New and Future Developments in

Microbial Biotechnology and

Bioengineering. Aspergillus System
Properties and Applications - eBook
PDF
Visit to download the full and correct content document:
https://ebooksecure.com/download/new-and-future-developments-in-microbial-biotec
hnology-and-bioengineering-aspergillus-system-properties-and-applications-ebook-pd
f/
New and Future Developments in Microbial
Biotechnology and Bioengineering
New and Future
Developments in Microbial
Biotechnology and
Bioengineering
Aspergillus System Properties and Applications

Edited by
Vijai Kumar Gupta

AMSTERDAM • BOSTON • HEIDELBERG • LONDON

NEW YORK • OXFORD • PARIS • SAN DIEGO
SAN FRANCISCO • SINGAPORE • SYDNEY • TOKYO
Elsevier
Radarweg 29, PO Box 211, 1000 AE Amsterdam, Netherlands
The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, United Kingdom
50 Hampshire Street, 5th Floor, Cambridge, MA 02139, United States
Copyright © 2016 Elsevier B.V. All rights reserved.
No part of this publication may be reproduced or transmitted in any form or by any means, electronic or
mechanical, including photocopying, recording, or any information storage and retrieval system, without
permission in writing from the publisher. Details on how to seek permission, further information about the
Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance
Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/permissions.
This book and the individual contributions contained in it are protected under copyright by the Publisher (other
than as may be noted herein).
Notices
Knowledge and best practice in this field are constantly changing. As new research and experience broaden our
understanding, changes in research methods, professional practices, or medical treatment may become necessary.
Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using
any information, methods, compounds, or experiments described herein. In using such information or methods
they should be mindful of their own safety and the safety of others, including parties for whom they have a
professional responsibility.
To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability
for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or
from any use or operation of any methods, products, instructions, or ideas contained in the material herein.
British Library Cataloguing-in-Publication Data
A catalogue record for this book is available from the British Library
Library of Congress Cataloging-in-Publication Data
A catalog record for this book is available from the Library of Congress
ISBN: 978-0-444-63505-1

For Information on all Elsevier publications

visit our website at https://www.elsevier.com

Publisher: John Fedor

Acquisition Editor: Kostas Marinakis
Editorial Project Manager: Sarah Watson
Production Project Manager: Anitha Sivaraj
Designer: Greg Harris
Typeset by MPS Limited, Chennai, India
List of Contributors
A.M. Abdel-Azeem University of Suez Canal, Ismailia,
Egypt
M.A. Abdel-Azeem University of Sinai, North Sinai, Egypt
H.S. AL-Maliki The State University of New Jersey, New
Brunswick, NJ, United States
J.W. Bennett The State University of New Jersey, New
Brunswick, NJ, United States
M. Cereia Universidade de São Paulo, Ribeirão Preto,
SP, Brazil
F.J. Contesini University of Campinas, Campinas, São
Paulo, Brazil
R.R. de Melo University of Campinas, Campinas, São
Paulo, Brazil
M. Dimarogona National Technical University of Athens,
Athens, Greece
C.S. Farinas Embrapa Instrumentation, São Carlos, SP,
Brazil; Federal University of São Carlos, São Carlos,
SP, Brazil
F.J. Fernández Spanish National Science Council (CIB-
CSIC), Madrid, Spain
A.C. Flores-Gallegos Universidad Autónoma de Coahuila,
Saltillo, Coahuila, México
B. Gajaraj Jain University, Bengaluru, India
Bharath Ganesan K.S. Rangasamy College of Technology,
Erode, Tamil Nadu, India
S. Gómez Spanish National Science Council (CIB-CSIC),
Madrid, Spain
R. Hung The State University of New Jersey, New
Brunswick, NJ, United States
N.A. Khan N.D. University of Agriculture and Technology,
Faizabad, UP, India
D. Kumar N.D. University of Agriculture and Technology,
Faizabad, UP, India
M. Kumar Bihar Agricultural University, Sabour Bhagalpur,
Bihar, India; Amity University, Noida, Uttar Pradesh, India
Ravi Ranjan Kumar Bihar Agricultural University,
Sabour Bhagalpur, Bihar, India
Ranjeet Ranjan Kumar Division of Biochemistry, Indian
Agricultural Research Institute, New Delhi, India
V. Kumar Amity University, Noida, Uttar Pradesh, India
F. Lara-Victoriano Universidad Autónoma de Coahuila,
Saltillo, Coahuila, México
S. Lee The State University of New Jersey, New
Brunswick, NJ, United States
S. Lee Rutgers The State University of New Jersey, New
Brunswick, NJ, United States
M. Michel-Michel Universidad Autónoma de Coahuila,
Saltillo, Coahuila, México
K. Mikawlrawng University of Delhi, Delhi, India
M.T. Mohesien University of Damietta, New Damietta,
Egypt
G. Molina University of Campinas, Campinas, São Paulo,
Brazil; Universidade Federal dos Vales do Jequitinhonha e
Mucuri, Diamantina, Minas Gerais, Brazil
A. Mukherjee Special Centre for Molecular Medicine,
Jawaharlal Nehru University, New Delhi, India
V.K. Nadumane Jain University, Bengaluru, India
N.A. Nafady Assuit University, Assiut, Egypt
P. Pandey N.D. University of Agriculture and Technology,
Faizabad, UP, India
G.M. Pastore University of Campinas, Campinas, São
Paulo, Brazil
K.K. Pennerman The State University of New Jersey,
New Brunswick, NJ, United States
M.G. Pereira Universidade de São Paulo, Ribeirão Preto,
SP, Brazil
M.L.T.M. Polizeli Universidade de São Paulo, Ribeirão
Preto, SP, Brazil
A.G. Rodrigues Martin-Luther University Halle-Wittenberg,
Halle, Germany
R. Rodríguez-Herrera Universidad Autónoma de
Coahuila, Saltillo, Coahuila, México
xi
xii List of Contributors
F.M. Salem University of Suez Canal, Ismailia, Egypt
H.H. Sato University of Campinas, Campinas, São Paulo,
Brazil
A.S.A. Scarcella Universidade de São Paulo, Ribeirão
Preto, SP, Brazil
Md. Shamim N.D. University of Agriculture and
Technology, Faizabad, UP, India; Bihar Agricultural
University, Sabour Bhagalpur, Bihar, India
S. Siddiqui Integral University, Lucknow, Uttar Pradesh,
India
K.N. Singh N.D. University of Agriculture and Technology,
Faizabad, UP, India
S. Singh Lovely Professional University, Phagwara, Punjab,
India
E.A. Soliman University of Suez Canal, Ismailia, Egypt
D. Srivastava N.D. University of Agriculture and Technology,
Faizabad, UP, India
P. Teotia Chaudhary Charan Singh University, Meerut,
Uttar Pradesh, India
E. Topakas National Technical University of Athens,
Athens, Greece
A. Varma Amity University, Noida, Uttar Pradesh, India
F. Veana-Hernandez Universidad Autónoma de Coahuila,
Saltillo, Coahuila, México
M.C. Vega Spanish National Science Council (CIB-CSIC),
Madrid, Spain
P. Venkatachalam Jain University, Bengaluru, India
A.C. Vici Universidade de São Paulo, Ribeirão Preto, SP,
Brazil
Chapter 1
Biodiversity of the Genus Aspergillus
in Different Habitats
A.M. Abdel-Azeem1, F.M. Salem1, M.A. Abdel-Azeem2, N.A. Nafady3, M.T. Mohesien4 and E.A. Soliman1
1
University of Suez Canal, Ismailia, Egypt, 2University of Sinai, North Sinai, Egypt, 3Assuit University, Assiut, Egypt, 4University of Damietta,
New Damietta, Egypt
INTRODUCTION
Members of the genus Aspergillus are cosmopolitan and
prevalent components of different ecosystems in a wide
range of environmental and climatic zones (Klich, 2002a;
Lević et al., 2013), because they can colonize a wide variety
of substrates. Species belonging to the genus Aspergillus are
widely distributed throughout the world biomes, for exam-
ple, soil (Hill et al., 1983; Klich, 2002a; Abdel-Azeem and
Ibrahim, 2004; Conley et al., 2006; Jaime-Garcia and Cotty,
2010), salterns (Butinar et al., 2011; Balbool et al., 2013),
agroecosystems (Bayman et al., 2002; Horn, 2003; Jaime-
Garcia and Cotty, 2006; Abdel-Azeem et al., 2007; Marín
et al., 2012; Muthomi et al., 2012), polar (Arenz et al., 2014),
living plants, animals and lichens (Yu et al., 2012; Salem and
Abdel-Azeem, 2014; Tripathi and Joshi, 2015), stones (Tang
et al., 2012), water-related (Sivakumar et al., 2006; Bonugli-
Santos et al., 2015), fossil records (Thomas and Poinar, 1988;
Dörfelt and Schmidt, 2005), and human (Horré et al., 2010;
Marguet et al., 2012; Findley et al., 2013).
The occurrence of Aspergillus species is controlled by
several factors including microclimate, the availability of
substrates, as well as water activity and complex ecological
interactions (Mouchacca, 1995; Grishkan and Nevo, 2010;
Pettersson and Leong, 2011). Survival in different environ-
mental and geographical habitats can be related to meta-
bolic diversity, high reproductive capacity, and competitive
capabilities of Aspergillus strains in nature (de Vries and
Visser, 2001; Horn and Dorner, 2002; Shehu and Bello,
2011; Mehl and Cotty, 2013).
The genus Aspergillus consists of about 339 species,
including both pathogenic and beneficial species (Samson
et al., 2014). Several species are pathogenic to plants, ani-
mals, and humans (eg, Aspergillus fumigatus, Aspergillus
terreus) and/or produce different types of toxins, such
as aflatoxins and ochratoxins (eg, Aspergillus flavus,
Aspergillus ochraceous). On the other hand, several spe-
cies are widely used in different industrial applications, for
New and Future Developments in Microbial Biotechnology and Bioengineering. DOI: http://dx.doi.org/10.1016/B978-0-444-63505-1.00001-4
© 2016 Elsevier B.V. All rights reserved.
example, production of foods, drinks, organic acids, and a
large variety of enzymes (eg, Aspergillus niger, Aspergillus
aculeatus, Aspergillus oryzae). The broad relevance and
economic importance of the genus have pushed it to the
forefront of fungal research, with one of the largest aca-
demic and industrial research communities dedicated to this
genus. We searched major names of interest of Aspergillus
species in both the web of Google Scholar and Research
Gate on July 17, 2015. Results showed that A. niger came
first by 307,000 and 79,900 recorded hits followed by
A. fumigatus (199,000 and 55,500), A. oryzae (82,900 and
25,200) and A. flavus (79,000 and 43,100), respectively.
The aim of this chapter is to give an overview of the
studies aimed at the investigation of Aspergillus biodiver-
sity in a wide variety of different ecological habitats.
