and
Eaglesham, 1973; Ikram and Broughton, 1980; Ahmad and Hassouna, 1981). The
reaction can be performed directly on nodule crushes, a unique advantage enabling
many nodules to be screened rapidly. An intrinsic limitation of these methods in
studying the diversity of nodule occupants is that they can only provide informa-
tion on those rhizobia that cross-react with the antisera at one’s disposal, not on
those that do not react.
116 N. AMARGER
C. CHEMICAL COMPOSITION
Different methods based on the chemical composition of bacteria are of com-

mon use for classification and identification purposes. Several of them have been
tested for their ability to differentiate rhizobia. Whole-cell composition of bacte-
ria has been approached by pyrolysis mass spectrometry (PyMS), a procedure in
which bacterial cells are pyrolyzed under vacuum and the compounds produced
are ionized and analyzed by a mass spectrometer. PyMS provides a simple method
for discriminating very closely related strains wherever apparatus is available.
It has been used successfully for characterization of nodule isolates from alfalfa
(Goodacre et al., 1991) and Lupinus spp. (Barrera et al., 1997) and for identification
of inoculant strain in nodule isolates from soybeans (Kay et al., 1994). Whole-
cell extracts of protein separated by sodium dodecyl sulfate–polyacrylamide gel
electrophoresis (SDS–PAGE) show a large number of bands. Comparison of the
patterns obtained from different isolates provides information about the relatedness
of these isolates. Although visual comparison may be sufficient for rapid compar-
ison, numerical analysis of the patterns allows quantification of the resemblance,
and grouping of the patterns can then be made. SDS–PAGE of whole proteins has
been used as an initial approach often associated with a physiological or sero-
logical method to describe the diversity of nodule isolates of clovers (Dughri
and Bottomley, 1983; Demezas and Bottomley, 1984; Dughri and Bottomley,
1984; Zahran, 1992), alfalfa (Jenkins and Bottomley, 1985a,b), common bean
(Arredondo-Peter and Escamilla, 1993), soybean (Noel and Brill, 1980; Kamicker
and Brill, 1986), and tropical plants (Moreira et al., 1993). It is now more often
utilized as one of the several methods used to determine relationships between
rhizobial isolates (Batzli et al., 1992; de Lajudie et al., 1994; Madrzak et al., 1995;
van Rossum et al., 1995; Irisarri et al., 1996; Tan et al., 1997).
Enzyme electrophoresis, which separates allelic forms of enzyme molecules by
differences in their surface charge, reveals the polymorphism of the gene loci cor-
responding to the different enzymes analyzed. Electrophoretic mobility variants
thus give an indication of the number of alleles at a particular gene locus. In multi-
locus enzyme electrophoresis (MLEE), mobility variants of several housekeeping
enzymes are used to characterize strains (each distinct allelic variant profile is
designated an electrophoretic type or ET) and to assess genetic relatedness among
isolates or ETs and genetic variations among populations (Selander et al., 1986).
MLEE has been employed to explore genetic diversity and genetic structure of
various rhizobial populations. Strains from collections or field populations have
been characterized on the basis of allele profiles at a number of polymorphic
enzyme loci that varied from 3 to 28 depending on the study. So far, the method
has been used to study fast-growing rhizobia isolated from a few leguminous
species, clover, pea, common bean, and alfalfa (for references see Section IV),
and slow-growing rhizobia from lupines, seradella, and siratro (Bottomley et al.,
1994; Barrera et al., 1997). MLEE has also been used to study the genetic diversity
of nonsymbiotic rhizobia isolated directly from the rhizosphere of common beans
(Segovia et al., 1991) and of Lotus corniculatus (Sullivan et al., 1995). The method
can be highly discriminatory. It is useful to provide information of genetic variation
within a species. Its main advantage is that it relies on the polymorphism of a
number of gene loci dispatched all over the chromosome and thus gives an image
of the overall genome, with the exception, however, of the symbiotic genome.
Lipopolysaccharides (LPS) are important components of the external cell wall
of gram-negative bacteria. Variability of the sugar components and in the numbers
of the O-specific side chains of LPS molecules result in different migration pat-
terns when gram-negative bacteria are applied to SDS–PAGE. Such LPS patterns
have been used successfully for determining the diversity of some fast-growing
(de Maagd et al., 1988; Casella et al., 1992; Zahran, 1992; Lindström and Zahran,
1993) and slow-growing rhizobia (Alves and Lemos, 1996; Santamaria et al., 1997;
Jayaraman and Das, 1998). Since LPS molecules are only present in gram-negative
bacteria, LPS profiling can be performed directly on individual nodule squashes,
which is a significant advantage when large numbers of nodule isolates have to be
studied (Santamaria et al., 1998).
Fatty acids are constituents of phospholipids and of LPS of the outer membrane
of gram-negative bacteria. The ability of fatty acid methyl ester (FAME) analy-
sis to discriminate among strains belonging to the known diversity of fast- and
slow-growing rhizobia has been determined by several authors (Mac Kenzie and
Child, 1979; Jarvis and Tighe, 1994; So et al., 1994; Graham et al., 1995; Jarvis
et al., 1996, 1998; Dunfield et al., 1999). Principal component analysis distin-
guished clusters of strains that corresponded to the known species or to groups
already formed by other methods, suggesting that FAME analysis could form the
basis of a rapid method of identification.
D. SYMBIOTIC CHARACTERISTICS
Because of their agricultural importance, the variability in symbiotic proper-

ties of rhizobia was the first to be described. Wide differences among rhizobia
relative to their ability to produce nodules on any one legume species and to
their effectiveness or ability to aid plant growth through nitrogen fixation have
long been recognized. Rhizobia can thus be characterized by the range of hosts
that they nodulate. Some rhizobia appear highly specific and nodulate only plants
belonging to a single genus or to some species within a genus. For instance,
rhizobia isolated from Galega spp. induced nodules only on Galega spp. plants
(Lindström, 1989); rhizobia associated with European species of clovers will
not nodulate clover species native to mid-Africa (Vincent, 1974). Other rhizo-
bia are symbionts of multiple legume species, e.g., some Brazilian isolates from
118 N. AMARGER
common bean (Martinez-Romero et al., 1991) or some fast-growing isolates from

soybean (Krishnan and Pueppke, 1994). The range spectrum of one of these lat-
ter strains, Rhizobium fredii USDA 257, and of Rhizobium sp. NGR 234, iso-
lated from Lablab purpureus (Trinick, 1980), has recently been the subject of
an extensive investigation (Pueppke and Broughton, 1999). USDA 257 and NGR
234 were able to nodulate 135 and 232 of the 452 legume species tested, re-
spectively. When the N2-fixing effectiveness of the rhizobium–host association is
taken into account, a further complex pattern of specificity (effectiveness speci-
ficity) is usually observed. According to the host genotype, levels of effective-
ness of a strain can vary from fully effective to fully ineffective. Effectiveness
specificity is observed within broad as well as narrow host range strains. For
instance, the broad host range strain NGR 234 formed effective nodules with
only 135 of the 232 legume species it nodulated (Pueppke and Broughton, 1999)
and the narrow host range strains isolated from Galega officinalis were effective
with this host plant, but ineffective with Galega orientalis and vice versa (Lind-
ström, 1989). The abilities of a strain to form nodules and to fix nitrogen with a
range of leguminous hosts are important practical characteristics and are crucial
characteristics for the description of any rhizobium. However they are difficult
to establish because they are multiple. For this reason, the range of host plants
tested is most often limited to the plant from which the strain was isolated and
in the best cases, to the few species known to be nodulated by closely related
strains.
E. DEOXYRIBONUCLEIC ACID
As they have the advantage of being independent of gene expression, methods

based on the analysis of genomic DNA have, since 1970, provided the main basis
for defining groups of closely related strains of microorganisms. The methods,
which were first limited in their applicability since they were laborious and time
consuming, have become, with the advent of molecular techniques, more and more
diversified and easy to use. As their development proceeded they have been adapted
to rhizobial characterization and utilized to reveal the diversity of nodule isolates.
Early genetic evidence (Higashi, 1967) suggested that some symbiotic genes
had an extrachromosomal location. Physical evidence of the presence of relatively
small plasmids (<100 kb) in fast-growing rhizobia was given some years later by
Sutton (1974). The subsequent development of extraction methods allowed the
detection of larger plasmids (up to 500 kb) in different fast- and slow-growing
rhizobia (Nuti et al., 1977) and of extralarge (>1000 kb) plasmids, also called
megaplasmids, in rhizobia isolated from alfalfa (Rosenberg et al., 1982). Since
then, large and/or extralarge plasmids have been shown to be common compo-
nents of the fast-growing rhizobium genome and can constitute up to 50% of the
cell genome (Prakash and Atherly, 1986; Sobral et al., 1991). Their number varies
from 1 to 10. In these fast-growing rhizobia, most of the genes involved in the sym-
biotic function are located on one, sometimes several, plasmid(s) named symbiotic
plasmids or pSyms. The presence of large plasmids in slow-growing rhizobia is
more difficult to establish. Although strains carrying plasmids have been described
(Nuti et al., 1977; Gross et al., 1979), the presence of plasmids is not a general
feature of the slow-growing rhizobia. When they are present, the amount of ge-
netic information that they carry is limited and does not include genes essential in
symbiotic function.
Plasmid content of fast-growing rhizobia can be easily revealed by agarose
gel electrophoresis of cell lysates. The in-well lysis procedure first developed by
Eckhardt (1978) and subsequently modified (Hirsch et al., 1980; Rosenberg et al.,
1981; Hynes et al., 1986; Wheatcroft et al., 1990) permits detection of plasmids
with extremely large molecular size. With this method, overall composition and ap-
proximate size of each plasmid present in every strain can be established at the same
time for a large number of strains. Variation in plasmid profiles of field isolates from
diverse leguminous crops has been reported. In rhizobia isolated from alfalfa, one
or two bands corresponding to the megaplasmids could be observed in all the iso-
lates (Bromfield et al., 1987; Brockman and Bezdicek, 1989; Shishido and Pepper,
1990; Hartmann and Amarger, 1991). Additional plasmid bands ranging in size
from approximately 50 to 300 kb were visualized in many of these isolates. Their
number in these cases was most often limited to 1 or 2 but could reach 4. Megaplas-
mids were not observed in isolates from clovers, pea, fababean, and lentil, but the
number of large plasmids with size ranging from 50 to 700 kb varied from 2
to 10 and was commonly higher than 4 (Tichy and Lotz, 1981; Glynn et al.,
1985; Thurman et al., 1985; Harrison et al., 1987, 1988, 1989; Brockman and
Bezdicek, 1989; Hynes and McGregor, 1990; Laguerre et al., 1992; Zahran, 1992;
Hirsch et al., 1993; Moenne-Loccoz et al., 1994; van Berkum et al., 1995; Castro
et al., 1997b; Handley et al., 1998; Moawad et al., 1998; Wilson et al., 1998).
An equivalent variability in the number of large plasmids was observed in isolates
from common bean but in addition a megaplasmid could be detected in some iso-
lates (Pepper et al., 1989; Geniaux et al., 1993; Laguerre et al., 1993b; Amarger
et al., 1994; Geniaux et al., 1995; Sessitsch et al., 1997a; Aguilar et al., 1998b;
Mhamdi et al., 1999). Other rhizobia analyzed by plasmid profiling represent
isolates from Hedysarum spp. (Mozo et al., 1988; Struffi et al., 1998), Astragalus
sinicus (Zou et al., 1997), soybean (Gross et al., 1979), Amorpha fructicosa (Wang
et al., 1999b), Onobrychis sativa (Gigova et al., 1997), and diverse tree and shrub
legumes (Thomas et al., 1994; Kuykendall et al., 1996; Milnitsky et al., 1997;
Santamaria et al., 1997). Isolates with identical plasmid profiles were usually
found to be very closely related genotypically. Whereas isolates with similar chro-
mosomal background may yield different plasmid profiles, different chromosomal
genotypes do not share common plasmid profiles. Plasmid profiling has proven
120 N. AMARGER
a practical and reliable method to rapidly screen and characterize fast-growing

rhizobia at the subspecies level.
The key tools that have allowed major progress to be made in analysis of genomic
DNA are, on one hand, restriction endonucleases that cleave DNA at specific sites
they recognize and, on the other hand, Taq polymerase that allows amplification
of DNA sequences by polymerase chain reaction (PCR). Restriction endonucle-
ases specifically cleave DNA into fragments whose number and length depend
on the number and position of the recognition sequences. Electrophoresis of the
cleaved DNA allows the separation of the fragments according to their size and
gives a pattern of bands, which can be stained by ethidium bromide. Modifications
in DNA sequences generate restriction fragment-length polymorphism (RFLPs)
which will give new patterns of bands; the similarities between these patterns are
thus, a measure of the relatedness of isolates. DNA restriction profiles have been
used to distinguish among rhizobia that nodulate peas (Hynes and OConnell, 1990;
Laguerre et al., 1992), Galegae spp. (Lindström et al., 1990), alfalfa (Mielenz et al.,
1979; Hartmann and Amarger, 1991), and clover (Glynn et al., 1985). Although this
type of fingerprinting is rather simple and gives a complete image of total DNA, its
utilization is limited because of the complexity of the patterns generated. Simpli-
fied patterns can be obtained by using restriction endonucleases that rarely cut and
pulse-field gel electrophoresis (PFGE), which enables large fragments to be sepa-
rated. This method has been used to study the genomic diversity of slow-growing
soybean isolates belonging to the same serotype (Sobral et al., 1990; Ramirez
et al., 1997) and to fingerprint nodule isolates from a leguminous tree (Haukka and
Lindström, 1994). The method gave a good resolution of isolates primarily at the
subspecies level. Another way to obtain simplified patterns is by transfer of the sep-
arated DNA fragments to nitrocellulose or nylon membranes (Southern, 1975) fol-
lowed by hybridization with a specifically labeled DNA probe. Such RFLP patterns
provide information about defined DNA regions. Depending on the level of con-
servation of their sequences, DNA probes will allow RFLP analyses of more or less
divergent isolates. As ribosomal RNA genes (rDNA) are highly conserved among
bacteria, hybridization of total DNA digest with rDNA probes generates RFLP pat-
terns from bacteria belonging to remote species, each species being characterized
by one or several rDNA RFLP patterns (ribotype). Such a probe has permitted dis-
crimination of different species of rhizobia among nodule isolates of common bean
(Geniaux et al., 1993; Sessitsch et al., 1997a; Mhamdi et al., 1999). More specific
DNA fragments have also been used as hybridization probes to identify restriction
polymorphism in the homologous gene regions of nodule isolates. They include
chromosomally located regions such as the lac and LPS gene regions of Rhizobium
leguminosarum (Young and Wexler, 1988; Cava et al., 1989), symbiotic plasmid
regions such as various nitrogen fixation (nif ) or nodulation (nod ) genes (Quinto
et al., 1982; Ruvkun et al., 1982; Russel et al., 1985; Watson and Schofield, 1985;
Young and Wexler, 1988; Demezas et al., 1991), plasmid regions such as replication
genes (Rigottier-Gois et al., 1998), or repeated sequences distributed over the en-
tire genome such as IS sequences (Wheatcroft and Watson, 1987; Bromfield et al.,
1995; Mazurier et al., 1996) or RS␣ and RS␤ (Hartmann et al., 1992; Minamisawa
et al., 1992). By allowing characterization of different parts of a single genome,
RFLP fingerprinting with different probes can provide information on the relation-
ships between these different parts whether they are located on the same replicon or
not. For instance, by comparing the repartition of pSym types within the host strain
nonsymbiotic genome, it has been shown that transfer of symbiotic genes, located
either on plasmids or on the chromosome, has occurred in nature (Schofield et al.,
1987; Young and Wexler, 1988; Laguerre et al., 1993c; Sullivan et al., 1995). The
degree of specificity has not been determined for all the different probes used in
RFLP analysis, but some of them, such as those including lac region or nod genes,
were found to be specific enough to position isolates in a given taxon (Harrison
et al., 1988; Laguerre et al., 1993a). Although restriction enzymes used in conjunc-
tion with DNA probes have proven to be very potent tools to demonstrate sequence
divergences and, thus, to reveal diversity in the housekeeping, symbiotic, and plas-
mid genomes, their use is decreasing with the development of more rapid methods
based on PCR. These methods present a wide spectrum of application and several
of these methods have been used for detecting genetic diversity among rhizobia.
In order to get an image as complete as possible of the genome in its totality,
amplification of multiple DNA fragments of variable lengths distributed over the
entire genome has been obtained by using as primers either short arbitrary oligonu-
cleotides, which produce randomly amplified polymorphic DNA (RAPD), or
naturally occurring repetitive sequences interspersed throughout the genome,
which amplify definite segments included between copies (rep-PCR). Using
gel electrophoresis, the amplified fragments are separated according to their
size and yield a complex pattern of bands of variable intensity that indicates
the polymorphism of total DNA. The quantification of the similarity between
the generated fingerprints can be performed by numerical analysis, which en-
ables groupings of the isolates to be made by similarity levels. Several arbi-
trary primers of 10 to 15 nucleotides in length have been used to differenti-
ate the genomes of a range of rhizobia belonging to different taxa (Harrison
et al., 1992; Dooley et al., 1993; Lunge et al., 1994; Richardson et al., 1995;
Selenska-Pobell et al., 1995; van Rossum et al., 1995; Laguerre et al., 1996;
Nathan et al., 1996; Paffetti et al., 1998; Agius et al., 1997; Harrier et al.,
1997; Sikora et al., 1997; Gonzalez Andrés and Ortiz, 1998; Handley et al.,
1998; Hebb et al., 1998; Teaumroong and Boonkerd, 1998; Wilson et al.,
1998; Young and Cheng, 1998). Among the diverse repetitive sequences iden-
tified in bacteria, two of them, the repetitive extragenic palindromic (REP) se-
quence and the enterobacterial repetitive intergenic consensus (ERIC) with, to
a lesser extent, the BOX element, are at the basis of the development of rep-
PCR fingerprinting in rhizobia (de Bruijn, 1992; Judd et al., 1993; Nick and
122 N. AMARGER
Lindström, 1994; Madrzak et al., 1995; Schneider and de Bruijn, 1996; Ibekwe
et al., 1997; Laguerre et al., 1997; Niemann et al., 1997; Sessitsch et al., 1997a;
Del Papa et al., 1999; Santamaria et al., 1999). Rep-PCR fingerprinting can
be performed directly on DNA extracted from crushed nodules, which allows
rapid processing of large numbers of nodule samples. The RAPD and rep-PCR
fingerprinting methods were comparable in their ability to resolve differences
among isolates at the subspecies level and approximately equivalent to protein
fingerprinting.
To study the polymorphism of defined regions of the genome, specific primers
can be used to amplify these regions and yield enough DNA to perform restriction
analysis (PCR–RFLP). This type of analysis, first limited by the availability of the
DNA sequences necessary to the design of primers, is, with the increasing number
of sequences available, becoming applicable to a more and more diversified
number of gene and intergene spacer (IGS) regions. Ribosomal DNA sequences,
because of their importance in phylogenetic investigations, were the first to be de-
termined. They contain both conserved regions that can be used to define primers
and variable regions that can be used to differentiate strains. PCR–RFLP analysis
of the 16S rRNA genes, which enables differentiation of rhizobia at the species
level (Laguerre et al., 1994, 1997), is now of common use for rapid assignation
of nodule isolates to a given species. PCR–RFLP of a more divergent region, the
spacer region between the 16S and the 23S rDNA, can differentiate rhizobia within
a species with a resolution level intermediate between 16S-RFLP analysis and
rep-PCR fingerprinting (Nour et al., 1994a; Laguerre et al., 1996). Differentiation
of rhizobial isolates based on their symbiotic genome can be performed by
PCR–RFLP analysis of conserved symbiotic genes such as certain nod and nif
genes (Eardly et al., 1992; Laguerre et al., 1996; Haukka et al., 1998). PCR–RFLP
analysis of repC sequences of the plasmid replication region has also been used as a
tool to reveal the diversity of this region within and among rhizobia (Rigottier-Gois
et al., 1998). As for conventional RFLP analysis using DNA probes, comparison
of the polymorphism observed in PCR-amplified chromosomal and plasmid genes
can provide information on the relationships between the different replicons that
bear these genes. For instance, RFLP analysis of nod and repC sequences, amplified
from pea rhizobia representing different chromosomal genotypes, has given cir-
cumstantial evidence that pSym and cryptic plasmid transfer had occurred within
the population studied (Rigottier-Gois et al., 1998). PCR-based techniques can
also be used, like molecular probes, for the detection of groups of rhizobia. Pairs
of primers are then designed for amplification of regions of DNA specific to the
rhizobia that have to be detected. Specific pairs of primers able to differentiate be-
tween different rhizobial species that nodulate common bean, symbiotic genomes
associated with different plant–host specificity, or groups of plasmid replication
sequences have been designed from 16S–23S IGS (de Oliveira et al., 1999), nif H
(Aguilar et al., 1998a), or repC (Turner et al., 1996) sequences, respectively.
In this first part, we have seen that the development of molecular techniques and
of automated systems for more traditional techniques has introduced new sources
of data for bacterial characterization. When applied to isolates from legume nod-
ules, these methodologies have revealed a vast diversity in any character studied.
Yet, the symbionts of only very few nodulated legumes have been explored. For
each character considered, the data generated and analyzed using computerized
methods of numerical analysis and clustering have provided estimation of inter-
strain similarity and difference among each set of isolates studied. Groups that
were considered homogenous appeared diverse in many respects. Although clus-
tering using different methods did not always agree, new groups of isolates could
be made at different levels of similarity. In order to be conveniently handled, this
new diversity needs to be structured. To do this, the classification is permanently
adapted. The current organization of rhizobia into groups or taxa is given in the
next part.
III. RHIZOBIUM SYSTEMATICS
A. FROM CROSS-INOCULATION GROUPING TO

POLYPHASIC TAXONOMY
The bacteria that nodulate leguminous plants were classified for a long time
in the unique genus Rhizobium. The observation that rhizobia from a given
host would nodulate a limited number of legume species led to the concept
of “cross-inoculation groups,” defined by Fred (1932) as “groups of plants
within which the root nodule organisms are mutually interchangeable.” These
authors gave the status of species to 6 of the 16 groups they recognized. Four
species, Rhizobium meliloti, Rhizobium trifolii, R. leguminosarum, and Rhizobium
phaseoli, were constituted of fast growers, and two, Rhizobium japonicum and
Rhizobium lupini, of slow growers. Deficiencies of the cross-inoculation concept
for delineating rhizobial species accumulated over the years (Wilson, 1944). Lange
(1961), in a study that further demonstrated the weakness of a classification scheme
based solely on symbiotic features, proposed the application of a taxonomic sys-
tem based on Adansonian principles. The application was taken on by Graham
(1964), who proposed major taxonomic changes within the Rhizobiaceae. Two
of these changes were retained by Jordan (1982, 1984) in his modified classifi-
cation: the creation of the genus Bradyrhizobium for slow-growing rhizobia and
of three biovars (bv.), viciae, phaseoli, and trifolii, within R. leguminosarum in
place of the former species R. leguminosarum, R. phaseoli, and R. trifolii. The
inclusion of Agrobacterium spp. in the genus Rhizobium was not retained. Since
then, new species have been created at a pace that has accelerated over the past
124 N. AMARGER
few years. These species were first created mainly on the basis of a few phenotypic
and genetic characters. Following the development of molecular techniques and
the proposal of minimum standards (Graham et al., 1991), more diverse criteria
have been progressively included in the description of new species.
Today, in order to delineate a new bacterial taxonomic unit, the polyphasic
approach is recommended (Vandamme et al., 1996). This approach requires the
integration of genotypic, phenotypic, and also phylogenetic information. It should
lead to the constitution of hierarchical groups of strains with increasing degrees of
similarity between strains. The species represents the basic unit of the classification
scheme and is defined as a group of strains sharing at least 70% of DNA–DNA
relatedness measured under specified conditions (Stackebrandt and Goebel, 1994).
A designated type strain serves as the reference for the species. So far, no general
rules have been specified on the degree of similarity that bacteria should share to
belong to the same genus. The grouping of species into genera relies mainly on
phylogenetic data based on 16S rDNA sequencing.
B. PHYLOGENY AND TAXONOMY
With the agrobacteria and phyllobacteria, the rhizobia form the Rhizobiaceae
family within the alpha subclass of the Proteobacteria (Stackebrandt et al., 1988).
Reviews on the phylogeny and taxonomy of rhizobia have been recently pub-
lished (Martinez-Romero and Caballero-Mellado, 1996; Young, 1996; Young and
Haukka, 1996; van Berkum and Eardly, 1998). The phylogenetic relationships
among bacteria are mainly inferred from analysis of the 16S ribosomal sequences.
Figure 1 shows a phylogenetic tree based on 16S ribosomal sequences of rhizobium
species and of some related bacteria. The currently recognized species, their prin-
cipal host legumes, and references to the article in which they were first published
are listed in Table I.
−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−→
Figure 1 Phylogenetic tree, based on aligned sequences of the small subunit ribosomal RNA
genes, showing relationships among rhizobia and some related bacteria in the alpha-subdivision of
the Proteobacteria. The tree is constructed by the Neighbour-Joining method. Bootstrap probability
values (100) greater than 80% are indicated at the branch point. Abbreviations: Ag., Agrobacterium;
Al., Allorhizobium; Az., Azorhizobium; B., Bradyrhizobium; Bl., Blastobacter; M., Mesorhizobium; R.,
Rhizobium; Rhp., Rhodopseudomonas, and S., Sinorhizobium. The genbank accession numbers are,
top to bottom, as follows: D30778, X67221, L11661, S46917, D25312, Z35330, U69638, X87273,
M69186, M65248, U35000, X70405, X70404, X70403, X70401, D32226, M59060, U86344, X67228,
X67223, X67225, Y17047, X67231, X68387, X68390, D12783, L39882, D12786, U71079, U50165,
UO7934, X67229, U50164, L38825, U50166, Y14158, D12797, AFO41442, D12794, L26167,
D12793, AF025852, U71078, U28939, U28916, U47303, U89831, X67227, U29388, X67224,
X67234, X67223, U38469, U89823, U89816, U89819, U89817, U89818, and U86343.
126 N. AMARGER
Table I
Current List of Rhizobium Species
Species Principal host legumes References
Rhizobium Frank (1889)