METHODOLOGY OF STUDYING
ASPERGILLUS BIODIVERSITY
Phenotypic Studies
Microscopic features of Aspergillus and its teleomorphs
are an important part of the species concept. However,
many debatable taxonomic schemes in several sections
of the genus have resulted due to the occurrence of much
morphological variation. Phenotypic characters of asper-
gillum-like spore-bearing structure include conidial head
shape (presence or absence of metulae, ie, uniseriate or
biseriate), color, shape, texture and dimension of stipes,
vesicles, conidia, and Hülle cells if present. Morphological
characteristics, such as colony growth rates on identifica-
tion media, texture, sporulation rate, production of sclerotia
or cleistothecia, colors of mycelia, sporulation, diffusible
pigments, exudates and reverses, and physiological charac-
teristics (temperature, water activity) have been used with
aforementioned criteria for charcterizing species. The pre-
liminary identification of species can be performed with
the aid of taxonomic keys and descriptions available in the
3
4 SECTION | I Biology and Biodiversity
literature (Thom and Church, 1926; Thom and Raper, 1945;
Raper and Fennell, 1965; Christensen and States, 1982;
Christensen, 1981,1982; Gams et al., 1985; Samson and
Gams, 1985; Pitt, 1985; Klich and Pitt, 1988; Kozakiewicz,
1989; Samson and Pitt, 2000; Klich, 2002b; McClenny,
2005; Varga and Samson, 2008; Pitt and Hocking, 2009;
Samson et al., 2010; Hubka et al., 2013). Furthermore, all
these phenotypic features have to be determined by trained
mycologists under standardized laboratory conditions
to obtain an accurate identification (Okuda et al., 2000).
However, without professional expertise this may often lead
to an incorrect description, therefore, the use of biochemi-
cal and molecular methods is recommended.
Secondary Metabolite Profiling and
Chemotaxonomy
Aspergilli have a variety of biochemical characteristics
that classify them as Eumycota. Their cell walls containing
polysaccharide (chitin and glucan); ergosterol; fatty acid
profile dominated by C16 and C18 chain lengths; and pro-
duction of trehalose and polyols (Wessels, 2005).
Guarro et al. (1999) recommended other chemical
markers or patterns of metabolites, secondary metabolite
profiles, in conjunction with morphology and physiology
approaches for further classification of Aspergillus. Raper
and Fennell (1965) did not use any physiological, chemical,
or biochemical characters, but in later physiological tests
(Klich and Pitt, 1988) and secondary metabolites (Frisvad,
1989; Frisvad et al., 1998, 2004, 2007; Samson et al., 2004)
have been introduced in the taxonomy of Aspergillus.
Secondary metabolites have been the molecules most
often used in species recognition due to their high species
specificity (Frisvad, 1989; Larsen et al., 2005). All species
produce a unique combination of different types of small
organic compounds of mixed biosynthetic origin and even
unique to a single species (Frisvad et al., 2007). Recently
various studies have shown that major genomic differences
between Aspergillus species are often related to the num-
ber and similarity of polyketide and nonribosomal peptide
synthase genes (Galagan et al., 2005; Nierman et al., 2005;
Pel et al., 2007). Hence, secondary metabolites are indeed
excellent phenotypic characters for species recognition.
Chemotaxonomy by using fatty acid profiles have been
used extensively for bacteria and the characterization of
microbial communities (Zelles, 1999; Kirk et al., 2004).
In comparison with bacteria, fewer different fatty acids are
produced by fungi (Lechevalier and Lechevalier, 1988), and
by the end of the 20th century fatty acids analyses were
increasingly used to distinguish different fungi (Welch,
1991; Stahl and Klug, 1996; Nemec et al., 1997; Silva et al.,
1998; Guarro et al., 1999). Fatty acid methyl esters (FAME)
prepared in most methods and analyzed by gas chroma-
tography (GC) or gas chromatography–mass spectrometry
(GC-MS) and multivariate programs have been developed
to apply fungal Fatty Acid data in routine taxonomy and
identification work (Stahl and Klug, 1996). Few studies
concerning the Fatty Acid methodology have been applied
as a taxonomic tool for discriminating amongst Aspergillus
(Fraga et al., 2008). Glassbrook (2008) studied the bio-
chemical markers for the detection and classification of
Aspergillus. In his study, reference strains of different
Aspergillus species, Penicillium chrysogenum, Candida
albicans, and Cryptococcus neoformans were characterized
using liquid chromatography–mass spectrometry (LC-MS)
and gas chromatography–mass spectrometry (GC-MS) bio-
chemical profiling techniques in order to find specific small
molecules, peptides, or biochemical profiles that can be
used in addition to established methods to detect and clas-
sify Aspergilli to the species level.
Thus in various scenarios detection of a unique mixture
or in some cases one or a few biomarkers can be used for
species recognition based on the chemical nature of such
small organic molecules which can be detected by differ-
ent spectroscopic tools. These spectroscopic techniques
(Infrared (IR), Ultra Violet (UV), Fluorescence Detection
(FLD), Mass Spectroscopy (MS), and Nuclear Magnetic
Resonance (NMR), UV, FLD, MS, and NMR) give com-
plementary structural information, and are often used in a
combined setup in connection with either gas or liquid chro-
matography (Nielsen et al., 2004).
In the last decade, other tools concerning chemoinformat-
ics have been developed and applied in order to deal with
large amounts of spectroscopic data that can be generated
from analysis of numerous fungal taxa (Nielsen et al., 2004;
Larsen et al., 2005). The use of electronic nose technologies,
a similar but very different approach for species recognition
combined with neural network analysis as a kind of “black
box” approach for detection of fungal growth, is associated
with certain kinds of feed or foodstuffs (Karlshøj et al., 2007).
Protein profiles, as a diagnostic tool, are not used exten-
sively in the taxonomy of genus Aspergillus (Glassbrook,
2008). By using electrophoretic techniques different protein
patterns will be observed and they directly related to the
diversity of the coding genes and may indicate specific differ-
ences or similarities between examined species (Mitterdorfer
et al., 2002). One-dimensional polyacrylamide gel electro-
phoresis (PAGE) of proteins has been used to compare dif-
ferent species of Aspergillus (Rath, 2001; Leila et al., 2010;
Khosravi et al., 2012). Several investigators (Khosravi et al.,
2012 Nealson and Garber, 1967; Nasuno, 1971, 1972a,b,
1974; Kurzeja and Gabber, 1973; Cruickshank and Pitt, 1990;
Sugiyama and Yamatoya, 1990; Yamatoya et al., 1990) have
studied enzyme profiles of a limited number of Aspergillus
isolates. Slab polyacrylamide gel electrophoresis method was
introduced by Saito et al. (1991) for the identification of the
alkaline proteinases of A. flavus and Aspergillus parasiticus,
but the result was not good enough.
 Biodiversity of the Genus Aspergillus in Different Habitats Chapter | 1
Ubiquinone (coenzyme Q) is a lipid component of the
mitochondrial electron transport chain and has been used
as a taxonomic criterion for yeast and filamentous fungi
(Yamada et al., 1989; Yaguchi et al. (1996)). The num-
ber of isoprene units attached to the benzoquinone varies,
and such differences in ubiquinone structure are excellent
indicators in the classification of genera and subgeneric
taxa in bacteria and yeasts. In addition, the isoprene units
of ubiquinone were highly correlated with morphologi-
cal and physiological characters in the infrageneric taxa
of Aspergillus (Kuriashi et al., 1990). Sugiyama et al.
(1991) and Matsuda et al. (1992) reported that three major
ubiquinone systems (Q-9, Q-10, and Q-10(H2)) occurred
in Aspergillus and the ubiquinones were useful indicators
for classification. Kuriashi et al. (1990) studied the ubiqui-
none systems in Aspergillus in relation to the taxonomy of
Raper and Fennell (1965), who subdivided Aspergillus into
uniseriate species, uniseriate or biseriate species, and biseri-
ate species. Their study showed that nearly all species hav-
ing Hülle cells possessed only the Q-10(H2) system while
xerophilic species had Q-9 or Q-10. Yamatoya et al. (1990)
determined the ubiquinone systems of 27 isolates assigned
to Aspergillus sect. Flavi. Coenzyme Q systems for 190
(teleomorphic and anamorphic) isolates, and three samples
of Dendrosphaera eberhardtii fruit bodies, which belonged
to Eurotiales, Onygenales, and related taxa have been deter-
mined by Kuraishi et al. (2000).
Several biochemical and physiological techniques have
been introduced to improve Aspergillus taxonomy, one of
which is isoenzyme patterns (Cruickshank and Pitt, 1990;
Yamatoya et al., 1990). It generally is most successful at
distinguishing species and has been used to make recom-
mendations on the separation or combination of species
(Micales et al., 1992). Differences in isozyme banding
patterns have been used to separate species of Aspergillus
(Kurzeja and Gabber, 1973).
Cruickshank and Pitt (1990) used polyacrylamide gel
electrophoresis to examine several kinds of exoenzymes (pec-
tinases, ribonucleases, amylases, and proteases) from six iso-
lates of Aspergillus. They found that four isolates (A. flavus,
A. parasiticus, Aspergillus tamarii, and Aspergillus nomius)
produced distinct patterns. On the other hand, A. oryzae
produced very similar patterns to those of A. flavus, and
patterns of Aspergillus sojae were very similar to those of
A. parasiticus. In the above-mentioned studies taxonomic
relationships could be elucidated, but until now isozyme pro-
files have not provided a practical system for identification
because isoenzyme patterns could not be used to distinguish
the domesticated species from their wild types.
Frisvad et al. (2007) discussed the particular interest of
using mycotoxins, as secondary metabolites with bioac-
tive properties, in taxonomy of Aspergillus species. As an
important chemotaxonomic marker, aflatoxins have been
used by several investigators (Frisvad et al., 1998; Klich
5
et al., 2000; Seifert and Levesque, 2004; Varga et al., 2004;
Frisvad et al., 2007) in taxonomic studies of aflatoxin-
producing taxa of Aspergillus.
Evolution of the Approach: Polyphasic
Taxonomy of Aspergillus
The polyphasic taxonomy takes into account all available
phenotypic and genotypic data and integrates them in a con-
sensus type of classification. Phylogenetic species recogni-
tion is increasingly being used with the internal transcribed
spacers of the nrDNA (ITS) now accepted as the official
DNA barcode for fungi (Schoch et al., 2012). Sequencing
of genomic regions widely applied to the identification of
a large number of Aspergillus species and the results of
these techniques are generally well correlated with mor-
phological and physiological characteristics (Rodrigues
et al., 2011). Genomic regions that are sequenced for the
identification of Aspergillus species include the ITS (inter-
nal transcribed spacer) region (White et al., 1990), β-tubulin
(BenA) gene (Glass and Donaldson, 1995), and calmodu-
lin (CaM) gene (Carbone and Kohn, 1999). The nuc rDNA
internal transcribed spacer rDNA region (ITS1-5.8S-ITS2)
is the official DNA barcode for fungi because it is the most
frequently sequenced marker in fungi and has primers that
work universally (Schoch et al., 2012). In contrast, BenA is
easy to amplify, in comparison with the RNA polymerase
II second largest subunit (RPB2), but has been reported to
vary in the number of introns and amplification of paralo-
gous genes sometimes resulting from PCR (Peterson, 2008;
Hubka and Kolarik, 2012).