R. leguminosarum Frank (1889)
bv. viciae Lathyrus, Lens, Pisum, Vicia Jordan (1984)
bv. trifolii Trifolium Jordan (1984)
bv. phaseoli Phaseolus Jordan (1984)
R. galegae Galega Lindström (1989)
R. tropici Phaseolus, Leucaena Martinez-Romero et al. (1991)
R. etli Segovia et al. (1993)
bv. phaseoli Phaseolus Segovia et al. (1993)
bv. mimosae Mimosa affinis Wang et al. (1999a)
R. gallicum Amarger et al. (1997)
bv. gallicum Phaseolus, Onobrychis Amarger et al. (1997)
bv. phaseoli Phaseolus Amarger et al. (1997)
R. giardinii Amarger et al. (1997)
bv. giardinii Phaseolus, Leucaena Amarger et al. (1997)
bv. phaseoli Phaseolus Amarger et al. (1997)
R. hainanense Desmodium spp., Stylosanthes, . . . Chen et al. (1997)
R. huautlense Sesbania herbacea Wang et al. (1998)
R. mongolense Medicago ruthenica van Berkum et al. (1998)
Bradyrhizobium Jordan (1982)
B. japonicum Glycine max Jordan (1982)
B. elkanii Glycine max Kuykendall et al. (1992)
B. liaoningensis Glycine max Xu et al. (1995)
Sinorhizobium de Lajudie et al. (1994)
S. meliloti Medicago, Melilotus, Trigonella Dangeard (1926)
S. fredii Glycine max, . . . Scholla and Elkan (1984)
S. teranga de Lajudie et al. (1994)
bv. acaciae Acacia Lortet et al. (1996)
bv. sesbaniae Sesbania Lortet et al. (1996)
S. saheli de Lajudie et al. (1994)
bv. sesbaniae Sesbania Haukka et al. (1998)
bv. acaciae Acacia Haukka et al. (1998)
S. medicae Medicago Rome et al. (1996)
Mesorhizobium Jarvis et al. (1997)
M. loti Lotus Jarvis et al. (1982)
M. huakuii Astragalus sinicus Chen et al. (1991)
M. ciceri Cicer arietinum Nour et al. (1994)
M. mediterraneum Cicer arietinum Nour et al. (1995)
M. tianshanense Glycine max, Glycyrrhiza, . . . Chen et al. (1995)
M. plurifarium Acacia, Leucaena de Lajudie et al. (1998b)
M. amorphae Amorpha fruticosa Wang et al. (1999b)
Azorhizobium Dreyfus et al. (1988)
Az. caulinodans Sesbania rostrata Dreyfus et al. (1988)
Allorhizobium de Lajudie et al. (1998a)
Al. undicicola Neptunia natans de Lajudie et al. (1998a)
In the phylogenetic tree (Fig. 1), rhizobia are found intermingled with nonrhi-
zobia showing that the current division in genera is not entirely supported by the
16S rRNA phylogeny. The genus Azorhizobium forms a separate branch and is dis-
tantly related to other rhizobia. Bradyrhizobium clusters with a number of bacteria
placed in different genera. This genus includes the slow-growing, alkali-producing
rhizobia that are not assigned to any species (referred to as Bradyrhizobium sp.
followed by the genus name of the host in parentheses) and the extraslow- and
slow-growing symbionts of soybean distributed in three species. Unnamed strains
may be more closely related to Bradyrhizobium japonicum strains than strains
of B. japonicum are to each other. In all these strains the symbiotic genes are
chromosomally located. The current genus Rhizobium is heterogenous. It har-
bors two monophyletic groups and one species, Rhizobium giardinii, phylogenet-
ically distinct from the lineages that contain other rhizobia. The group composed
of six species, R. leguminosarum, Rhizobium tropici, Rhizobium etli, Rhizobium
gallicum, Rhizobium hainanense, and Rhizobium mongolense, is considered to
represent the true genus Rhizobium. R. tropici has been subdivided into two types,
A and B, which are genotypically and phenotypically different. Agrobacterium
rhizogenes is part of this cluster. The other monophyletic group is composed of
two closely related species, Rhizobium galegae and Rhizobium huautlense. Its
phylogenetic position is not determined with certainty but appears to be distant
from the other Rhizobium species. The genus Sinorhizobium, initially proposed
to accommodate fast-growing soybean isolates (Chen, 1988), has been amended
based on the results of a polyphasic approach and contains the former R. meliloti
and R. fredii as well as three new species, Sinorhizobium saheli, Sinorhizobium
teranga, and Sinorhizobium medicae. It represents a coherent phylogenetic group.
In Sinorhizobium and Rhizobium isolates, most of the symbiotic genes are plas-
mid borne. The genus Mesorhizobium is another monophyletic group, recently
created on the basis of distinct genetic as well as phenotypic characters. It harbors
the former Rhizobium loti, Rhizobium ciceri, Rhizobium mediterraneum, Rhizo-
bium tianshanense, and Rhizobium huakuii and two new species, Mesorhizobium
amorphae and Mesorhizobium plurifarium. Strains from this genus are acid pro-
ducers but the growth rate of some of them is intermediate between that of Bradyrhi-
zobium and Rhizobium strains. The location of the symbiotic genes is chromoso-
mal in Mesorhizobium loti and plasmidic in M. plurifarium and Mesorhizobium
huakuii. Allorhizobium has been created recently based on the results of a polypha-
sic study. It belongs to the Agrobacterium lineage.
As the 16S ribosomal sequences of rhizobia accumulate, sequence variability
within species is observed and may be even greater than variability between species
(Fig. 1). Sequence heterogeneity is also found between copies of the rRNA genes
within a single genome (Haukka et al., 1996; Wang et al., 1999b). 16S rRNA
sequencing may thus be limited for species delineation; it is, however, very useful
for determining the closest relatives of an isolate and for assigning species to
128 N. AMARGER
genera. In the current classification, the division in species still reflects, in most
cases, differences in symbiotic characteristics. However, some species within the
genera Rhizobium and Sinorhizobium are symbiotically heterogeneous. Transfer
of a plasmid is supposed to be at the origin of this heterogeneity, since isolates
within these species may differ by the symbiotic plasmid they harbor, a plasmid
which confers distinct host specifity. Biovars have been created to account for this
difference in symbiotic specificity within species. So far, four species of Rhizobium
and two of Sinorhizobium have been subdivided into biovars (Table I). Recently,
transfer of symbiotic genes located on the chromosome has also been demonstrated
in Mesorhizobium species (Sullivan et al., 1995; Sullivan and Ronson, 1998).
Therefore, it might be possible to identify distinct host range determinants within
species of Bradyrhizobium or Mesorhizobium.
Comparison of phylogeny of 16S rRNA to that of nod or nif genes (Young
and Johnston, 1989; Lindström et al., 1995; Ueda et al., 1995; Laguerre et al.,
1996; Haukka et al., 1998; Wernegreen and Riley, 1999) also suggests a nonpar-
allel evolution of symbiotic genes and of the rest of the genome within genera.
This reinforces the hypothesis that the symbiotic genes have moved within major
rhizobial lineages.
C. IDENTIFICATION
As long as the basis of the classification was the growth rate and the isolation
host of rhizobia, identification of nodule isolates was simple. Since it has become
apparent that a given legume genotype can be nodulated by rhizobia belonging
to different species and/or different biovars, identification requires more thorough
characterization.
FAME analysis and miniaturized phenotypic fingerprinting are largely used
in bacterial identification. Their potential as identification methods for rhizobia
has been demonstrated (references in Section II), but these methods need further
development before they can be routinely used for rhizobial identification. In the
absence of rapid tests which would allow straightforward identification of isolates,
the most direct approach for identifying an isolate is, first, to place it in the 16S
rRNA phylogenetic framework and then to determine its levels of identity with
existing reference strains. RFLP analysis of amplified DNA sequences provides
a simple and reliable alternative to sequencing in the estimation of phylogenetic
position for identification purposes. Methods that give good levels of resolution
at the subspecies level and allow estimation of relatedness to type strains in-
clude MLEE, RFLP with hybridization probes, and PCR–RFLP of 16S–23S IGS.
Whole-cell protein, RAPD, or rep-PCR analyses can be useful but are often too
discriminatory. Nodulation tests are necessary to ascertain the symbiotic position
of isolates within the identified species or within one of its biovars. Phylogeny of
common nod genes can also be used, as a complement or in place of a nodulation

test, to position isolates at the symbiotic level.
IV. NATURAL POPULATIONS OF RHIZOBIA
Rhizobia are saprophytic bacteria representing only a small fraction of the soil
microflora and do not possess properties selective enough to allow their quantitative
recovery from soil by direct plating. Successful isolation of rhizobial populations
directly from soil has only been performed in a few cases by using different mul-
tistep procedures (Jarvis et al., 1989; Soberon-Chavez and Najera, 1989; Segovia
et al., 1991; Laguerre et al., 1993a; Bromfield et al., 1995; Louvrier et al., 1996;
Sullivan et al., 1996; Hartmann et al., 1998). The quasitotality of the rhizobial pop-
ulations described so far has thus been recovered from the nodules of leguminous
plants that had grown in the field, in pots containing soil samples, or in any axenic
device in which the plants were inoculated with a soil suspension. They represent
the result of the selection exerted by the plant from the soil rhizobia under the
tested growth conditions and do not correspond to a random sample of the specific
rhizobia present in the soil.
The size of the soil population capable of nodulating a given leguminous plant
can be assessed by the indirect plant infection method (Vincent, 1970). This method
allows estimation of most probable numbers (MPN) of specific rhizobia when the
numbers in soil are at least 10 g−1. For lower numbers, semiquantitative estimates
can be made by growing the host plant on sand mixed with increasing amounts of
soil. The size of rhizobial populations in agricultural soils varies widely. In most
soils, different populations of rhizobia can coexist at an average density of 102 to
104 g−1. Population density sometimes appears to be correlated with different fac-
tors that include soil pH, base saturation, soil texture, organic matter content, mean
annual rainfall, or temperature. However, most often the differences in population
densities cannot be explained by simple variations in environmental parameters.
The cultivation of the plant host generally induces a transient increase in the spe-
cific rhizobium population. The density of the specific rhizobia can then reach up
to 107 g−1 of soil under the crop but decreases more or less rapidly after harvesting.
A. DIVERSITY OF POPULATIONS
Many studies have been conducted on the diversity of populations of rhizobia

that can be recovered from different legume species. Only those related to the pop-
ulations of rhizobia that nodulate the most widespread legume crops are considered
here.
130 N. AMARGER
1. Rhizobia That Nodulate Clovers
So far, all the rhizobia isolated from nodules of plants belonging to the genus
Trifolium are classified in the bv. trifolii of R. leguminosarum. However, R. legu-
minosarum bv. trifolii may not be the only symbiont of the Trifolium species since,
in a collection of strains isolated from different species of Trifolium, Eardly (1993)
has detected strains, the 16S rDNA alleles of which were identical to that of R.
etli. The host specificity of bv. trifolii is restricted to plants of the genus Trifolium.
Some specificity in effectiveness has been observed among strains of bv. trifolii
and effectiveness host-groups have been delineated (Vincent, 1974).
Clover species occur naturally or have been introduced in most parts of the world
and their microsymbionts are usually detected in soils of pH in the range of 4.5 to
8, in numbers varying from 102 to 105 g−1. Low numbers of rhizobia (<102 g−1 )
are most often found in soils with pH below 5 (Rice et al., 1977; Hagedorn, 1978;
Harrison et al., 1989; Nazih et al., 1993; Slattery and Coventry, 1993) or in soils
contaminated with heavy metals (Coventry and Hirth, 1992; Chaudri et al., 1993;
Giller et al., 1993).
The different methods of characterization used to study clover rhizobial pop-
ulations have each indicated the existence of heterogeneous groups of isolates
within these populations. MLEE and RFLP analyses with chromosomal probes
were found equivalent in their ability to differentiate strains of R. leguminosarum
bv. trifolii (Demezas et al., 1991, 1995). Although the correlation was not so tight
between serotypes and ETs, the great majority of serotypes was restricted to very
similar ETs (Leung et al., 1994a). Analysis of plasmid content (Harrison et al.,
1989; Laguerre et al., 1992) and rep-PCR or Biolog fingerprinting (Leung et al.,
1994a; Strain et al., 1994; Laguerre et al., 1996) were shown to group isolates shar-
ing the same ET or the same chromosomal RFLP. Thus, besides differentiation of
isolates, MLEE and RFLP analysis with chromosomal probes should provide a
reasonable estimation of the overall genetic relatedness among isolates composing
clover nodulating populations.
When presented with the same soil population of rhizobia, different species
or cultivars of clover can select different samples of the strains that it con-
tains. For instance, a greater number of serotypes were identified in nodules
of perennial species, including Trifolium repens or Trifolium pratense, than in
nodules of Trifolium subterraneum and four other annual species grown on
the same soil (Valdivia et al., 1988; Leung et al., 1994b). Also, Dughri and
Bottomley (1984) found differences in the distribution of unique and common ETs
among populations isolated from different cultivars of T. subterraneum. In con-
trast, preferential nodulation was not observed among four cultivars of T. repens
(Harrison et al., 1987) and was weak among four cultivars of T. pratense (Hagen
and Hamrick, 1996b). Depending on the clover genotype used to trap the rhi-
zobia, the image obtained from soil populations may therefore appear different
and the actual diversity in R. leguminosarum bv. trifolii present in soils may be
underestimated.
Variations in the diversity of populations of clover rhizobia can also be observed
among sites. Hagen and Hamrick (1996a) found high levels of genotypic diversity
among eight populations of red clover rhizobia collected over 2 years from five sites
in two 1500-km distant regions of the eastern United States. Among the 912 isolates
analyzed, they distinguished 272 ETs, 64% of which were represented by unique
isolates. Within a same region, a variable fraction of multiple isolates was shared
between two or more populations and caused, with the variable frequency of unique
ETs, differentiation among populations. Differences among populations increased
with geographical distances, suggesting that geographic separation contributed to
the differenciation of populations. Most of the total genetic variation was found
on individual plants (58%) and among plants within populations (20%).
Strain et al. (1994) compared the diversity of populations of clover rhizobia
isolated from two sites 1-km distant. These sites differed by the natural abun-
dance of clover species and by the densities of R. leguminosarum bv. trifolii in
soil. The levels of genetic diversity (average level of polymorphism of the chro-
mosomal loci for the different enzymes studied) of the populations were nearly
identical, indicating that low-density populations are not necessarily of little ge-
netic diversity. Nevertheless, the populations were genotypically differentiated.
Among the 70 and 198 isolates analyzed, 28 and 54 distinct ETs were identified
respectively and only 1 was common to both sites. This difference between the
two populations was attributed to the different nature of the vegetation present
at each site. The overall genotypic diversity detected among populations of white
clover rhizobia from soils sampled in 10 different sites in the UK was more limited
(Harrison et al., 1989). Only 10 ETs represented more than 98% of the 721 isolates
analyzed. Many ETs were common to two or more sites and the populations at
different sites differed more in the occurrence and relative frequencies of common
ETs than in the presence of unique ETs. Much of the total genetic variation was
found within populations. The between-site variations observed might have been
influenced by geographical separation. The strain polymorphism was reduced at
sites that were acidic, suggesting a better tolerance of particular genotypes to acid
soils. Seven of the genotypes common to at least five of these sites had also been
identified in another UK population isolated from white clover by Young (1985).
Four of these genotypes common to several UK sites were shared with the two
Oregon populations described by Strain (Strain et al., 1994), one of them being
also found in a population isolated from red clover in France (Mazurier, 1989).
In addition, the French population and the UK population described by Young
(1985) shared their dominant genotype, a genotype that had also been identified
by Strain et al. (1994) from the two Oregon populations. The conservation of some
genotypes among the different populations suggests migration over large distances
and implies that these genotypes were particularly competitive in a wide range of
132 N. AMARGER
situations and/or that limited genetic exchanges among genotypes had taken place
at each site.
Environmental factors other than pH can also modify the diversity of populations
of clover rhizobia. Soil contamination by heavy metals has been reported to induce
decreases in the diversity and effectiveness of populations of rhizobia isolated from
subclover (Castro et al., 1997a) and white clover (Hirsch et al., 1993). In this latter
case, a single group of very closely related rhizobia ineffective with white clover
had survived in the contaminated soil, causing the decrease in the effectiveness of
the population.
The levels of genetic diversity found in the different studies, which ranged from
0.346 (Hagen and Hamrick, 1996b) to 0.559 (Demezas et al., 1995), were similar
to that calculated by Demezas et al. (1991) from strains of diverse origins (0.58),
showing that the chromosomal diversity is high within R. leguminosarum bv. trifolii
as a whole. A great part of the genetic variation within the species was maintained
within populations and individual plants.
The genetic diversity of symbiotic gene regions has been investigated by RFLP
analysis, using bv. trifolii-specific probes, for two Australian (Schofield et al.,
1987; Demezas et al., 1995), one French (Laguerre et al., 1993b), and several
Californian (Wernegreen et al., 1997) populations. The plasmid-encoded Sym
region was polymorphic in all the populations and different Sym types could
be identified in similar chromosomal backgrounds. Similar Sym types were re-
stricted to identical or related chromosomal backgrounds with the exception of
two Sym types, one in the population described by Schofield et al. and one in
the French population, that were each found in two different chromosomal back-
grounds. This suggests that, although pSym tranfers occur among isolates, there
are limitations in these transfers between major R. leguminosarum bv. trifolii chro-
mosomal types.
2. Rhizobia That Nodulate Plants of the Genera Pisum,

Vicia, Lens, or Lathyrus
Nodule isolates of plants belonging to the genera Pisum, Vicia, Lens, or Lathyrus
are classified in the bv. viciae of R. leguminosarum. Cross-infection seems the rule
within this biovar. One exception is a genotype of pea collected in the Middle East,
Pisum sativum cv. Afghanistan (Lie, 1978), the nodulation of which is restricted to
strains of R. leguminosarum bv. viciae, harboring a specific nod gene, nodX, com-
mon to the Middle East strains but absent in the European strains. The effectiveness
behavior of strains of R. leguminosarum bv. viciae is quite diverse. It is frequent
to isolate strains effective with a host plant and ineffective with others, but no
subdivision of the bv. could be delineated on the basis of effectiveness specificity.
Rhizobium leguminosarum bv. viciae are found in many soils. Similarly to R.
leguminosarum bv. trifolii, they can be absent or present in low numbers in acid
soils. In two surveys conducted in Denmark (Engvild, 1989) and France (Amarger,
1980) on 44 and 57 soils, respectively, R. leguminosarum bv. viciae were not
detected (<10g−1 ) in three Danish and seven French soils, all of pH below 5.
Their densities in the soils of higher pHs varied from 103 to 105 g−1.
Different host plant species have been shown to display different selectivity
toward the chromosomal genotypes of R. leguminosarum bv. viciae present in a
soil (Turco and Bezdicek, 1987; Mazurier, 1989; Louvrier, 1995; Handley et al.,
1998). When peas and lentils (Mazurier, 1989) or peas and fava beans (Louvrier,
1995) were grown in the same soil, less chromosomal genotypes were detected
in the nodules of peas than in the nodules of lentils or fava beans. However, the
difference was not significant in one site, where the diversity of the population
directly isolated from soil (Louvrier, 1995) was low, indicating that genotype-
specific selection is dependent on the soil population diversity. Nevertheless, these
results as a whole suggest that peas are more selective and may give a more
distorted image of the real soil population of R. leguminosarum bv. viciae than the
other species. Actually, the high genetic diversity (0.66) and variability in plasmid
content and serology observed by van Berkum et al. (1995) among fava bean
strains from diverse origins suggest that fava bean is not a selective host. Highly
diverse populations of R. leguminosarum bv. viciae can also be found at low soil
temperatures. Two populations of cold adapted rhizobia were isolated from two
Lathyrus species, Lathyrus japonicus and Lathyrus pratensis, growing in northern
regions of Quebec by Drouin et al. (1996). They had a genetic diversity of 0.45
each and were genotypically very diverse and highly differentiated.
Environmental factors can influence the composition of populations of pea rhi-
zobia. Differences in topographic position within fields in a hilly region of eastern
Washington state caused variations among populations of pea rhizobia (Mahler and
Bezdicek, 1978; Brockman and Bezdicek, 1989). Populations of nodule isolates
recovered from peas grown on the south slopes and ridge tops differed from those
recovered from peas grown on the north slopes and bottom lands in the distribu-
tion of serogroups and in the diversity of plasmid profile groups within serogroups.
These differences were attributed to the differences in soil microclimate that was
warmer and dryer on the south slopes and ridge tops than on the north slopes and
bottomlands. Labes et al. (1996) also identified an effect of the slope position
on the composition of populations of pea rhizobia while the northern or southern
exposure had no impact. The application of slurry induced changes in these popu-
lations, some groups of isolates disappeared, and new groups were detected in the
slurry-polluted soil.
At the two Oregon sites studied by Strain et al. (1994), the results obtained with
populations of R. leguminosarum bv. viciae were similar to those found with bv.
trifolii. Despite wide differences in the numbers of soil rhizobia present at each site,
the genetic diversity of the populations recovered from plant nodules was similar.
However, the composition of these two populations differed, which was attributed
134 N. AMARGER
to the absence of host plants at one site. In a field population of pea rhizobia,
Young et al. (1987) observed that although the population on each individual
plant was highly diverse, ET frequencies were similar from plant to plant, which
indicated a remarkably homogenous distribution of the rhizobial genotypes within
the soil. A majority of these genotypes had been previously identified at another
field 25 km distant (Young, 1985) and the authors suggested that genetic exchange
was infrequent relative to the rate of migration between the two populations. Some
of these genotypes were also shared with R. leguminosarum bv. viciae populations
from France (Laguerre et al., 1992) and Oregon (Strain et al., 1995), indicating
that the rates of migration of these genotypes were indeed rather high.
The diversity in the plasmid-encoded symbiotic region has been studied in sev-
eral populations of R. leguminosarum bv. viciae isolated from nodules of peas
(Engvild et al., 1990; Young, 1988) or from peas and lentils (Laguerre et al.,
1992) and in two populations isolated directly from soils (Louvrier et al., 1996).
High levels of polymorphism were observed in each population. The distribution
of this polymorphism across chromosomal backgrounds differed greatly among
the populations. Whereas this distribution was at random in the two populations
directly isolated from soil, more or less strong associations between Sym types
and chromosomal backgrounds were detected in the populations isolated from
nodules. Each chromosomal type but one was associated to a different Sym type in
the population studied by (Engvild et al., 1990). In the other populations, several
Sym types were associated to a same chromosomal background. Identical Sym
types were found in different chromosomal backgrounds, in greater proportion
in the French (Laguerre et al., 1992) than in the UK population (Young, 1988).
These findings suggest that pSym transfer occur in natural populations but that
opportunities for such transfers differ greatly between sites. At the sites where
populations of R. leguminosarum belonging to the three biovars, trifolii, viciae,
phaseoli, had been isolated (Laguerre et al., 1992; Young, 1988) some chromo-
somal genotypes were shared among the three biovars, giving additional evidence
of pSym transfer among populations of R. leguminosarum.
3. Rhizobia That Nodulate Phaseolus spp.
The symbionts of Phaseolus spp., to which I collectively refer as Phaseolus rhi-