Isolates of Aspergillus species usually produce a diverse
range of extrolite (secondary metabolites) that are charac-
teristic of the different groups of sections of Aspergillus.
For example, production of kojic acid characterized species
of Aspergillus section Flavi (Varga et al., 2011), while peni-
cillic acid (small acidic molecules) produced by most spe-
cies of Aspergillus section Circumdati (Frisvad et al., 2004).
Production of a specific extrolite is considered an efficient
identification tool for allocating a species of Aspergillus
to section but some extrolites, for example, ochratoxin A,
are produced by species in different sections, for example,
Flavi, Circumdati, and Nigri (Frisvad et al., 2004, 2011;
Varga et al., 2011; Samson et al., 2014). Various poly-
phasic studies have been carried on different sections of
Aspergillus by several researches (Hong et al., 2005; Varga
et al., 2007a; Houbraken et al., 2007; Silva et al., 2011;
Samson et al., 2007, 2014). Samson et al. (2014) recom-
mended an updated qualitative database on the verified
production of secondary metabolites to identify isolates of
Aspergillus up to species level.
Current knowledge pertaining to the diversity, detec-
tion, and distribution of Aspergillus taxa is still rudimen-
tary. Obviously, improvements in traditional approaches
6 SECTION | I Biology and Biodiversity
combined with other biochemical/serological methods
and incorporation of various molecular techniques (DNA-
based) have provided new data on these aspects but, for a
clearer picture and a better understanding, a combination of
all approaches (polyphasic) is essential. There is a need to
unravel the taxonomic diversity of speciose groups (Jeewon
and Hyde, 2007).
ASPERGILLUS DIVERSITY IN DIFFERENT
HABITATS
Desert
By definition a “desert” is a region that receives extremely
low rains—less than 250 mm/year—far less than the amount
required to support the growth of most plants. Approximately
one-third of the earth’s land surface is desert, with an area
more than 52,000 square kilometers (Fig. 1.1).
Deserts are extreme environments where intense solar
radiation, limited nutrients, low organic matter content,
and restricted water availability present formidable chal-
lenges for fungi inhabiting these areas. Desert soils gener-
ally are characterized by low propagule densities but high
species diversity (Christensen, 1981; Mouchacca, 1995).
Studies on mycobiota of soils may be dated back to 1886
when Adametz started his pioneer study by isolation and
naming 4 species of yeasts and 11 species of filamentous
fungi including Aspergillus (Watanabe, 2002). Species of
Aspergillus are common and they may account for up to
20% of the total species isolated in the desert (Christensen
and Tuthill, 1985).
The number of mycological studies on desert soil is
rather limited in comparison with other ecological habitats.
FIGURE 1.1 Map shows the generalized location of Earth’s ten largest deserts on the basis of surface area (http://geology.com/records/largest-desert.shtml).
Several authors assume the diversity of microbes including
fungi is low compared to soil in moderate or tropical regions
and they suggest these extreme ecosystems as suitable in
situ models to study the relationship between phylogenetic
biodiversity and function (Adams et al., 2006).
Desert mycobiota of Egypt have been the target of many
studies, namely: Montasir et al. (1956a,b), Mahmoud et al.
(1964), Besada and Yusef (1968), Moubasher and Moustafa
(1970), Moubasher and El-Dohlob (1970), Salama et al.
(1971), Mouchacca (1971, 1973a,b, 1977, 1982); Naguib
and Mouchacca (1970-1971), Mouchacca and Nicot (1973),
Mouchacca and Joly (1974, 1976), Samson and Mouchacca
(1974, 1975), Moubasher et al. (1985, 1988, 1990), Nassar
(1998), Abdel-Hafez et al. (1989a,b, 1990), Abdel-Sater
(1990, 2000), Abdel-Hafez and El-Maghraby (1993),
Abdel-Azeem and Ibrahim (2004), and Abdel-Azeem
(1991, 2009).
Moubasher and Moustafa (1970) surveyed the Egyptian
soil fungi with special reference to Aspergillus, Penicillium,
and Penicillium-related genera in 32 soil samples collected
from different localities in Egypt. They met 16 species of
Aspergillus and the highest population and occurrence were
recorded for A. niger, A. terreus, A. flavus, and Aspergillus
sydowii, respectively.
Mouchacca and Joly (1976) studied the biodiversity of
genus Aspergillus in arid soils of Egypt. They collected 31
soil samples from the western desert of Egypt. They col-
lected 14 soils (set A) from regions receiving very weak to
null winter rains and 17 (set B) samples from regions that
benefit from an appreciable amount of wintry precipitation.
In their study the taxonomic distribution is hardly affected
by the dimensions of soil sand components, while regional
localization exerts a certain influence. Twenty-seven species
 Biodiversity of the Genus Aspergillus in Different Habitats Chapter | 1
of Aspergillus were isolated, some are practically omnipres-
ent (A. niger, A. flavus group), others develop preferen-
tially in set A soil (Aspergillus nidulans, Aspergillus ustus,
A. ochraceous, and possibly A. fumigatus groups) and/or
have distribution positively affected (Aspergillus flavipes
and A. terreus) or perhaps negatively (A. fumigatus group)
due to soil reclamation.
In their extensive survey of Sinai terricolous fungi,
Abdel-Azeem and Ibrahim (2004) and Abdel-Azeem (2009)
recorded 17 species of Aspergillus. They recorded A. aluta-
ceous, Aspergillus candidus, Aspergillus clavatus, A. flavus,
A. fumigatus, Aspergillus japonicus, A. niger, A. ochraceous,
A. sydowii, Aspergillus tamerii, A. terreus, A. ustus,
Aspergillus versicolor, Aspergillus wentii, Emericella nidu-
lans, Eurotium amstelodami, and Eurotium chevalieri.
Six taxa are introduced to the genus Aspergillus as novel
taxa based on type materials collected from Egyptian deserts
namely: Aspergillus egyptiacus Moubasher and Moustafa
(1972) (as Aspergillus aegyptiacus), Aspergillus floriformis
Samson and Mouchacca (1975), Aspergillus pseudodeflec-
tus Samson and Mouchacca (1975), Emericella deserto-
rum Samson and Mouchacca (1974), Emericella purpurea
Samson and Mouchacca (1975), and Eurotium xerophilum
Samson and Mouchacca (1975).
Few investigations have been made on soil mycobiota in
Libya. Naim (1967a,b) studied rhizosphere and soil fungi
of Artemisia herba-alba and fungi under citrus trees in
Tripoli, Libya. Youssef (1974) studied the fungal flora of
Libyan soil. He examined 16 different localities in Libya for
their fungal microflora. El-Said and Saleem (2008) studied
soil fungi at the western region of Libya. Mansour (2010)
studied the distribution and occurrence of various groups
of fungi in different kinds of soils in the eastern region
of Libya. Result showed that the most abundant species
were Aspergillus flavus, A. fumigatus, A. niger, Aspergillus
ochraceus, A. terreus, and A. ustus. For more details con-
cerning the checklist of Libyan fungi check El-Buni and
Rattan (1981).
Mycobiota of Algerian, Tunisian, and Moroccan deserts
do not receive that much attention from mycologists and
hence few studies have been published concerning the myc-
obiota of these deserts. Recently mycobiota of three chotts
located in the northeast of Algerian Sahara have been stud-
ied by Dendouga et al. (2015). They isolated 327 colonies
of fungi and Aspergillus was one of the most common gen-
era isolated in this study.
Studies on micromycetes of desert soils of the Kingdom
of Saudi Arabia showed that Aspergillus amstelodami,
Aspergillus chevalieri, Aspergillus ruber, A. ochraceous,
A. fumigatus, A. flavus, A. sydowii, A. terreus, and A. ustus
are the most common species (Fathi et al., 1975; Ali, 1977;
Ali et al., 1977; Abdel-Hafez, 1982a,b,c, 1994; Hashem,
1991, 1995; Arif and Hashem (1988); Barakat, 1999;
Abdulmoniem and Saadabi (2006); Abou-Zeid and Abd
7
El-Fattah, 2007). Also, the teleomorph genera Emericella
(E. nidulans) and Eurotium with E. amstelodami and
E. chevalieri are common in Saudi Arabian desert soils.
Tolba et al. (1957), Al-Doory et al. (1959), Ismail and
Abdullah (1977), and Abdullah et al. (1986) studied soil
microfungi from different localities in Iraq. In these stud-
ies genus Aspergillus accounted for about 16% of the total
species isolated. Aspergillus fumigatus was the most com-
mon species, being isolated from 70% of the sampling sites
examined. Aspergillus candidus and A. niger were in the
second and third positions in frequency, being isolated from
60% and 50% of the sampling sites examined, respectively.
Imran and Al Rubaiy (2015) studied the molecular ecologi-
cal typing of environmental isolates of A. terreus collected
from the desert region in Iraq.
In Syria various species of Aspergillus were recorded by
various investigators, such as: Sizova et al. (1967), Baghdadi
(1968), Abdel-Hafez et al. (1983), and Abdel-Kader et al.
(1983). Aspergillus niger, A. sydowii, A. flavus, A. wentii,
and A. clavatus were the most prevalent species. Aspergillus
kassunensis as a new species added to genus Aspergillus
was introduced by Baghdadi (1968) from Syrian soil.
Al-Subai (1983) and Moubasher (1993) concluded
that Aspergillus was consistently the most common genus
in Qatari soils. Moubasher (1993) isolated fungi from 11
desert soil samples out of 42 samples representing differ-
ent ecological habitats of Qatar. Aspergillus contributed by
23 species and 5 varieties, of which A. terreus, A. flavus,
A. versicolor, and A. niger were the most frequent species.
Halwagy et al. (1982) found Aspergillus, Alternaria, and
Drechslera constituted 16%, 5%, and 3% respectively of
the total species isolated from desert soils in Kuwait. They
recorded Aspergillus terreus, A. fumigatus, and A. niger with
frequencies of occurrence of 70%. El-Said (1994) studied
soil mycoflora of Bahreen (Bahrain) in which 39 species
belonging to 20 genera were isolated from 50 soil samples
on different isolation media. Aspergillus flavus, A. fumigatus,
A. niger, A. sydowii and A. terreus, Eurotium amstelodami,
and E. chevalieri were the most common species.
Mycobiota of the northern part of the Negev desert
(Rayss and Borut, 1958; Borut, 1960; Guiraud et al., 1995;
Steiman et al., 1995) represented by 159 species belonging
to 58 genera in which 16 of them under genus Aspergillus.