zobia, have long been recognized as a group of rhizobia that shows considerable
genetic and phenotypic diversities (Graham, 1964; Gibbins and Gregory, 1972;
Jarvis et al., 1977; Beynon and Josey, 1980; Roberts et al., 1980; Crow et al.,
1981; Robert and Schmidt, 1985; Pinero et al., 1988), but it is only in recent years
that this group, which formed the species R. leguminosarum bv. phaseoli, has been
partitioned into different species. To date, five Rhizobium species have been recog-
nized as microsymbionts of Phaseolus vulgaris (Table I), but potentially there are
others (van Berkum et al., 1996; Herrera-Cervera et al., 1999). The host specificity
of strains belonging to the bv. phaseoli of R. leguminosarum, R. etli (formerly type I

strains), R. gallicum, and R. giardinii is restricted to plants of the genus Phaseolus.
Rhizobium tropici (formerly type II), R. gallicum bv. gallicum, and R. giardinii
bv. giardinii have wider host ranges, probably not fully described yet, that include
Leucaena spp. in addition to P. vulgaris. Moreover, R. tropici isolates also form
nodules on a wide spectrum of other tropical legumes (Hernandez-Lucas et al.,
1995) and R. gallicum bv. gallicum nodulates Onobrychis spp. (Amarger et al.,
1997), Vigna unguiculata, and Gliricidia spp. (Sessitsch et al., 1997a). Strains
of R. giardinii bv. giardinii are totally ineffective with their two known hosts,
P. vulgaris and Leucaena spp. P. vulgaris is a highly promiscuous leguminous
plant that under laboratory conditions nodulates with many other classified and
unclassified rhizobia (Graham and Parker, 1964; Eardly et al., 1985; Sadowsky
et al., 1988; Bromfield and Barran, 1990; Michiels et al., 1998), which in most
cases form ineffective nodules. Field nodulation of common bean is often sparse
and nodules may even be absent on roots of field-grown beans (Graham, 1981).
However, soil populations of Phaseolus rhizobia commonly range in size from 102
to close to 106 g−1 (Amarger, 1980; Robert and Schmidt, 1983; Kucey and Hynes,
1989; Anyango et al., 1995; Aguilar et al., 1998b).
Great genetic heterogeneity has been found among populations of rhizobia re-
covered from nodules of soil- or field-grown Phaseolus. Many of the popula-
tions analyzed, even those originating from single sites, were composed of several
species, in numbers up to 5 (Herrera-Cervera et al., 1999; Mhamdi, 1999). It
is noteworthy that the species identified in single-species populations was either
R. etli or R. leguminosarum and that, in populations composed of two or more
species, R. etli or R. leguminosarum bv. phaseoli were always present.
It is commonly accepted that P. vulgaris is native to the Americas and that
domestication of wild beans took place independently in Mesoamerica and in the
Southern Andes. R. etli bv. phaseoli, the predominant species found in the rhizobial
populations associated with wild beans from the southern Andes (Aguilar et al.,
1998b) and from Mexico (Souza et al., 1994), is supposed to have coevolved with
P. vulgaris.
Populations of rhizobia isolated from Mexico and considered to belong to R. etli
appear to be very diverse. The total genetic diversity found in three different studies
was similar (0.487, Souza et al., 1994; 0.504, Silva et al., 1999; and 0.531, Segovia
et al., 1991). The five populations collected over 2 years from nodules of wild and
cultivated common beans sampled in three locations by Souza et al. (1994) were
found to differ in terms of ETs and of allelic frequencies, among sites, and within
a site across years. The differences among sites were attributed to differences in
soil nutrient characteristics. On average, 50% of the genetic diversity in a sam-
pled population was present within plants. Populations isolated from cultivated
P. vulgaris and Phaseolus coccineus, grown in six traditionally managed plots
in close proximity, were also very diverse, globally and within each plot (Silva
136 N. AMARGER
et al., 1999). They were genotypically differentiated and, as for the populations de-
scribed by Souza et al. (1994), these differences in composition were related to soil
characteristics. The main proportion of the variability was found within the plant
level (70%) rather than at higher local geographical scales. P. coccineus plants were
found to nodulate with a wider range of genotypes than P. vulgaris. On the basis
of DNA fingerprinting and plasmid profile, Aguilar et al. (1998b) observed diver-
sity of R. etli isolates from wild beans at all levels of sampling, within plants and
within and among sites across north western Argentina. Highly diverse populations
of R. etli bv. phaseoli have been found not only in the Americas but also in tropical
regions of Africa and in Indonesia (Tjahjoleksono, 1993; Anyango et al., 1995).
Tjahjoleksono (1993) isolated populations of 100 isolates each from P. vulgaris
grown in two fields 40 km apart in Indonesia and in one field in Burundi. All isolates
were identifed as R. etli bv. phaseoli with the exception of two from Burundi which
were identified as R. tropici. Using RFLP fingerprinting with chromosomal probes,
42 genotypes were described. The two Indonesian populations were well differ-
entiated from each other and equally diverse; they counted 15 genotypes each and
shared only 1 of these genotypes. The Burundian population counted 13 genotypes,
none of which was closely related to the Indonesian ones. Anyango et al. (1995) iso-
lated from two Kenyan soils with contrasting pH rhizobia that could be considered
as R. etli bv. phaseoli and represented almost all (40/41) isolates from the near-
neutral soil and a small fraction (5/35) of the isolates from the acid soil. This species
has also been identified as a constituent of populations recovered from bean grown
in soils from Tunisia (Mhamdi et al., 1999), Spain (Herrera-Cervera et al., 1999)
Austria (Sessitsch et al., 1997a), and France (unpublished results), representing 5 to
58% of the isolates. Although numbers of isolates studied in these populations were
limited, various chromosomal genotypes were detected among the R. etli bv. phase-
oli isolates at each site. Collectively, these results show that R. etli is highly diverse
not only in the countries where it likely coevolved with Phaseolus spp., but also in
countries where Phaseolus spp. have been introduced for less than 5 centuries. This
high diversity might have been generated by frequent recombinations between the
limited numbers of R. etli bv. phaseoli genotypes introduced with the seeds or by
pSym transfer from these introduced genotypes to nonsymbiotic R. etli or R. etli
of different plant specificity previously present in the soils. The distribution of R.
leguminosarum bv. phaseoli, among the populations studied so far, is more limited.
It was a component of the Tunisian and Spanish populations (Herrera-Cervera et al.,
1999; Mhamdi et al., 1999) and has also been identified in South American pop-
ulations by Aguilar et al. (1998b). This species was found in the different French
populations analyzed in proportion varying from 2 to 100% (Geniaux et al., 1993;
Amarger et al., 1994), and was the only species recovered from the UK population
studied by Young (1985). The chromosomal diversity among isolates varied with
the sites. One chromosomal genotype that represented 98% of the UK population
was shared with isolates from two French populations (Geniaux et al., 1993).
The strains of collections first described as R. tropici, (Martinez-Romero et al.,

1991) were isolated from Brazil and Columbia. Eleven of the 35 ETs described
corresponded to Phaseolus isolates, the others being Leucaena isolates. In the
American populations of bean rhizobia studied since then and that do not include
Brazilian populations, R. tropici has only been detected as a scarce constituent: 1
isolate of 53 from a Mexican population (Vasquez Arroyo et al., 1998) and 1 of
64 isolates sampled in 17 sites in Argentina (Aguilar et al., 1998b). The same is
true for African populations isolated from near-neutral soil (Tjahjoleksono, 1993;
Anyango et al., 1995), but not for populations from acid soils either from Kenya
(Anyango et al., 1995) or from France (Amarger et al., 1994), in which R. tropici
was the predominant species. These different results suggest that when R. tropici
is present in soils, it is not competitive enough to nodulate beans in the presence of
R. etli or R. leguminosarum. However, when the densities of the competitive species
diminish due to acid pH, R. tropici, which is more tolerant to acidity (Graham et al.,
1994), can express its bean-nodulating capacity. R. gallicum has been identified
as a component, which represented 21 to 42% of the isolates, of the populations
studied in Austria (Sessitsch et al., 1997a), Spain (Herrera-Cervera et al., 1999),
and Tunisia (Mhamdi et al., 1999). It was also detected in collections of strains
isolated from Mexico (Sessitsch et al., 1997b) and from France (Amarger et al.,
1997), suggesting a wide distribution. In the Spanish population, MLEE analysis
distinguished two clusters of ETs only distantly correlated among the isolates
carrying 16S rDNA alleles similar to R. gallicum and that could correspond to
different species. The distribution of R. giardinii seems more limited. It formed
about one-third of the isolates in two French populations (Geniaux et al., 1993) and
5% in the Spanish and Tunisian populations (Mhamdi, 1999). In addition to four
of the recognized species of Phaseolus rhizobia, isolates with 16S rDNA RFLP
patterns matching Sinorhizobium fredii have been identified in the Spanish and in
the Tunisian populations. These S. fredii-like strains were not soybean symbionts,
as they did not have the capacity to nodulate American or Chinese soybeans,
but represent a new type of Phaseolus symbiont that forms effective nodules on
common bean.
In a recent study, Caballero Mellado et al. (1999) have shown that levels of
fertilization commonly used in agricultural fields in Mexico diminish the genetic
diversity among nodule isolates of some but not all P. vulgaris cultivars, almost
eliminating strains that diverged from the main R. etli group at a genetic distance
of 0.8.
Hybridization of digested genomic DNAs with R. etli bv. phaseoli nif H and
nodB gene probes has been used in several studies to characterize the symbiotic
genome. All isolates identified as R. etli or R. leguminosarum in the different
populations analyzed, a portion of the isolates identified as R. giardinii in the
French populations (Geniaux et al., 1993), or as R. giardinii or R. gallicum in
the Spanish population (Herrera-Cervera et al., 1999) appeared to contain three
138 N. AMARGER
copies of nif H, a characteristic of the bv. phaseoli described in the four species.
In contrast to the high chromosomal diversity observed among and within these
four species, only seven nif H hybridization patterns were detected among the
many chromosomal genotypes analyzed. Two of these patterns were represented
in the four species and in high proportions in every population analyzed. This sug-
gests that transfer of symbiotic genes has occurred within and between species.
This also implies that the symbiotic gene region carried by the pSym is remark-
ably stable and/or that there is a strong selective advantage for the few gene
arrangements that have been conserved in the course of evolution and among
diverse genetic backgrounds. The remaining portion of the isolates identified as
R. gallicum by Herrera-Cervera et al. (1999); the R. gallicum present in the pop-
ulations studied by Sessitsch et al. (1997) and Mhamdi (1999); the few R. tropici
identified by Anyango et al. (1995), Aguilar et al. (1998b), and Tjahjoleksono
(1993); and the S. fredii-like isolates found by Herrera-Cervera et al. (1999) and
Mhamdi (1999) all contained a single band hybridizing to the nif H probe. The
size of the band was the same for the isolates of the same species and corre-
sponded in both R. gallicum and R. tropici to that of the type strain, suggest-
ing conservation of the symbiotic genome within each species and over large
distances.
It appears from the different studies that the Phaseolus rhizobia are very diverse
at the species, intraspecies, and population levels. Paradoxically, the highly spe-
cialized symbiotic genome that likely coevolved with the plant is very conserved.
It can be expressed in different species backgrounds and succeeded to be ubiqui-
tous in less than 5 centuries. The populations of rhizobia present in the nodules
of Phaseolus spp. are highly differentiated between sites. It is difficult from the
available data to identify factors that might be involved in the distribution of the
different species among sites if we except low soil pHs that enhance the proportion
of R. tropici in the populations.
4. Rhizobia That Nodulate Plants of the Genera Medicago,

Melilotus, or Trigonella
Rhizobia isolated from nodules of plant species belonging to the related genera
Medicago, Melilotus, and Trigonella were classified in a single species, R. meliloti.
This species has been transferred to the genus Sinorhizobium (de Lajudie et al.,
1994). Nodulation and effectiveness interactions are commonly observed between
host plants and strains from this group, some species, like Melilotus sativa and
Melilotus alba, being promiscuous, others, like Melilotus laciniata, extremely
selective. Nodulation and effectiveness host groups have been defined to take
into account these interactions (Brockwell and Hely, 1966). The existence of
two major phylogenetic divisions (A and B) in Sinorhizobium meliloti has been
demonstrated by Eardly et al. (1990) and confirmed by Gordon et al. (1995).
Division A represented a cosmopolitan collection of strain from a variety of an-

nual and perennial medics, including alfalfa, and division B was restricted to strains
from annual species growing in the Mediterranean region. Isolates of division B
are now classified in the species S. medicae (Rome et al., 1996). Recently, R.
mongolense has been created to accommodate isolates from Medicago ruthenica
that also nodulate P. vulgaris. Some of these isolates form ineffective nodules with
M. sativa. In addition to these three species, strains that belong to an unrecognized
taxonomic group of Rhizobium (Eardly et al., 1992) have been recovered from al-
falfa grown in moderately acid soils (5.0 < pH < 6.0) in Oregon (Eardly et al., 1985)
and in central Argentina and Uruguay (Del Papa et al., 1999). Strains from this
group nodulate alfalfa and P. vulgaris but form ineffective nodules on both species.
Sinorhizobium meliloti is highly sensitive to acid conditions and its abundance
in soils decreased sharply as the pH of the soils decreased below pH 6. It reaches
undetectable levels at pH 5 (Rice et al., 1977; Amarger, 1980). For this reason, in-
oculation of alfalfa is a common practice in soils of pH < 6.5.The different methods
used to study field or soil populations of S. meliloti have all revealed considerable
levels of diversity in these populations. Phage typing has been used in separate stud-
ies to evaluate the composition of populations of S. meliloti isolated from different
host genotypes growing in field or in soils collected from different sites in Canada
(Bromfield, 1984; Thurman and Bromfield, 1988). In both studies, the variety and
distribution of phage types differed markedly between sites, a difference that was
attributed by the authors to differences in cropping histories. Significant variations
in the frequency of occurrence of particular phage types were observed both among
the three host species, M. sativa, Melilotus lupulina, and Melilotus alba, and the
two M. sativa cultivars tested by Thurman and Bromfield (1988) and Bromfield
et al. (1984) respectively, indicating host genotype variation in nodulation pref-
erences for specific soil rhizobia. However, this genotype-specific selectivity was
not revealed at each site. Variation in nodulation preferences was also observed
among plants in each species. This variation fluctuates with the species and ap-
peared to be related to pollination characteristics, less variation being present for
the self than for the cross-pollinated species. Site-dependent variations in the di-
versity of strains nodulating different varieties of M. sativa were also reported
by Paffetti et al. (1998), confirming that genotype-specific selectivity is related to
local characteristics that could be the composition of the soil population and/or
soil properties.
Direct evidence of host preference for specific members of soil populations of
R. meliloti has been given in two studies that have used IS typing to compare the
diversity of populations isolated directly from soil and from nodules of plant hosts.
The variety of IS genotypes from the two plant species studied by Bromfield et al.
(1995) was either similar, in the case of M. albus, or greater, in the case of M.
sativa, than that obtained directly from the soil. Eight of the 9 genotypes identi-
fied in soil were present in different proportions in the two plant species, showing
140 N. AMARGER
that the plants have selected the most abundant soil rhizobial genotypes. How-
ever, their relative distribution differed between the three populations, indicating
that they have been differently selected by the two plant species and that relative
abundance of rhizobial genotypes in nodules is not indicative of that in the soil
population. Moreover, the isolation of 7 additional genotypes from alfalfa indi-
cates that this plant has also selected less abundant and likely more competitive
genotypes. Marked difference in the diversity of the IS genotypes isolated from soil
relative to that sampled from nodules of alfalfa has also been observed in the popu-
lations studied by Hartmann et al. (1998). Although the level of diversity detected
among soil and nodule isolates were similar, 38 and 37 genotypes respectively,
only 11 genotypes were common to both populations. Moreover, the distribution of
these common genotypes varied among the two populations. These results confirm
that nodule populations are not representative of soil populations. They also show
that the population of S. meliloti present in the field was highly diverse and that
each method of sampling has revealed a different portion of the diversity. Chro-
mosomal and megaplasmid variations have been examined by Bromfield et al.
(1998) in samples of two field populations of S. meliloti in close proximity using
RFLP analysis with gene probes from the chromosome and from each of the two
megaplasmids (pnod and pexo). Comparative analysis of the polymorphisms ob-
served at the different chromosomal and megaplasmid loci have revealed that the
same chromosomal type commonly coexists with different pexo or pnod types and,
conversely, the same megaplasmid types occur with different chromosomal types.
This suggests that genetic exchanges of megaplasmids sequences have occurred
among isolates in the two populations. However, transfer of the pexo sequences ap-
peared more limited than the pnod ones since the distribution of the megaplasmid
loci across chromosomal backgrounds was random for the pnod plasmid and not
random for the pexo plasmid.
Measurements of symbiotic effectiveness of isolates representing the diversity
found in alfalfa populations has revealed high average levels of symbiotic effec-
tiveness (86 to 95%) relative to standard inoculant strains (Bromfield et al., 1987;
Shishido and Pepper, 1990; Hartmann and Amarger, 1991; Gandee et al., 1999).
5. Rhizobia That Nodulate Soybean
The soybean microsymbiont are presently classified in three species of Bradyrhi-

zobium, B. japonicum, Bradyrhizobium elkanii, and Bradyrhizobium liaoningense,
and in one species of fast-growing rhizobia, S. fredii. These four species are in-
digenous to Chinese soils. Their relative abundance in these soils is not well doc-
umented, but the four species have been detected in different Chinese provinces
(Chen et al., 1988; Xu et al., 1995). Slow- and fast-growing species can coex-
ist in a same soil and compete, more or less successfully, depending on soil and
soybean cultivar, for the formation of nodules (Dowdle and Ben Bohlool, 1986).
Since soybean rhizobia are not indigenous to Western countries, their introduction
in these countries necessitates the use of rhizobial inoculants. Soybean rhizobia
have thus been progressively introduced with the soybean crop, first in the United
States and later in South America, Europe, and Africa. Since the strains used in the
inoculants were bradyrhizobia, which appeared later to belong to the two species
B. japonicum and B. elkanii, the populations of soybean rhizobia that have devel-
oped in the soils of these countries are composed of one or both species. Since
soybean inoculation is routinely practiced, the established populations generally
result from several successive introductions.
Serological methods have been the most commonly utilized methods in studying
the diversity of soybean bradyrhizobial populations. Serogroups are correlated with
most groupings based on other phenotypic and genetic characteristics (reviewed by
Fuhrmann, 1993) and provide convenient means of characterizing nodule isolates.
However, no apparent correlation between symbiotic effectiveness and any of the
phenotypic or genotypic characteristics of soybean bradyrhizobia has been found
so far. Serological studies of field populations of soybean bradyrhizobia have
revealed considerable diversity within and among geographical locations in U.S.
soybean production areas. Serogroup 123 isolates (later identified as B. japonicum)
were the more prevalent in the upper Midwest (Damirgi et al., 1967; Ham et al.,
1971; Keyser et al., 1984; Kamicker and Brill, 1986; Weber et al., 1989) while
serogroups 31, 46, 6, 76, and 94 isolates (later identified as B. elkanii) were common
in Southern soils (Caldwell and Hartwig, 1970; Keyser et al., 1984; Fuhrmann,
1989; Weber et al., 1989; Mpepereki and Wollum, 1991; Ramirez et al., 1997). The
prevalence of distinct serogroups in these regions could be related to a certain extent
to the presence of strains of the corresponding serogroups in early inoculants used
in these regions (Weber et al., 1989). In some instances the serogroups present in
soybean nodules appeared related to soil properties such as soil nitrogen (Bezdicek,
1972) or pH (Damirgi et al., 1967; Ham et al., 1971). Whereas some studies have
demonstrated that soybean genotype can affect serogroup recovery (Caldwell and
Vest, 1968; Caldwell and Weber, 1970; Kvien et al., 1981), no cultivar effect
could be shown in other locations (Fuhrmann, 1989), indicating that such effects
are dependent on local parameters.
In Japan, where soybean is generally cultivated without inoculation, site-
dependent variations were observed in the distribution of Bradyrhizobium
species and of RS␣ and RS␤ genotypes among populations of soybean isolates
(Minamisawa et al., 1999), suggesting that bradyrhizobia have diversified in asso-
ciation with various factors found in individual fields. Highly diverse populations
of soybean rhizobia are also encountered in countries with a more recent history of
soybean cultivation. In tropical soils from Brazil, new genotypes adapted to local
environments have developed from early inoculant strains of B. elkanii (Neves
and Rumjanek, 1997). In Poland, although most of the isolates recovered from
different sites belonged to two serotypes, they were found to be highly diverse on
142 N. AMARGER
the basis of antibiotic resistance, protein content, rep-PCR patterns, and nif and
nod gene hybridization profiles, which suggests that they have diversified since
their release in the late 1980s (Madrzak et al., 1995). Surprisingly, in soils where
soybean has been inoculated with a single bradyrhizobial strain, strains differing
from the inoculant strain have been identified in nodule isolates from soybean cul-
tivated several years later (van Rensburg and Strijdom, 1985; C. Revellin, personal
communication). Although these strains might have been introduced with seeds,
they might as well result from genetic interactions between inoculant strain and
soil bradyrhizobia of different plant specificity or nonsymbiotic. On the whole,
these results show that, following the introduction of a limited number of strains
in field environment, highly diversified populations of soybean bradyrhizobia can
develop in relatively short periods of time.
B. POPULATION STRUCTURE
Great diversity is a common feature among rhizobial populations, no matter

which legume species they have been isolated from. Levels of genetic diversity,
estimated by MLEE analysis, are similar for the different rhizobial species studied,
with values near 0.50–0.55, and are retained in most of the individual populations.
Lower levels of genetic diversity found in some populations were generally asso-
ciated with specific soil conditions such as acid pH (Harrison et al., 1989) or high
nitrogen content (Souza et al., 1994; Caballero-Mellado, 1999). Most of the varia-
tion observed in a given population is found within individual plants (Young et al.,
1987; Souza et al., 1994; Hagen and Hamrick, 1996a, 1996b; Silva et al., 1999).
Thus, with the exception of recently introduced legume species, each legume plant
has access to a great proportion of the diversity found in its associated rhizobial
species. This genetic diversity has been associated with levels of linkage desequi-
librium (nonrandom association between the alleles of different enzyme loci) that
varies with the population studied.
Absence or low levels of linkage desequilibrium have been estimated in pop-
ulations of the species R. leguminosarum bv. viciae (Gordon et al., 1995), R.
leguminosarum bv. trifolii (Hagen and Hamrick, 1996a,b), nonsymbiotic R. etli
(Segovia et al., 1991), R. etli bv. phaseoli (Souza et al., 1994), and S. meliloti
(Bromfield et al., 1998), suggesting that frequent genetic exchanges have occurred
in these populations. Conversely, significant levels of linkage desequilibrium have
been found in other populations of R. leguminosarum bv. viciae (Young et al.,
1987; Strain et al., 1995), R. leguminosarum bv. trifolii (Harrison et al., 1989;
Demezas et al., 1991; Strain et al., 1995; Hagen and Hamrick, 1996a,b), R. etli
bv. phaseoli (Pinero et al., 1988; Souza et al., 1992; Souza et al., 1994; Silva
et al., 1999), and R. meliloti (Eardly et al., 1990; Gordon et al., 1995). They have
been interpreted as resulting predominantly from clonal reproduction, implying
that chromosomal recombination is a rare event in these populations. However,