Aspergillus fumigatus, Aspergillus sclerotiorum, and A. ver-
sicolor are the most common species in this region. Volz
et al. (2001) concluded that the majority of Israel soil fungi
(309 species—70%) belong to the division Ascomycota, but
only 56 species of them were found to have a perfect stage
in their life cycle. Concerning species diversity among gen-
era, they showed that Aspergillus recorded only 48 species
(15.53%) out of 309 species. Aspergillus niger, A. terreus,
A. ustus, and A. versicolor are the most widely distributed
species in Israel. Grishkan and Nevo (2010) isolated 185
species belonging to 76 genera from the soil of Makhtesh
8 SECTION | I Biology and Biodiversity
Ramon hot desert in Israel. Ten species of Aspergillus, nine
anamorphic and one teleomorphic, were isolated in which
A. fumigatus comprised a basic part of thermotolerant myc-
obiota obtained in this study.
Aspergillus as a xerotolerant and xerophilic genus can
grow at or below a water activity (aw) of 0 (Pettersson and
Leong, 2011). Several researchers have isolated genus
Aspergillus from desert soils in Argentina, Chile, and Mexico
(Giusiano et al., 2002, Piontelli et al., 2002, Samaniego-
Gaxiola and Chew-Madinaveitia, 2007). Conley et al. (2006)
studied the fungal content of Atacama desert, the driest and
oldest desert on Earth, without any record rainfall for dec-
ades. They reported 12 genera of fungi, with Aspergillus one
of them. Aspergillus flavus and A. fumigatus reported from
desert soils worldwide (Moubasher, 1993; Abdel-Hafez,
1981; Giusiano et al., 2002; Piontelli et al., 2002; El-Said and
Saleem, 2008) and Aspergillus carneus recorded exclusively
from desert soils in the Middle East (Abdullah et al., 1986;
Ali-Shtayeh and Jamous, 2000, El-Said and Saleem, 2008)
were missing in the Atacama soil.
Grishkan et al. (2015) examined the variations in micro-
fungal communities inhabiting different biological crust
types in the vicinity of the Shapotou Research Station in
the Tengger Desert, China. The mycobiota isolated from the
crusts sampled in 2011 and 2013 were composed of 123 and
67 identified species, respectively. Altogether 134 species
were isolated: 6 of Mucoromycotina, 22 of teleomorphic
(morphologically sexual) Ascomycota, and 106 of anamo-
rphic (asexual) Ascomycota. These species belonged to 66
genera, with the most common being Aspergillus (12 spe-
cies). Taxa of Aspergillus fumigatus, A. niger, A. nidulans,
and Aspergillus rugulosus dominated.
Klich (2002a) published her biogeography of Aspergillus
species in soil and litter and she concluded that there was no
overall trend in distribution of the members of the entire
genus by ecosystem, however, individual sections of the
genus appeared to have distinct distribution patterns. Most
members of sections Aspergillus, Nidulantes, Flavipedes,
and Circumdati occurred at greater than expected frequen-
cies in desert soils (Klich, 2002a). To conclude, in desert
environments, the pan-global stable Aspergillus spe-
cies are represented by A. niger, A. flavus, A. fumigatus,
A. ochraceus, A. terreus, A. sydowii, A. tamerii, A. ustus,
A. versicolor, A. wentii, Emericella nidulans, Eurotium
amstelodami, and E. chevalieri.
Salterns
When evaporation of seawater accompanied with hal-
ite (NaCl) concentrations of greater than 10% (m/w),
Thalassohaline hypersaline environments originated (Oren,
2002) and provide some of the most extreme habitats in
the world. They are common all around the globe, and
include, for example, marine ponds and salt marshes that
are subjected to evaporation, salt or soda lakes, and sea-salt
and manmade salterns (Trüper and Galinski, 1986).
Life-limiting parameters in salterns are many, for exam-
ple, variable water activities (aw), high concentrations
of NaCl, low oxygen concentrations as well as high light
intensity (Brock, 1979). Halotolerant and halophilic fungi
were first reported as active inhabitants of solar salterns by
Gunde-Cimerman et al. (2000). Later they were isolated by
several investigators (Butinar et al., 2005a, b, c; Cantrell
et al., 2006) from salterns around the world, for example,
La Trinidad in the Ebro River Delta and Santa Pola on the
Mediterranean coast of Spain, Camargue in France, and the
salterns on the Atlantic coast in Portugal, and in Namibia,
the Dominican Republic, and Puerto Rico.
After a decade of research into the fungal diversity in
salterns, together with new taxa, a number of fungal genera
with high diversities of halotolerant and halophilic species
have been described. Different species of genus Aspergillus
are among the filamentous fungi that appear with the high-
est frequencies in salterns (Butinar et al., 2011). The group
of filamentous fungi that have been isolated from differ-
ent salterns around the world is mainly represented by the
order Eurotiales by the teleomorphic genera Eurotium and
Emericella and the anamorphic Aspergillus and Penicillium
(Tresner and Hayes, 1971; Cantrell et al., 2006; Butinar
et al., 2011).
Global natural hypersaline waters are characterized by
certain taxa mainly of Aspergillus niger and Aspergillus
caesiellus, while hypersaline localities at higher envi-
ronmental temperatures are characterized by primarily or
exclusively taxa of A. ochraceus, A. flavus, Aspergillus
roseoglobulosus, and Aspergillus tubingensis. Butinar
et al. (2011) listed Aspergillus melleus, A. sclerotiorum,
and Petromyces alliaceus (holomorphic species) within
these taxonomic groups, although they have appeared only
locally. Both Aspergillus versicolor and A. sydowii have
also been identified as part of the fungal communities in the
hypersaline environments, even they are common in marine
environments and in dry foods. Aspergillus wentii, A. fla-
vipes, A. terreus, and particularly A. candidus have been
repeatedly isolated from Adriatic salterns, whereas A. peni-
cillioides, A. proliferans, and A. restrictus have been found
only sporadically at salinities below 10% NaCl. Aspergillus
fumigatus is common in arid environments (deserts) at high
temperatures, and has been found consistently in solar salt-
erns, although it is also most abundant at salinities below
10% NaCl (Moustafa, 1975; El-Dohlob and Migahed, 1985;
Moubasher et al., 1990; Abdel-Azeem, 2003; Abdullah
et al., 2010; Butinar et al., 2011; Balbool et al., 2013).
Six different species of the known teleomorphic food-
borne xerophilic genus Eurotium were repeatedly isolated
in a mycodiversity study of hypersaline waters: Eurotium
amstelodami, Eurotium herbariorum, and Eurotium repens
as indigenous taxa in hypersaline water, while Eurotium
 Biodiversity of the Genus Aspergillus in Different Habitats Chapter | 1
rubrum, E. chevalieri, and E. halotolerans are only imper-
manent inhabitants of brine at lower salinities (Butinar
et al., 2005c).
The representatives of genus Emericella, which are
recognizable by Hülle cells in the cleistothecial walls and
ornamented ascospore, have frequently been isolated from
dry substrata in hot and arid areas worldwide. These appear
to be well adapted to dry and warm climates (Samson and
Mouchacca, 1974) and low aw (Zalar et al., 2008). The
new taxa of soil representative of Emericella was isolated
also from desert saline soil as mentioned before (Samson
and Mouchacca, 1974, 1975), while two newly described
halotolerant species, Emericella filifera and Emericella
stella-maris, were reported from hypersaline water of the
Sečovlje salterns in Slovenia (Zalar et al., 2008). Emericella
striata was described as a new taxon from Lake Enriquillo
in Dominican Republic (Butinar et al., 2011).
To conclude, in hypersaline environments, the pan-
global stable taxa of genus Aspergillus are represented
by A. niger and E. amstelodami, and possibly also by
A. sydowii, A. candidus, and E. herbariorum, which are also
quite abundant, although more locally distributed (Butinar
et al., 2011).
Aspergillus Xerophily in Different Habitats
The most xerophilic of the anamorphic Aspergilli are spe-
cies in the section Restricti (Peterson, 2008), particularly
Aspergillus restrictus and A. penicillioides. The later is
regarded as an extreme xerophile (Andrews and Pitt, 1987),
as it grows restrictedly or not at all at high aw, optimally at
0.91–0.93 aw and is capable of growth down to at least 0.73
aw in experimental systems.
Aspergillus candidus is an important xerophilic species,
and has been reported from a wide range of commodities,
but rarely as a primary cause of spoilage. The most toler-
ant of the Aspergilli to low oxygen tensions is A. candidus
which can grow in 0.45% oxygen, which assists develop-
ment to high populations in stored grain. It produces a range
of secondary metabolites, but of these, only kojic acid is
regarded as a significant toxin. Aspergillus flavus and
A. parasiticus are perhaps the most widely reported food
spoilage fungi, since the discovery in the early 1960s of
their toxic carcinogenic metabolites, aflatoxins. Aspergillus
parasiticus appears to be widely distributed in foodstuffs in
the USA, Latin America, Africa, India, and Australia and
rarely in Southeast Asia (Pettersson and Leong, 2011).
Aspergillus niger, Aspergillus carbonarius, A. japoni-
cus, and A. aculeatus, as black Aspergilli, are widely dis-
tributed species. Aspergillus niger is widespread throughout
the tropical and temperate zones and was regarded as a
nontoxigenic species until it was demonstrated that certain
strains produce ochratoxin A and fumonisin B2 (Frisvad
et al., 2007). Aspergillus carbonarius is considered to be the
9
major producer of ochratoxin A. Aspergillus niger occurs in
a range of foods (eg, peanuts, cereals, oilseeds, spices, dried
fish, and meat products).
Aspergillus ochraceus and related species Aspergillus
westerdijkiae and Aspergillus steynii produce the myco-
toxin ochratoxin A. Like most Aspergilli, A. ochraceus is
tolerant of a wide range of pH, growing well between pH
3 and 10, and weakly at pH 2.2. It is common in dried and
stored products, has been reported in high numbers from
green coffee beans, and may be a source of ochratoxin con-
tamination in this commodity. It is less frequently reported
from cereals and cereal products. Sterigmatocystin is pro-
duced by Aspergillus versicolor, which considered as an
important species in the deterioration of stored grain and
a major source of volatile compounds. It has also been
implicated as one cause of the “Rio” off-flavor in coffee
(pungent, medicinal, or iodine-like taste, musty cellar-like
odor) due to formation of trichloroanisoles (Pettersson and
Leong, 2011).
Eurotium species are perhaps the epitome of xerophilic
fungi, being capable of rapid growth over wide tempera-
ture and aw ranges (minimum ∼ 0.70–0.72 aw), and hav-
ing a cosmopolitan distribution. There are four common
foodborne species of Eurotium: Eurotium amstelodami,
E. chevalieri, E. repens, and E. rubrum. All are similar
physiologically (halophilic xerophiles), and they appear
to occupy similar ecological niches, namely causing dete-
rioration of dried foods and also high-sugar products such
as confectionery, dried fruit, jams, and conserves (Butinar
et al., 2005c; Pettersson and Leong, 2011). Although most
Eurotium species are capable of growth at high water activ-
ity, they compete poorly in natural substrates at water activ-
ity values above 0.92.