in some of these populations (Eardly et al., 1990; Demezas et al., 1991; Gordon
et al., 1995; Strain et al., 1995), cluster analysis has revealed deeply diverging lin-
eages and absence or low levels of linkage desequilibrium within these lineages,
suggesting that the source of linkage desequilibrium was more likely reproductive
isolation than clonality. This type of population structure corresponds to the struc-
ture described by Maynard Smith et al. (1993) as reticulated. Recently Silva et al.
(1999) have observed reticulated structure in populations of R. etli bv. phaseoli.
The reticulated structure of these populations was associated with an epidemic
structure, which corresponds to populations “in which recombination occurs but
occasionally a highly successful genotype arises and increases in frequency to pro-
duce an epidemic clone” (Maynard Smith et al., 1993). It can also generate linkage
desequilibrium in populations. It is therefore possible that genetic recombination
occurs more frequently in rhizobial populations than first estimated.
The differences observed in the composition of populations directly isolated
from soils and isolated from nodules have shown that populations isolated from
nodules are not representative of soil populations (Bromfield et al., 1995; Hartmann
et al., 1998). Same conclusions are drawn when comparing the composition of pop-
ulations isolated from different genotypes growing on the same soil. Structures of
rhizobial populations that have been inferred from the populations studied to date
are therefore not the structures of the actual populations of rhizobia present in the
soils but rather structures of portions of these populations that have been differ-
ently filtered by plants. In populations directly isolated from soil, frequent genetic
exchanges are suggested both by the absence of linkage desequilibrium (Segovia
et al., 1991) and by the random distribution of Sym types across chromosomal
genotypes (Louvrier et al., 1996). It seems therefore likely that the linkage des-
equilibrium observed in nodulating populations of rhizobia result more from the
plant selectivity than from the absence of recombination in the soil populations.
This suggests that genetic recombination plays an important role in generating
diversity within populations of rhizobia.
V. INTRODUCTION OF RHIZOBIA INTO SOIL
Rhizobia are very widespread as a result of the natural distribution and of the
cultivation of legumes. Despite this, there are still soils where strains of rhizobia
specific for a legume crop are absent or present in low numbers. Such situations
are encountered when a new crop is introduced into a region where symbiotically
related legumes are absent, e.g., soybean in western Europe (Obaton and Rollier,
1970; Madrzak et al., 1995), or where the soil or environmental conditions are
detrimental for the occurrence or the survival of a rhizobial species, e.g., acid soils
144 N. AMARGER
for R. leguminosarum bv. trifolii (Nazih et al., 1993), S. meliloti (Amarger, 1980),
alkaline soils for Bradyrhizobium sp. Lupinus (Amarger, 1980), and high tem-
peratures for chickpea rhizobia (Ruppela et al., 1974). Introduction of rhizobial
strains may also be desirable where soils harbor populations of rhizobia com-
posed of a majority of strains symbiotically ineffective with a particular legume
(Gibson et al., 1975; Hagedorn, 1978; Bottomley and Jenkins, 1983; Quigley
et al., 1997; Moawad et al., 1998) or when better performing strains become
available (Bosworth et al., 1994).
A. INOCULATION
Legume inoculation is an agricultural practice that has been used for more than
a century to introduce rhizobia into the soil at sowing (for review see Brockwell,
1977; Date and Roughley, 1977; Roughley, 1988; Smith, 1992; Somasegaran and
Moben, 1994; Brockwell and Bottomley, 1995). Commercial inoculants are pro-
duced in many countries. Their quality depends on both the number of rhizobia
they contain and their effectiveness in fixing nitrogen with the target host. Besides
symbiotic effectiveness, strain selection usually takes into consideration other char-
acters such as genetic stability, ability to survive in inoculant, to persist in soil, and
to compete in nodule formation with soil rhizobia. Inoculants are produced in pow-
der, granular, or liquid forms. They can be applied directly on the seed, which is
the traditional and most commonly used means of inoculation, on mineral granules
(Wadoux, 1991), or into the seedbed (Hynes et al., 1985). Their quality, evaluated
by enumeration of viable rhizobia, is highly variable and it appears that a high
proportion of those presently on the market in countries where quality controls are
not systematically practiced are of poor quality (Catroux et al., 1999). However,
high-quality inoculants are produced and are available in powdered or liquid form
in North America, Europe, and some other countries. They can be stored one year
at room temperature and provide at least 106 viable rhizobia per seed for soybean.
Assuming minimal losses during inoculation, high-quality inoculants introduce in
soil approximately 2 × 1011 to 4 × 1011 rhizobia ha−1, which represents about 1%
of the numbers of rhizobia present in the top 20 cm of soil containing 104 g−1
rhizobia (Catroux and Amarger, 1992).
B. SOIL COLONIZATION BY INOCULANT RHIZOBIA
When soils are devoid of rhizobia, the inoculant strain nodulates the host legume
and multiplies in the nodules and, upon nodule senescence, high numbers of viable
cells are released into the soil. For soybean, numbers of 106 bradyrhizobia per gram
of soil are commonly observed after cropping (Brockwell et al., 1987; Lagacherie,
1978; Hiltbold et al., 1985). The introduced rhizobia most often become a persistent
if not permanent component of the soil microflora. Soybean rhizobia present in
early inoculants are still prevalent members of the soil rhizobial populations in
many parts of the United States (Weber et al., 1989). The survival of the introduced
rhizobia depends on how the strain responds to or resists the conditions that prevail
in the soil. Multiple abiotic and biotic factors (reviewed recently by Sadowsky and
Graham, 1998) can affect the persistence of rhizobia in soils.
When soils contain indigenous rhizobia, the inoculant rhizobia have to com-
pete with these indigenous rhizobia for the formation of nodules. Very often they
are not successful and cannot be recovered from the plant nodules (Dudman and
Brockwell, 1968, Ham, 1980; Carter et al., 1995). Although in these cases inocula-
tion has failed, it does not mean that introduction of the inoculant rhizobia in soils
has also failed. Due to methodological problems, quantitative data on the presence
of inoculant bacteria in soils containing indigenous rhizobia are scarce. Two sep-
arate studies have shown that marked strains of R. leguminosarum bv. viciae, field
released as inoculants in soils containing indigenous rhizobia, could survive at
the level of the indigenous population for several years, whether they have formed
nodules or not (Hirsch and Spokes, 1994; Amarger and Delgutte, 1990). In another
field experiment, a nonsymbiotic strain of R. leguminosarum was also found to
survive in numbers similar to those of the indigenous populations, several years
after its release (Hirsch, 1996). These findings provide evidence that inoculant
strains can persist in soils containing indigenous rhizobia even if they have not
multiplied in nodules. However, such an introduction was found to depend on local
conditions, since a same strain, inoculated to a pea crop in three different European
countries, behaved differently and 1 year after release was only detectable at one
site (Hirsch, 1996).
C. INTERACTIONS WITH INDIGENOUS RHIZOBIAL POPULATIONS
Evidence that strains, once introduced into soils, can exchange genetic informa-
tion with indigenous bacteria has been given recently by Sullivan et al. (1995). A
single strain of M. loti was introduced as an inoculant of a Lotus corniculatus crop
in a New Zealand soil devoid of M. loti. Seven years later, the mesorhizobia re-
covered from the other nodules were genotypically diverse. Despite their diversity
all strains contained a chromosomally integrated symbiotic region identical to the
original inoculant strains. Since then, transfer of a 500-kb symbiosis island from
an M. loti strain to at least three genomic species of nonsymbiotic mesorhizobia
has been demonstrated in laboratory matings (Sullivan et al., 1996). These findings
give evidence that the inoculant bacteria have generated, in the field environment,
146 N. AMARGER
a new diversified population of Lotus-nodulating mesorhizobia by lateral transfer

of their chromosomal symbiotic genes to nonsymbiotic mesorhizobia.
Knowing that transfer of symbiotic genes is not limited to rhizobia with plasmid-
encoded symbiotic genes, we can speculate that gene transfer also occurs be-
tween bradyrhizobia. This could explain why, in soils which were devoid of in-
digenous soybean rhizobia, bradyrhizobia different from the original inoculant
strain were recovered from soybean nodules (van Rensburg and Strijdom, 1985;
C. Revellin personal communication). We can also suppose that the high diver-
sity presently found in populations of soybean bradyrhizobia, which was intro-
duced less than a century ago, has developed from genetic exchanges between
inoculant strains and local bradyrhizobia. The presence of conserved DNA re-
gions around nif genes among different genotypes of soybean bradyrhizobia iden-
tified in Japanese populations would support this hypothesis (Minamisawa et al.,
1999).
Although there is circumstancial evidence of pSym transfer within and between
species of Rhizobium, evidence that such transfers occur in the field is still missing.
Transfer of a marked conjugative pSym from an R. leguminosarum bv. viciae strain,
released as an inoculant, to indigenous R. leguminosarum was not detected in field
experiments (Amarger and Delgutte, 1990; Hirsch and Spokes, 1994), nor was the
acquisition of genetic elements from indigenous bacteria by introduced inoculant
strains (Hirsch, 1997). This suggests that plasmid transfers in natural environments
are not frequent (<10−5), at least in the absence of selection pressure for the
plamid-encoded characters.
D. AGRICULTURAL IMPLICATIONS
It has to be realized when performing inoculation that the inoculant strain has
the potential to become a permanent member of the soil microflora and to exchange
genetic information with the soil-resident bacteria, even in case of inoculation fail-
ure. Once introduced into the soil, the inoculant rhizobia may form an ineffective
symbiosis with a subsequent crop presenting the same nodulation specificity. They
may also exchange genes with soil bacteria and create new rhizobia less effective
than the original inoculant strain with the inoculated crop. In both cases they are
likely to form a barrier to the introduction of more effective strains. Presently,
there are no reliable means of estimating the potentialities of a strain to persist
in soil and to exchange genes with other bacteria. It is therefore neither possible
to evaluate the risks that are taken when inoculation is performed nor to prevent
them. Although there is no clear evidence of the existence of any critical agricul-
tural problem caused by the practice of legume inoculation, which suggests that
the risks taken were not too high, the problem should be considered and more
knowledge on the behavior of rhizobia in soil has to be acquired.
VI. CONCLUDING REMARKS
Since 1985, molecular and genetics analyses have considerably increased our
understanding of legume–rhizobium symbiosis and there is evidence that these
processes benefit legume productivity through targeted selection or modification
of one or both partners. However, more knowledge on the ecology of rhizobia is
needed for reasoned exploitation of such organisms. Progress in molecular biology
has allowed the development of bacterial characterization methods more reliable
than the phenotypic methods used previously. The application of these methods
to rhizobia has revealed a huge diversity in any characters studied. Such diversity
is difficult to handle and although species are created at an accelerated pace, they
accommodate only a part of this diversity. These numerous species, created on the
basis of polyphasic taxonomy, are difficult to distinguish and there is an increasing
need for simple standardized methods that could be used easily for identifying and
classifying the large numbers of rhizobial isolates that are required in ecological
studies. Nevertheless, progress in the determination of the composition of rhizobial
populations has been made. The diversity of rhizobia recovered from nodules of
a single legume crop, and even of a single plant, is great. Many legume species
are nodulated by several rhizobial species or genera. This diversity gives the plant
multiple opportunities to nodulate with effective rhizobia. Its agricultural drawback
is that it is likely to present a barrier to the establishment of inoculant strains in
the legume crop ecosystem. However, populations studied up to now represent
almost exclusively populations isolated from nodules. Although they are those of
interest in agriculture, they represent only a portion of the rhizobial populations
present in soils. In order to control introduction of new strains and/or better manage
indigenous strains, we need to acquire knowledge on the identity and behavior of
rhizobia in soils. Methods for direct isolation from soil, in situ localization, and
identification need to be developed. Finally, and likely most importantly, we have
gained evidence that genetic exchanges occur in field conditions and can build
rhizobial diversity in just a few years. We have now to determine how frequent
these exchanges are and which conditions favor or repress them. For the past
century, through introduction of new legume crops and rhizobial inoculation in
most parts of the world, humans have actively participated in the spread of the
existing rhizobial diversity and the creation of a new one, the challenge now is to
manage this diversity.
ACKNOWLEDGMENTS
The author is grateful to Louise Nelson for reviewing the Chapter and to Gisèle Laguerre for
constructing the phylogenetic tree.
148 N. AMARGER
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Index
A morphology, 7
taxonomy, 7–8
AM fungi, see Arbuscular mycorrhizal fungi AURY, see Apparent urea relative yield
Ammonia
acidification of soils, 66–67 B
emission factors for nitrogen fertilizers,
88–90, 100–101 Barley, dwarfing gene mutants
emission measurement following nitrogen breeding application, 45
fertilizer application gibberellin-insensitive mutants, 41
agronomic evaluations, 83–84 molecular mapping, 51–52
field measurements phytochrome mutants, 42
chamber measurements, 82–83 quantitative trait loci and linkage mapping,
micrometeorological methods, 79 52–53
overview, 78–79
summary of studies, 80–81 C
wind tunnel measurements, 79, 82
laboratory measurements, 85–86 Clover rhizobia, see Rhizobium leguminosarum
solution emissions, 86–87, 100 bv. trifolii
urease inhibitor studies, 87–88 Competitor, soil fungus classification, 3
environmental factors in soil release, 68–69, Corn, dwarfing gene mutants
99 gibberellin-insensitive mutants, 41
fertilizer sources, 67–68, 101 gibberellin-sensitive mutants, 39
volatilization
barium soils, 70–71 D
bicarbonate–carbonate equilibrium and soil
pH, 70 Dwarfing genes
calcium addition studies, 71–73 agronomic importance, 36, 56
calcium soils, 70–73, 77, 100 breeding application
fertilizer type effects, 75 barley, 45
ionization equilibrium, 69 challenges, 45–47
magnesium soils, 70–71 foxtail millet, 45
modeling, 75–78 oat, 43, 45, 47
pH role, 74–75 pearl millet, 45–46
rate of fertilizer application effects, rice, 43, 47
73 sorghum, 45
Apparent urea relative yield (AURY), wheat, 43, 46–48
calculation, 84 cereal improvement genes, 44
Arbuscular mycorrhizal (AM) fungi cloning and sequencing, 54–55
abundance and distribution, 8–10 gibberellin mutants
agroecosystem role, 10–12 biosynthetic pathway, 38, 55–56
host preference, 8, 10 insensitive mutants
interactions at root with pathogenic and barley, 41vigor effects, 46
nonpathogenic fungi, 14–15 corn, 41
169
170 INDEX
Dwarfing genes (continued) Foxtail millet, dwarfing gene breeding