Agricultural
Globally the majority of the research which has involved
the isolation and identification of Aspergillus strains from
various agricultural and horticultural crop fields in differ-
ent agro-climatic zones was undertaken in order to evaluate
them for mycotoxin production (Klich, 2002b). Therefore,
only a limited number of studies deal with biodiversity of
the genus Aspergillus in specific crop fields or agroecosys-
tems. Climatic factors, followed by edaphic and spatial pat-
terning, are the best predictors of soil fungal richness and
community composition at the global scale (Tedersoo et al.,
2014). Biotic (plant species and their growth stage, micro-
bial competition) and abiotic factors (soil physico-chemical
characters, application of pesticides and/or fertilizers) as
well as the geographical position affected populations and
diversity of fungal communities in agroecosystems (Kredics
et al., 2014).
In her biogeographic study of Aspergillus species in
soil and litter, Klich (2002a) found that five species of
10 SECTION | I Biology and Biodiversity
Aspergillus reported in over 100 studies were A. fumiga-
tus, A. versicolor, A. terreus, A. flavus, and A. niger var.
niger. With one exception, these five species occurred at the
expected frequencies in all of the biomes; A. terreus occurred
at greater than expected frequencies in cultivated soils and
less than expected frequencies in forest soils. In many parts
of Egypt several investigators studied soil fungi from cul-
tivated soil, for example, Abdel-Hafez (1974), Moubasher
and Abdel-Hafez (1978), and Abdel-Azeem (2003). They
found taxa belonging to Aspergillus, Penicillium, Fusarium,
Mucor, and some dematiaceous Hyphomycetes were the
most common in various types of Egyptian soils. Mazen
and Shaban (1983) studied the fluctuation of soil fungi in
wheat fields and found that the most common fungi isolated
were Aspergillus represented by five species Aspergillus
niger, A. terreus, A. fumigatus, A. flavus, and A. versicolor.
Abdel-Hafez and coworkers (2000) isolated 118 species in
addition to seven varieties belonging to 51 genera from cul-
tivated and desert soils in Egypt. The results obtained from
the three soil types were basically similar, and the most
common Aspergillus species were A. flavus, A. flavus var.
columnaris, A. fumigatus, A. niger, Aspergillus sydowii, and
A. terreus.
Hafez (2012) made an ecological comparison on soil
and rhizospheric fungi of maize and wheat plants in differ-
ent areas in Minia Governorate in Egypt. She isolated 28
fungal species from wheat belonging to 18 genera and that
13 species were isolated from maize belonging to 9 genera.
Aspergillus was the most dominant in both rhizospheric and
nonrhizospheric soils and was represented by four species:
A. niger, A. terreus, A. flavus, and A. ustus.
Fusaria and other fungi associated with rhizosphere and
rhizoplane of lentil and sesame at different growth stages
from cultivated soil in Egypt have been studied by Abdel-
Hafez et al. (2012). They isolated 16 Fusarium species and
three Aspergillus species (A. flavus, A. niger, and A. ochra-
ceous) were isolated.
Abdel-Azeem et al. (2007) studied the effects of long-
term heavy metal contamination on diversity of terricolous
fungi and nematodes in an agroecosystem in Egypt as a case
study. They collected 100 soil samples in a randomized way
to represent different stages of land reclamation during the
period from September (2004) to February (2005). These
profiles represented different land use periods of 0–20 years.
Isolated species belonged to 21 genera. The prevailing gen-
era were Aspergillus (12 species including anamorph stages
of one Emericella and one Eurotium species; 52.63% of
the total isolates). They found that the most abundant spe-
cies were: Aspergillus niger var. niger, (21.15% of the total
isolate number), Trichoderma pseudokoningii (12.65%),
A. flavus (9.4%), and A. fumigatus (8.63%).
Aspergillus taxa distributed in different altitudes (24 m
above sea level to 2000 m above sea level) of the eastern
Himalayas were studied by Devi and Joshi (2012). They
recorded Aspergillus versicolor in samples collected from
1–500 m above sea level, Aspergillus nomius (500–1000 m),
A. niger (1000–1500 m), A. fumigatus, A. flavus, A. terreus,
and Aspergillus awamori (1500–2000 m).
Aspergillus species are able to produce a range of myco-
toxins, including, for example, aflatoxins, ochratoxins,
fumonisins, and patulin. Aflatoxins are mainly produced by
members of Aspergillus section Flavi, and they contaminate
various agricultural products in several parts of the world
(Baranyi et al., 2013).
Taxonomically, based on Aspergillus species, myco-
toxins in fruits can be divided into three major groups:
(1) Aflatoxins produced by A. flavus, A. parasiticus, and
A. nomius; (2) Ochratoxin A produced by A. ochraceus,
A. carbonarius, A. niger aggregate, A. tubingensis, A. scle-
rotiorum, Aspergillus sulphureus, A. aculeatus, A. japoni-
cus var. aculeatus, Aspergillus alliaceus, A. melleus, and
other species; and (3) Other toxic metabolites produced by
a variety of Aspergillus spp., the most important of these
being sterigmatocystin, produced by A. flavus, A. flavipes,
A. nidulans, and A. versicolor; cyclopiazonic acid, pro-
duced by A. flavus, A. tamarii, and A. versicolor; aflatrem,
produced by A. flavus; citrinin, produced by A. flavipes, A.
carneus, A. niveus, and A. terreus; and patulin, produced
by A. terreus (Gill-Carey, 1949; Raper and Fennell, 1965;
Semeniuk et al., 1971; Ciegler, 1972; Hesseltine et al., 1972;
Buchanan et al., 1975; Durley et al., 1975; Lee et al., 1975;
Mislivec et al., 1975; Sommer et al., 1976; Moss, 1977;
Gallagher et al., 1978; Stack and Mislivec, 1978; Gorst-
Allman and Steyn, 1979; Anke et al., 1980; Cole and Cox,
1981; Davis, 1981; Wicklow and Cole, 1982; Turner and
Aldridge, 1983; Cole, 1984; Dorner et al., 1984; Scudamore
et al., 1986; Kurtzman et al., 1987; Vesonder et al., 1988;
Betina, 1989; Kim et al., 1993; Doster et al., 1996; Varga
et al., 1996; Richard et al., 1999; Giridhar and Reddy,
2001; Sage et al., 2002, 2004; Battilani and Pietri, 2002;
Bayman et al., 2002; Serra et al., 2003; Magnoli et al., 2004;
Iamanaka et al., 2005; Medina et al., 2005; Perrone et al.,
2006; Roussos et al., 2006; Barkai-Golan, 2008).
Fourteen species assigned to three sections of the genus
Aspergillus are responsible for acute aflatoxicosis epidem-
ics that occurred recently in several parts of Asia and Africa
leading to the deaths of several hundred people. Taxa dis-
tributed among three sections: Flavi (A. flavus, Aspergillus
pseudotamarii, A. parasiticus, A. nomius, Aspergillus bom-
bycis, Aspergillus parvisclerotigenus, Aspergillus miniscle-
rotigenes, Aspergillus arachidicola, Aspergillus togoensis),
section Nidulantes (Emericella astellata, Emericella vene-
zuelensis, Emericella olivicola), and section Ochraceorosei
(Aspergillus ochraceoroseus, Aspergillus rambellii) (Varga
et al., 2009; Rank et al., 2011). Potential aflatoxin-produc-
ing A. flavus isolates were also identified in other agricul-
tural products including stored wheat, onions, grapes, and
rice, and in cattle feed (Krnjaja et al., 2008). Aflatoxins
 Biodiversity of the Genus Aspergillus in Different Habitats Chapter | 1
were also detected in sunflower flour samples (Masic et al.,
2003) and in spices in Serbia (Saric and Skrinjar, 2008).
Several Aspergillus species are also able to produce
patulin, including species assigned to Aspergillus sections
Clavati (Varga et al., 2007b) and Terrei (Varga et al., 2005).
These species frequently occur in cereals and cereal prod-
ucts (Lopez-Diaz and Flannigan, 1997; Abramson et al.,
1987). The most well-known species A. clavatus can be
isolated mainly from soil and dung, but it also occurs in
stored products (mainly cereals) with high moisture con-
tent, for example, inadequately stored rice, corn, and millet
(Flannigan and Pearce, 1994). Aspergillus clavatus isolates
appeared to be particularly well adapted for growth during
malting (Flannigan and Pearce, 1994).
Polar
Around 2.3% of the world’s fungal biota exists in the Arctic
and fungi in this region have been isolated from various
substrates and habitats (Ivarson, 1965; Reeve et al., 2002;
Säwström et al., 2002; Callaghan et al., 2004; Ozerskaya
et al., 2009; Pathan et al., 2009). More than 1000 species
and over 400 genera of nonlichenized fungi have been
reported from Antarctic regions (including the sub-Antarc-
tic) (Bridge and Spooner, 2012; Arenz et al., 2014) including
genus Aspergillus. The genus Aspergillus is also mesophilic
to thermotolerant, yet some spores of Aspergillus and its
associated teleomorphs are found in Arctic regions (Gunde-
Cimerman et al., 2005). The presence of “cosmopoli-
tan” species such as Alternaria, Penicillium, Aspergillus,
Cladosporium, and others may be referred to their wide
dispersal potential and ubiquitous association with human
activities and material (Ruisi et al., 2007).
However, fungal diversity in Arctic soils has been inves-
tigated only to a limited extent. Krishnan et al. (2011) iso-
lated 28 isolates of fungi from bird-forming soil, pristine, and
human-impacted soils collected from the Fildes Peninsula,
King George Island, Antarctica, without any Aspergillus spe-
cies. Singh et al. (2012a,b) studied filamentous soil fungi
from Ny-Ålesund, Spitsbergen, and they isolated 19 species
under 14 genera. Aspergillus were represented by three spe-
cies, namely: A. aculeatus, A. flavus, and A. niger. Similarly,
other genera seem to be absent in cold ecosystems, for exam-
ple, Byssochlamys and its anamorphic state Paecilomyces.
Aspergillus species in general grow poorly below 12°C, and
thus may have been recovered as spores in cold ecosystems
(Gunde-Cimerman et al., 2003) because they are common as
marine spores, transported by wind or birds, or are carried
around due to human activity (Frisvad, 2008).
Water-Related
Shearer et al. (2007) estimated fungal biodiversity in fresh-
water, brackish, and marine habitats based on reports in the
11
literature. In their study they covered the ecological groups
including fungi and taxa formerly treated as fungi, exclu-
sive of yeasts, in freshwater, brackish, and marine habitats.
They have reported approximately 3047 taxa from aquatic
habitats thus far. The largest taxonomic group of fungi in
aquatic habitats is comprised of teleomorphic and anamo-
rphic Ascomycota, followed by the Chytridiomycota.
Marine fungi are an ecological rather than a taxonomic
group and comprise an estimated 1500 species, exclud-
ing those that form lichens (Hyde et al., 1998). Obligate
marine fungi grow and sporulate exclusively in the marine
or estuarine environment; facultative marine species may
grow in marine as well as in freshwater (or terrestrial) habi-
tats (Kohlmeyer and Kohlmeyer, 1979). A case in point is
Aspergillus sydowii, isolated from diseased sea fans and
causing the disease in laboratory experiments (Geiser et al.,
1998).
Boutaiba (1997) studied the fungal flora of Lake El
Golea in Algeria. He studied their taxonomy, ecology, and
metabolite production. He isolated A. niger, A. terreus,
A. sydowii, A. repens, A. ochraceous, A. fumigatus,
A. flavus, A. candidus, and A. wentii.
Singh et al. (2012a,b) investigated fungal diversity in
two sediment cores w40 cmbsf (cm below seafloor) at a
depth of w5000 m in the Central Indian Basin (CIB), by cul-
ture-dependent as well as culture-independent approaches.
This resulted in recovering a total of 19 culturable fungi
and 46 operational taxonomic units (OTUs), respec-
tively. The majority of the fungi belonged to Ascomycota,
within no single species dominating. It included members
of filamentous fungi such as Aspergillus sp., Eurotium
sp., Cladosporium sp., Pleospora sp., Chaetomium sp.,
Ascotricha sp., Penicillium sp., and Sagenomella sp.
Zhang et al. (2014) investigated the composition and
abundance of fungal community in the deep-sea sediments
of the Pacific Ocean. They identified 12 Ascomycetes
belonged to 6 genera (Aspergillus, Aureobasidium, Candida,
Exophiala, Fusarium, and Periconia). Aspergillus was rep-
resented only by two species A. sydowii and A. vitricola.
Abdel-Azeem et al. (2015) studied the occurrence and
diversity of mycobiota in heavy metal-contaminated sedi-
ments of a Mediterranean coastal lagoon, El-Manzala, Egypt.
They found that the prevailing genera were Aspergillus (11
species including anamorph stages of 2 Emericella species;
36.66% of the total isolates), Penicillium (4 species includ-
ing anamorph of Talaromyces; 13.33%), and the remain-
ing taxa were represented only by two to one species each.
Aspergillus niger, A. flavus, and A. terreus showed the high-
est percentage of frequency of occurrence.
Mangrove
Mangroves are an assortment of tropical and subtropical
trees and shrubs which have adapted to the inhospitable
12 SECTION | I Biology and Biodiversity
zone between sea and land: the typical mangrove habitat
is a muddy river estuary (Kathiresan and Bingham, 2001;
Hogarth, 2007). Mangles are considered a dynamic ecotone
and approximately 25% of the world’s coastline is domi-
nated by mangroves distributed in 112 countries encom-
passing an area of 18,000,000 ha (Spalding et al., 1997).
The biodiversity of biota associated with mangle ecosystem
is well known for animals and plants, but poorly known for
fungi (Khalil et al., 2013).
Species diversity of fungi, seasonal variation and fre-
quency of occurrence in Muthupettai mangroves, on the
east coast of Tamil Nadu, India, was studied at two different
seasons by Sivakumar et al. (2006). A total number of 118
fungal species were isolated, of which maximum 94 species
from sediment samples followed by water with 83 species
in which genus Aspergillus came first as the common genus
followed by Penicillium, Curvualria, and Alternaria.
Tariq et al. (2008) studied the rhizosphere fungi of four
different species of mangrove plants collected from coastal
areas in Pakistan. They found that A. flavus, A. fumigatus,
and A. niger were common in the rhizosphere soil of the
four species of mangrove plants sampled.
Behera et al. (2012) studied the diversity of soil fungi
from mangroves of Mahanadi delta, Orissa, India. Twenty-
two fungal species and A. oryzae, A. niger, A. flavus, and
Aspergillus albus were recorded as occasionally frequent.
Madavasamy and Pannerselvam (2012) studied the
phylloplane fungi of green, senescent, and brown leaves
of Avicinnia marina. Recovered taxa included Aspergillus
candidus, A. flavus, A. luchueusis, A. niger, A. sydowii,
A. fumigatus, and A. sulphureus out of a total of 22 species.
The mycobiota composition of the mangrove soil located
in costal area at the Red Sea in Egypt was investigated in
24 soil samples that were collected (Khalil et al., 2013).
Aspergillus flavus, A. niger, A. versicolor, and A. fumigatus,
were recorded at high species frequency in more than 15
cases out of 24.
Living Plants, Lichens, and Animals
Endophytes colonize symptomlessly the living, internal tis-
sues of their host, even though the endophyte may, after an
incubation or latency period, cause disease (Petrini, 1991).
In literature the term “fungal endophytes” is normally used
to describe fungal organisms, which in contrast to mycorrhi-
zal fungi, reside entirely within the host tissues and emerge
during host senescence (Rodriguez et al., 2001; Rodriguez
and Redman, 2008).
Endophytic fungi have been classified into two groups
based on differences in taxonomy, evolution, plant hosts, and
ecological functions into clavicipitaceous, able to infect only
some species of grasses, and nonclavicipitaceous, which are
found in the asymptomatic tissues of bryophytes, ferns, gym-
nosperms, and angiosperms (Rodriguez et al., 2009).
There are 1.3 million species of endophytic fungi alone,
the majority of which are likely found in tropical ecosys-
tems (Verma et al., 2014). There has been great interest in
endophytic fungi as potential producers of novel biologi-
cally active products (Schulz et al., 2002; Wildman, 2003;
Strobel and Daisy, 2003; Tomita, 2003; Urairuj et al., 2003;
Spiering et al., 2006; Manoharachary et al., 2013).
Unique species of endophytic fungi with a wide range of
potential practical applications in plant protection as repel-
lents, insecticides, antimicrobials, anthelmintics, and ver-
micides have been found (Strobel et al., 2008; Vega et al.,
2008). In the last 5 years, there has been evidence of the
use of endophytes for producing anticancer, antimicrobial,
and antioxidant compounds, and also in a biotransformation
process (Pimentel et al., 2011; Salem and Abdel-Azeem,
2014).
Species of Aspergillus as a member of nonclavicipita-
ceous endophytes attracted the attention of researchers as
effective producers of bioactive metabolites. Such studies
may result in the description of new Aspergillus species,
for example, Zhao et al. (2009) described Aspergillus niger
var. taxi as a new species variant of taxol-producing fungus
isolated from Taxus cuspidata in China.
Endophytic fungi Aspergillus clavatus isolated from the
Azadirachta indica plant have also been reported to synthe-
size silver nanoparticles, which have significant antibacte-
rial and antifungal activity (Verma et al., 2010). Endophytic
Aspergillus fumigatus, isolated from Juniperus communis
as a novel source of the anticancer prodrug deoxypodophyl-
lotoxin, has been isolated and chemically characterized by
Kusari et al. (2009).
Mustafa et al. (2013) exploited some Egyptian endo-
phytic taxa for extracellular biosynthesis of silver nanopar-
ticles. They isolated endophytic fungi from medicinal plants
in arid Sinai. Their results showed that Zygomycota repre-
sented by two species (9.5% of the total species number),
teleomorphic Ascomycota (3 species, 14.2%), anamorphic
Ascomycota (16 species, 76.19%). The prevailing genera
were Aspergillus (3 species including anamorph stages of
one Eurotium species; 14.28% of the total isolates), and
Alternaria (2 species, 9.5%). The remaining taxa were rep-
resented only by one species each. The most abundant spe-
cies were: Alternaria alternata (41.6%), Nigrospora oryzae
(38.3%), and Chaetomium globosum (11.1%). A total 13
species belonging to 11 genera were screened for the pro-
duction of AgNPs. They recorded that Aspergillus niger
synthesized AgNPs in a moderate rate in comparison with
other taxa.
Zhang et al. (2012a,b) isolated indolyl diketopipera-
zines (1–6) from the endophytic fungus Aspergillus tama-
rii of Ficus carica and examined its antiphytopathogenic
potentiality in vitro for the first time. Thirty-nine fungal
metabolites, including two new alkaloids, of endophytic
fungus Aspergillus fumigatus isolated from the stem bark of
 Biodiversity of the Genus Aspergillus in Different Habitats Chapter | 1
Melia azedarach and their antifungal, antifeedant, and toxic
activities were tested by Li et al. (2012).
Palenicia (2012) studied endophytic associations of
species in the Aspergillus section Nigri with Zea mays and
Arachis hypogea and their mycotoxins. He developed a sys-
tem to identify black Aspergilli from peanut and maize in
the southeastern United States. His survey indicated that
A. niger species complex is predominant in maize and pea-
nut fields.
Raghunath et al. (2012) screened Aspergillus niger iso-
lated from Taxus baccata for the production of lovastatin
on a solid state fermentation. The presence of lovastatin
was confirmed by different techniques, for example, spec-
troscopic method, nuclear magnetic resonance (NMR), thin
layer chromatography (TLC), and high-performance liquid
chromatography (HPLC) methods.
Silva et al. (2011) studied endophytic fungi from
Laguncularia racemosa (Brazilian mangrove) and their
antimicrobial potential. They recovered six isolates of
A. niger out of 70 endophytic strains.
Guatam (2014) isolated endophytic fungi from leaf seg-
ments of five medicinal plants collected from Mandi dis-
trict, Himachal Pradesh, India. Aspergillus niger, A. flavus,
A. clavatus, and A. variecolor were isolated with 14 species
belonging to 15 genera out of a total of 373 fungal strains.
Eight medicinal plants (Achillea fragrantissima,
Artemisia herba-alba, Chiliadenus montanus, Origanum
syriacum, Phlomis aurea, Tanacetum sinaicum, Teucrium
polium, and Thymus decussates) were screened for their
content of endophytic fungi on different altitudes by Salem
and Abdel-Azeem (2014) in Saint Katherine Protectorate,
South Sinai, Egypt. Salem and Abdel-Azeem isolated 32
genera belonging to 75 species in which nine species of
Aspergillus, namely, A. alliaceus, A. bisporus, A. candidus,
A. flavus, A. fumigatus, A. japonicus, A. niger, A. terreus,
and A. versicolor were recovered.
Yu et al. (2012) studied the diversity of endozoic fungi
in South China Sea sponges and their potential in synthesiz-
ing bioactive natural products suggested by PKS gene and
cytotoxic activity analysis. They isolated 14 genera and
Aspergillus was the predominant component in the culturable
fungal community and was represented by Aspergillus insu-
licola, A. penicillioides, A. terreus, A. oryzae, and E. rubrum.