rice, 40–41 application, 45
wheat, 40–41 Fungus, see also Arbuscular mycorrhizal fungi;
sensitive mutants Rhizosphere; specific fungi
corn, 39 additional nonpathogenic fungi in
dominance of genes, 40 classification
oat, 40 abundance and distribution, 12–13
rye, 39 agroecosystem role, 13
wheat, 39 definition, 12
signal transduction, 38–39 functional group classification in soil, 3–4
historical perspective of breeding, pathogens
37–38 clinical pathogens
molecular mapping abundance and distribution, 4–5
barley, 51–52 agroecosystem role, 5
oat, 52 definition, 4
rice, 51 interactions at root
wheat, 51 arbuscular mycorrhizal fungi, 14–15
ortholog identification by comparative co-infection, 13
mapping, 54 island biogeography theory, 16–17
phytochrome mutants nonpathogenic fungi, 15–16
barley, 42 subclinical pathogens
oat, 42 abundance and distribution, 6
transgenic plants, 42 agroecosystem role, 6–7
pleiotropic effects definition, 5–6
Dw genes in oat, 49–50 pest control
morphology and physiology, 49–50 biological control
Rht genes in wheat, 48–50 obstacles to implementation, 21
rice, 49 role, 20–21
yield, 50 strategies
quantitative trait loci and linkage mapping integrated pest management, 18–20
barley, 52–53 proactive pest management, 19
oat, 53–54 single-tactic approach, 18, 20
rice, 53 symbiosis versus pathogenicity in soil, 2
rye, 53 Fusarium oxysporum
Dw genes, see Dwarfing genes disease-suppressive soils, 23
interactions at root between pathogenic and
E nonpathogenic fungi, 15–16
proactive pest management, 19
Emission factors, nitrogen fertilizers
ammonia, 88–90, 100–101 G
nitrous oxide, 97–98, 101
Gibberellin, dwarfing gene mutants
F biosynthetic pathway, 38, 55–56
insensitive mutants
FAME analysis, see Fatty acid methyl ester barley, 41
analysis corn, 41
Fatty acid methyl ester (FAME) analysis, rice, 40–41
rhizobia, 117, 128 vigor effects, 46
Fava bean rhizobia, see Rhizobium wheat, 40–41
leguminosarum bv. viciae sensitive mutants
INDEX 171
corn, 39 comparative studies by fertilizer type,

dominance of genes, 40 90–96, 101
oat, 40 seasonal effects, 92–93, 102
rye, 39 tropical forest soils, 91
wheat, 39 microbial production in soil, 78
signal transduction, 38–39 modeling of soil release, 78
nitrification inhibitor effects on soil emissions
I DCD, 97
N-serve, 96
Integrated pest management (IPM), advantages nitrapyrin, 96–97
and limitations in pest control, 18–20 soil type effects, 96, 102
IPM, see Integrated pest management relative emission assessment for fertilizers,
Island biogeography theory, application to 95
root–fungal interactions, 16–17 soil emission consequences, 67–68
L O
Legume, see Rhizobia Oat, dwarfing gene mutants

Lentil rhizobia, see Rhizobium leguminosarum breeding application, 43, 45, 47
bv. viciae gibberellin-ssensitive mutants, 40
Lipopolysaccharide (LPS), rhizobia analysis, molecular mapping, 52
117 phytochrome mutants, 42
LPS, see Lipopolysaccharide pleiotropic effects, 49–50
quantitative trait loci and linkage mapping,
M 53–54
Maize, see Corn P

Mass spectrometry, pyrolysis mass spectrometry
of rhizobia, 116 Pea rhizobia, see Rhizobium leguminosarum bv.
MLEE, see Multilocus enzyme electrophoresis viciae
Multilocus enzyme electrophoresis (MLEE), Pearl millet, dwarfing gene breeding application,
rhizobia characterization, 116, 128, 142 45–46
Phytochrome, dwarfing gene mutants
N barley, 42
oat, 42
Nitric oxide transgenic plants, 42
emission measurement following nitrogen Plasmid, rhizobia analysis, 118–120
fertilizer application, 99, 102 Proactive pest management, advantages and
microbial production in soil, 78 limitations in pest control, 19
modeling of soil release, 78
soil emission consequences, 67 R
Nitrogen fertilizer gaseous emissions, see
Ammonia; Nitric oxide; Nitrous oxide Randomly amplified polymorphic DNA
Nitrogen fixation, see Rhizobia (RAPD), rhizobia analysis, 121–122, 128
Nitrous oxide RAPD, see Randomly amplified polymorphic
emission factors for nitrogen fertilizers, DNA
97–99, 101 Restriction fragment length polymorphism
emission measurement following nitrogen (RFLP), rhizobia analysis, 120–122, 128
fertilizer application RFLP, see Restriction fragment length
chamber studies, 91 polymorphism
172 INDEX
Rhizobia, see also individual species tolerance to external factors in classification,

assessment of soil populations, 129 114
benefits of symbiosis, 110–111 Rhizobium etli, genetic diversity, 133–134,
definition, 112 137–138
discovery, 110 Rhizobium gallicum, features, 137–138
fatty acid methyl ester analysis, 117, 128 Rhizobium giardinii, features, 137–138
gel electrophoresis analysis Rhizobium leguminosarum bv. phaseoli
denaturing gel electrophoresis of proteins, genetic diversity, 134–135, 137–138
116 host specificity, 134–135
lipopolysaccharide analysis, 117 species, 134–135
multilocus enzyme electrophoresis, Rhizobium leguminosarum bv. trifolii
116–117, 128, 142 characterization, 130
plasmids, 118–120 chromosomal diversity, 132
genetic exchanges, 147 clover host specificity, 130
growth rates, 112 geographic effects on root populations,
host range, 111, 117–118, 147 131–132
identification, 128–129 heavy metal effects on population, 132
introduction into soil pH dependence, 130
agricultural implications, 146 Rhizobium leguminosarum bv. viciae
colonization, 144–145 colonization, 145
indigenous population interactions, environmental factors in pea colonization, 133
145–146 host specificity, 133
inoculation, 144 pH dependence, 132–133
overview, 143–144 Rhizobium meliloti, features, 139
legume association and agricultural Rhizobium tropici, features, 137–138
importance, 110–111 Rhizosphere, see also Fungus; Rhizobia
metabolism and substrate utilization assays, divisions, 3
113–114 research prospects
phage susceptibility, 115 biological control, 23–24
population structure, 142–143 disease-suppressive soils, 23
pyrolysis mass spectrometry for chemical identification of root fungi, 22
composition analysis, 116 molecular biology, 22
randomly amplified polymorphic DNA pesticides, 24
analysis, 121–122, 128 root colonization, 23–24
restriction fragment length polymorphism Rht genes, see Dwarfing genes
analysis, 120–122, 128 Ribosomal RNA (rRNA), gene analysis in
ribosomal RNA gene analysis, 122, 124, rhizobia, 122, 124, 127
127 Rice, dwarfing gene mutants
serological characterization, 115 breeding application, 43, 47
soybean rhizobia gibberellin-insensitive mutants, 40–41
bradyrhizobia, 141–142 historical perspective of breeding, 37–38
serogroups, 141 molecular mapping, 51
species, 140–141 pleiotropic effects, 49
systematics quantitative trait loci and linkage mapping, 53
biovars, 128 rRNA, see Ribosomal RNA
historical perspective, 123–124 Ruderal, soil fungus classification, 3
phylogeny, 124–125, 127–128 Rye, dwarfing gene mutants
species list, 125 gibberellin-ssensitive mutants, 39
taxonomy, 124, 126 quantitative trait loci and linkage mapping, 53
INDEX 173
S U
Single-tactic approach, advantages and Urease inhibitor, effects on ammonia

limitations in pest control, 18, 20 volatilization, 87–88
Sinorhizobium meliloti
host specificity, 138–139 W
pH dependence, 139
symbiotic effectiveness, 140 Wheat, dwarfing gene mutants
Sorghum, dwarfing gene breeding application, breeding application, 43, 46–48
45 gibberellin-insensitive mutants, 40–41
Soybean rhizobia, see rhizobia gibberellin-sensitive mutants, 39
Stress-tolerant, soil fungus classification, 3 historical perspective of breeding, 37–38
Survivors-escapes, soil fungus molecular mapping, 51
classification, 3 pleiotropic effects, 48–50

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Agronomy

Martin Alexander Ronald Phillips

Kenneth J. Frey Larry P. Wilding

Prepared in cooperation with the

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International Standard Book Number: 0-12-000773-8

PRINTED IN THE UNITED STATES OF AMERICA

INTERACTIONS AMONG ROOT-INHABITING FUNGI AND

DWARFING GENES IN PLANT IMPROVEMENT

A REVIEW OF THE EFFECT OF N FERTILIZER TYPE

IV. Ammonia Emission Factors for Nitrogen Fertilizers . . . . . . . . . . . . . . . . . . . 88

RHIZOBIA IN THE FIELD

N. AMARGER (109), Laboratoire de Microbiologie des Sols, Institut National de

David M. Sylvia1 and Dan O. Chellemi2

application of island biogeography theory to the understanding of fungal species

of the complex ecology of root-inhabiting fungi so that researchers will be in a

II. FUNCTIONAL DIVERSITY IN THE ROOT ZONE

A. CLASSIFICATION SCHEMES FOR FUNCTIONAL GROUPS

Winogradsky (1924) attempted to classify soil microorganisms on the basis of

Clinical pathogens can be deﬁned as root-inhabiting fungi that cause visual

2. Abundance and Distribution

Clinical pathogens are found throughout the ecological range of terrestrial

2. Abundance and Distribution

Subclinical root-inhabiting fungi are distributed throughout the range of terres-

Subclinical pathogens also function as regulators of plant density, though to

D. ARBUSCULAR MYCORRHIZAL FUNGI

2. Abundance and Distribution

Max. root Max. spore Species

Aeschynomene Florida 30b 3 6 Medina et al. (1988)

We are becoming increasingly aware of the important multifunctional roles of

Figure 1 The relationship between stability of 1- to 2-mm-size aggregates and immunoreactive

Fixen, 1991); however, addition of phosphorus fertilizer to very low phosphorus

E. ADDITIONAL NONPATHOGENIC FUNGI

This group comprises a diverse collection of fungi capable of invading and

2. Abundance and Distribution

Information on the abundance of nonpathogenic root-inhabiting fungi in natu-

The role of nonpathogenic root-inhabiting fungi in ecosystems is complex. As

III. INTERACTIONS AMONG

A. INTERACTIONS AMONG PATHOGENS

B. INTERACTIONS OF AM FUNGI WITH PATHOGENIC

Often inoculation with AM fungi prior to challenging with a pathogen is neces-

(1997) found no evidence of a localized defense response when G. intraradices

C. INTERACTIONS BETWEEN PATHOGENIC AND

Competition for nutrients by nonpathogenic strains of F. oxysporum can regulate

D. APPLICATION OF ISLAND BIOGEOGRAPHY THEORY

Understanding the interactions within the community of root-inhabiting fungi

IV. OPPORTUNITIES FOR PEST CONTROL

A. CURRENT AND FUTURE CONTROL STRATEGIES

2. Integrated Pest Management

Several fundamental obstacles exist which hinder the widespread adaptation of

3. Proactive Pest Management

Proactive pest management is a strategy which seeks to minimize intervention

B. ROLE OF BIOLOGICAL CONTROL

Biological control of root pathogens can provide substantial beneﬁts to growers

C. OBSTACLES TO IMPLEMENTING BIOLOGICAL CONTROL

Many obstacles remain which impede the widespread adoption of biological

The biology of the rhizosphere is extremely complex due to the interplay of

Advances in grain yield due to the introduction and efﬁcient manipulation of

II. THE BIOCHEMICAL BASIS OF

been classiﬁed as GA-sensitive or -insensitive, respectively. There have also been

Another class of GA mutants is of those that do not respond to the exogenous

III. DWARFING GENES AND THEIR USE

IV. BREEDING CHALLENGES AND