Genus Aspergillus was associated with more than 30
species of sponge all over the world (Abrell et al., 1996;
Varoglu and Crews, 2000; Lin et al., 2003; Gao et al., 2008;
Proksch et al., 2008; Ein-Gil et al., 2009; Li and Wang,
2009; Lee et al., 2010; Liu et al., 2010; Menezesa et al.,
2010; Paz et al., 2010; Ding et al., 2011; Wiese et al., 2011;
Zhou et al., 2011; Thirunavukkarasu et al., 2012; Yu et al.,
2012). The most common species of Aspergillus recorded
in those studies were A. aculeatus, A. insuetus, A. niger,
A. ostianu, A. sclerotiorum, A. ustus, A. versicolor, and
Eurotium cristatum (Suryanarayanan, 2012).
13
Bai et al. (2014) characterized two new aromatic butyro-
lactones, flavipesins A (1) and B (2), two new natural prod-
ucts (3 and 4), and a known phenyl dioxolanone (5) from
marine-derived endophytic fungus Aspergillus flavipes.
Different species from the genus Aspergillus are cited as
marine-derived producers of enzymes (Bonugli-Santos
et al., 2015). Aspergillus terreus was most frequently iso-
lated as an endosymbiont from green, brown, and red
seaweeds namely: Caulerpa scalpelliformis, Helimeda
macroloba, Ulva lactuca, Ulva fasciata, brown Lobophora
variegata, Padina gymnospora, Stoechospermum margi-
natum, Sargassum ilicifolium, Portieria hornemanni, and
Gracilaria edulis, respectively (Suryanarayanan et al.,
2010). Marine-derived fungi such as Aspergillus spp.,
apart from dominating the endosymbiont assemblage of
seaweeds (Suryanarayanan et al., 2010) also dominate the
fungal consortium of marine invertebrates collected from
different localities such as the great Barrier Reef, North
Sea, the Mediterranean, and the Caribbean (Höller et al.,
2000) attesting to their adaptation to occupy such a niche
as the inner tissues of seaweeds or marine animals. Such
a widespread occurrence of marine-derived fungi may be
indicative of their passive migration from terrestrial habitats
(Alva et al., 2002).
However, since these fungi are better adapted to marine
environments than their terrestrial conspecifics (Zuccaro
et al., 2004; König et al., 2006) and survive in seaweeds
which produce antifungal metabolites (Kubanek et al.,
2003; Lam et al., 2008), it is likely that they are not cas-
ual residents of the seas but have coevolved with the
seaweeds (Zuccaro et al., 2004; Suryanarayanan et al.,
2010). Common endosymbiont of algae and seaweeds are
Aspergillus versicolor, A. terreus, A. niger, A. flavus, and
A. oryzae (Suryanarayanan, 2012).
Kelecom (2002) predicted a relationship between the
type of secondary metabolite and the source of fungus,
rather than the fungi themselves. The latter was exempli-
fied by the fungi in the genus Aspergillus that produce
fumiquinazoline derivatives if they are obtained from fish,
sesquiterpene nitrobenzoate derivatives if they originate
from algae, and indole diketopiperazine derivatives if they
are isolated from sponges.
To conclude, in association with seaweeds, the marine-
derived Aspergillus species are represented by Aspergillus
versicolor, A. terreus, A. niger, A. flavus, and A. ory-
zae (Belofsky et al., 1998; Lee et al., 2003; Zhang et al.,
2007a,b,c; Lin et al., 2008; Qiao et al., 2010).
Endolichenic fungi represent an important ecological
group of species that form associations with lichens, and to
extend the knowledge of their diversity within macrolichens,
Tripathi and Joshi (2015) isolated and identified the endoli-
chenic fungi from some healthy macrolichens of Kumaun
Himalaya. The majority of endolichenic fungi belonged to
anamorphic Ascomycota (Hyphomycetes), and the lowest
14 SECTION | I Biology and Biodiversity
were obtained from Zygomycetes. Aspergillus flavus and
A. niger were common as endolichenic species and were
recorded during various studies (Suryanarayanan et al.,
2005; Li et al., 2007; Tripathi et al., 2014a,b,c; Tripathi and
Joshi, 2015).
Air and Settled Dust
Over 225 species of fungi have been reported from indoor
environments, which represent a few of the proposed esti-
mate of 1.5 million species of fungi (McGinnis, 2007). The
most common allergenic fungal genera are Cladosporium,
Alternaria, Aspergillus, and Fusarium, where more than 80
genera of fungi have been linked with symptoms of respira-
tory tract allergies (Horner et al., 1995). Exposure to the
large concentration of conidia of the four genera is consid-
ered the main causative agent of aspergillosis (Anderson
et al., 1996), asthma and pneumonitis (Cuijpers et al., 1995;
Hu et al., 1997), allergic alveolitis, and toxicosis (Flannigan
et al., 1991).
Fröhlich-Nowoisky et al. (2012) studied the bioge-
ography and fungal diversity in the air. They found asco-
mycota species were represented by 67–85% of the total
isolated taxa and taxonomically distributed in four taxo-
nomic classes namely: Sordariomycetes, Dothideomycetes,
Eurotiomycetes, and Leotiomycetes, respectively. They
represent plant and animal pathogens, symbionts, sapro-
phytes, endophytes and epiphytes, and allergenic taxa (eg,
Cladosporium spp., Aspergillus spp.).
In the United States, Shelton et al. (2002) evaluated the
presence of indoor airborne fungi in 1717 buildings from
1996 to 1998, including hospitals, homes, schools, and
industries. They determined Aspergillus versicolor as the
predominant taxon, followed by A. flavus, A. fumigatus,
and A. niger. Studies of Samson et al. (2010) and Flannigan
et al. (2011) listed 100 fungal species common in indoor
environments. In these lists, A. fumigatus and A. sydowii
were common in the collected house dust.
As part of a worldwide survey of the indoor mycobiota,
dust was collected from nine countries (Australia, Indonesia,
Mexico, Micronesia, New Zealand, South Africa, Thailand,
United Kingdom, and Uruguay). Mycological analyses of
samples included the culture-dependent dilution-to-extinc-
tion method and culture-independent 454-pyrosequencing.
They found 2717 isolates out of the 7904 isolates were
identified as belonging to Aspergillus, Penicillium, and
Talaromyces, respectively (Visagie et al., 2014). Studies
showed that A. versicolor was considered to be very com-
mon in indoor environments and recently it was shown to
represent a species complex, with nine new species intro-
duced (Jurjević et al., 2012).
The diversity of air mycobiota showed the highest diver-
sity in countries that are also listed as biodiversity hotspots
of the world (Myers et al., 2000). This might refer to the
origin of at least a considerable proportion of these species
isolated from house dust as being from outdoors. However,
the prevalence of specific species commonly isolated from
indoor surveys suggests that the indoor environments do
select for the growth of specific species. In addition, much
of the metagenomics diversity may come from transient,
dormant, or dead spores (Visagie et al., 2014).
Júnior et al. (2012) studied the biodiversity of
Aspergillus spp. and Penicillium spp. residing in libraries
in Brazil. The genus Aspergillus was highlighted as one of
the principal airborne fungi present in indoor environments.
Aspergillus spp. were identified in 1277 (89.6%) samples
and Penicillium spp. in 148 (10.4%). The dry period exhib-
ited a greater number of isolates of the two taxa. Frequency
of species of 34 taxa of genus Aspergillus (anamorph and
teleomorph) isolated from library units in the dry (2009)
and wet season (2010) in the city of Cuiabá, MT, Brazil
were studied. The taxa belonged to 13 sections. Aspergillus
niger var. niger came first with a recorded 30.2% frequency
of occurrence, followed by A. flavus (19.7%).
In Egypt, Abdel-Azeem and Rashad (2013) studied
mycobiota of outdoor air that can cause asthma: a case
study from Lake Manzala, Egypt. They isolated a total
of 71,780 mold and 560 yeast colony-forming units from
600 exposures and the isolated taxa were assigned to 28
genera and 43 species. They found that the greater pres-
ence of fungal spores occurred in the summer. Aspergillus
niger, Cladosporium cladosporioides, Epicoccum nigrum,
Aureobasidium pullulans, Alternaria cheiranthi, P. chrys-
ogenum, Aspergillus fumigatus, and Alternaria alternata
were the predominant species. They found that Aspergillus,
Cladosporium, Penicillium, and Alternaria that had the
greatest frequencies in air of Lake Manzala are strongly
associated with allergic respiratory disease, especially
asthma, in Port Said and Ismailia governorates.
Decaying Wood and Mummies
Wood deterioration by fungi may occur from several sources.
These include the following: surface molds that cause local-
ized discoloration; stain fungi that penetrate deep into the
sapwood causing blue, gray, green, red, or other dark color-
ation; and wood-destroying fungi that decompose cell-wall
polymers (Blanchette, 1998). Many ascomycetous fungi,
such as Aspergillus nidulans, A. fumigatus, and A. oryzae,
Magnaporthe grisea, Neurospora crassa, and Fusarium
gramineum have a higher number of cellulases, with 34–44
hemicellulase encoding genes, and even 1–5 of the most
efficient cellobiohydrolases (Hatakka and Hammel, 2010).
Research on microbial and enzymatic degradation of wood
and wood components has provided a great deal of infor-
mation that has been useful in helping to protect and con-
serve historic and archeological wood. Ascomycetes fungi
(anamorphic and teleomorphic) usually cause soft-rot
 Biodiversity of the Genus Aspergillus in Different Habitats Chapter | 1
decay of wood with soft brown appearance and cracked and
checked when dry (Nilsson et al., 1989; Blanchette, 1995).
Two forms of soft rots were described by Blanchette (1995),
type I consisting of biconical or cylindrical cavities that are
formed within secondary walls, while type II refers to an
erosion form of degradation. The knowledge of lignocel-
lulose degradation by Ascomycetes is rather limited in com-
parison with other basidiomycetous fungi, and very little is
known about how they degrade lignin (Nilsson et al., 1989).
Zidan et al. (2006) studied the conservation of a wooden
Graeco-Roman coffin box and they isolated Paecilomyces
variotii, Penicillium aurantiogriseum, Aspergillus niger,
Aspergillus flavus, Aspergillus terreus, Emericella nidu-
lans, and Mucor racemosus. These fungi were found in
various parts of the coffin box, and their growth rate varied
from one part to the other.
In Latvia, during the period from 1996 to 2007, a total
of 300 private and public buildings, as well as more than 20
cultural monuments had been inspected regarding the dam-
age by wood decay basidiomycetes and discoloring micro-
fungi (Irbe et al., 2009). Wood decay fungi in constructions
occurred in 338 cases. Brown-rot damage occurred more
frequently (78.1%) than the white-rot (21.9%). Wood dis-
coloring fungi (molds and blue stain) on construction and
decorative materials were recorded in 55 cases where fre-
quent genera were Penicillium, Cladosporium, Aspergillus,
and Trichoderma.
Aspergillus candidus, A. ustus, and A. terreus were iso-
lated from two wooden masks dating back to the Greek-
Roman period in Egypt (Darwish et al., 2013). Abu Deraz
(2014) studied the soft rot fungi deteriorating archeologi-
cal wood in Al-Aqsa mosque, Jerusalem, Israel. He iso-
lated Aspergillus flavus, A. fumigatus, A. glaucus, A. niger,
A. ochraceopetaliformis, and Emericella nidulans. Both
A. flavus and A. niger showed high frequency of occurrence
in all examined samples.
Mummies have been widely investigated by phenotypic
and molecular techniques, particularly the study of ancient
bacteria and micromycetes. There are several well-known
examples showing the colonization of preserved bodies
by opportunistic fungi, such as the case of the restoration
of the body of Ramses II, performed in Paris in 1976–77.
The mummy showed a dense fungal population with spe-
cies belonging to the genera Aspergillus and Penicillium
(Mouchaca, 1985). In his study, Mouchacca isolated 21
species and one variety of Aspergillus from debris (D)
and abdominal materials (A) of Ramses II mummy. The
most common species of D and A were A. niger, A. flavus,
A. versicolor, A. sydowii, A. amstelodami, and A. restrictus.
Aspergilli also dominated the microbial communities of the
air and dust of the Egyptian mummy chamber at the Baroda
Museum in India (Arya et al., 2001).
Additionally, saprophytic fungi belonging to the genera
Monilia, Penicillium, Alternaria, Aspergillus, Rhizopus,
15
and Chrysosporium as well as saprophytic bacteria of the
genus Bacillus were isolated from a mummy from the col-
lection of the Archaeological Museum in Zagreb, Croatia
(Čavka et al., 2010). Fungal genera more related to the
mummy materials were: Botryotinia, Giberella, Didymella,
Fusarium, Verticillium, Tritirachium, Coprinus, and
Coniosporium (Piñar et al., 2013).
Microscopic fungi were isolated from different mate-
rials including muscles, bones, skin, and funeral clothes
from the mummified human remains of three members of
the Kuffner’s family and from the surrounding air environ-
ments in Slovakia by Šimonovičová et al. (2015). Their
hydrolytic abilities such as cellulolytic, lipolytic, and prote-
olytic keratinolytic were also assessed. The most commonly
isolated fungi, from human remains, belonged mainly to
the species of Aspergillus (A. candidus, A. calidoustus,
A. fumigatus, A. niger, A. sydowii, A. terreus, A. ustus,
A. venenatus, A. versicolor, and A. westerdijkiae).
Stones
The tiny pores and cracks in rocks which buffer microbial
communities from a number of physical stresses, such as
desiccation, rapid temperature variations, and UV radia-
tion is defined as endolithic environment. The diversity of
microorganisms in these ecosystems gained considerable
attention, but few culture-independent studies have been
carried out on the diversity of fungi to date.
Raghukumar et al. (1992) studied the endolithic fungi
from deep sea calcareous substrata from calcareous ani-
mal shells at 100–860 m depth in the Bay of Bengal. They
found that conidia of an isolate of A. niger obtained from
intertidal calcareous shells did not germinate above 1 atm.
Up to 512 Mu calcium was leached out upon growth of
A. restrictus on 1 g of calcareous shell substrata at 100 atm.
in 25 days.
The diversity of endolithic fungal communities in dolo-
mite and limestone rocks from Nanjiang Canyon in Guizhou
karst area was China studied by Tang et al. (2012). The most
common genus in the investigated carbonate rocks was
Verrucaria. Aspergillus and Penicillium were also identified
from the rock samples.
The diversity of culturable fungi associated with six
species of healthy South China Sea gorgonians was investi-
gated using a culture-dependent method followed by analy-
sis of fungal ITS sequences (Zhang et al., 2012a,b). A total
of 121 fungal isolates belonged to 41 fungal species from 20
genera. Of these, 30 species and 12 genera are new records
for gorgonians, and the genera Aspergillus and Penicillium
were the most diverse and common. Fourteen Aspergillus
were isolated, they were: Aspergillus carneus, A. flavus,
A. fumigatus, A. gracilis, A. insulicola, A. niger, A. nomius,
A. ochraceopetaliformis, A. penicillioides, A. sclertoiorum,
A. sydowii, A. terreus, A. tubingensis, and A. versicolor.
16 SECTION | I Biology and Biodiversity
Abu Deraz (2014) recovered seven species of endo-
lithic fungi from archeological stones of Al-Aqsa Mosque,
Jerusalem, Israel. Surface sterilized stones were incubated
on modified Czapek’s medium supplemented with calcium
carbonate, as sole carbon source, as described by Kurakov
et al. (1999). Five species of genus Aspergillus were com-
mon they were: A. flavus, A. fumigatus, A. niger, A. terreus,
and Emericella nidulans.
Human
The fungal biota in an environment (mycobiome) is an
important component of the human microbiome (Cui et al.,
2013). Every human has fungi as part of their microbiota,
however, the impact of fungi on human health is significant,
especially as a reservoir for pathogenic fungi when the host
is compromised and as a potential cofactor in inflammatory
diseases and metabolic disorders (Huffnagle and Noverr,
2013).
Findley et al. (2013) studied the skin mycobiota of
10 healthy Americans, six men and four women. Genera
included the potentially medically significant Candida,
Chrysosporium, Cryptococcus, and otherwise unnamed der-
matophytes assigned to the Arthrodermataceae. Common
saprobic genera such as Aspergillus, Cladosporium,
Epicoccum, Leptosphaerulina, Penicillium, Phoma, and
Rhodotorula were also frequently detected or isolated.
A survey of oral fungal genera has been carried out
by Ghannoum et al. (2010). They found that Candida and
Cladosporium were most common, present in 75% and
65% of participants, respectively. The fungi of the oral cav-
ity were previously believed to be few and relatively nondi-
verse based on culture-dependent or genus/species-focused
culture-independent methods of identification. In contrast
with fungal genera associated with local oral and invasive
diseases, they were Aspergillus, Cryptococcus, Fusarium,
and Alternaria, indicating that these genera are present in
the oral microbiome even during healthy state (Ghannoum
et al., 2010). Different studies (Schuster, 1999, Salonen
et al., 2000; Williams and Lewis (2006); Jabra-Rizk et al.,
2001; Seed, 2015) reported different genera of yeasts and
filamentous fungi, for example, Candida, Saccharomyces,
Penicillium, Aspergillus, Geotrichum, and Scopulariopsis
and the abundant presence of Candida, Aspergillus, and
Fusarium was recorded among the HIV-infected.
A large number of newly emerging pathogens have been
described, besides the most prevalent and well-known fun-
gal pathogens such as Candida albicans and Aspergillus
fumigatus (Horré et al., 2010; Marguet et al., 2012). The
lung mycobiome of healthy people is comprised of vari-
ous geni and species principally controlled by environment
agents including Aspergillus species (van Woerden et al.,
2013; Underhill and Iliev, 2014).
Aspergilloses are commonly caused by the fumiga-
tus, flavus, and niger groups of genus Aspergillus. Other
groups rarely act as agents of pulmonary disease, but it is
assumed that any species can cause hypersensitivity reactions
(Londero and Guadalupe-Cortés, 1990). Aspergillus species
responsible for pulmonary aspergillosis were A. amstelodami,
A. candidus, A. carneus, A. fischeri, A. flavus, A. fumigatus,
A. glaucus, A. niger, A niveus, A. phialiseptus, A. restric-
tus, A. sydowii, A. terreus, and A. versicolor (Londero and
Guadalupe-Cortés, 1990; Júnior et al., 2012). Finally, com-
mon taxa of Aspergillus and human biome are represented by
A. fumigatus, A. flavus, A. niger, and A. versicolor.
Fossils
Today there are reports of representatives of many different
groups of fungi in amber because the translucent nature of
the matrix makes it relatively easy to determine even very
delicate features useful in systematics, as well as those use-
ful in determining interactions with other organisms (Taylor
et al., 2015). Some examples including genus Aspergillus
have been recorded. Thomas and Poinar (1988) described
Aspergillus from a piece of Eocene amber originating from
the Dominican Republic as Aspergillus janus. Aspergillus
collembolorum, a novel species was introduced in 2005 by
Dörfelt and Schmidt when studying a piece of Baltic amber
(Tertiary, Eocene) which contains an inclusion of a spring-
tail (Collembola).
CONCLUSIONS
The studies discussed above reflect that the genus
Aspergillus can be characterized with high adaptability to
various ecological environments (Fig. 1.2). However, it is
important to mention that the results of any study aimed at
the examination of Aspergillus biodiversity should always
be evaluated in the context of the developmental stage of
Aspergillus taxonomy and the species identification meth-
ods available at the time of the publication of the respective
paper. Due to the constant development of the taxonomy of
the genus and the description of new species, more recent
examinations of a specific habitat may reveal higher bio-
diversity of the genus and refine the results of previous
studies. By introducing new techniques and methods in bio-
diversity studies during the past two decades, the amount of
information available about the distribution of Aspergillus
taxa is constantly growing, therefore it can be expected that
the biogeography of the genus will be understood more
deeply in the near future.
 Biodiversity of the Genus Aspergillus in Different Habitats Chapter | 1 17

Endozoic
A. flavipes
A. flavus
A. niger
A. oryzae
Polar A. penicillioides
A. aculeatus A. sclerotiorum
A. flavus A. terreus
A. niger A. ustus
Living plants,
A. versicolor
lichens and animals
Eurotium cristatum
E. rubrum A. alliaceus
A. bisporus
A. candidus
A. Clavatus
A. flavus,
A. fumigatus
A. japonicus
A. niger
A. tamarii
Desert
A. terreus
A. candidus
A. variecolor
A. flavipes
A. flavus A. versicolor
A. fumigatus
A. niger
A. ochraceous Agricultural
A. sydowii
A. terreus A. carbonarius
A. ustus A. flavipes
A. versicolor A. flavus
Emericella nidulans A. fumigatus
Eurotium amstelodami A. japonicus
E. chevalieri A. nidulans
A. niger
A. ochraceous
A. parasiticus
A. sydowii
A. tamarii
A. terreus
Water-related A. versicolor
environment
A. candidus
A. flavus Saltern
A. fumigatus A. candidus
A. niger A. flavus,
A. ochraceous A. fumigatus
A. repens A. niger
A. sydowii A. penicillioides
A. terreus A. restrictus
A. vitricola A. sydowii
A. wentii A. terreus
A. versicolor
A. wentii
Eurotiumamstelodami
E. chevalieri
E. halotolerans
E. herbariorum
E. repens
E.rRubrum
Petromyces alliaceus

FIGURE 1.2 Distribution of Aspergillus species among the different biomes of the world by Abdel-Azeem & Salem.
18 SECTION | I Biology and Biodiversity
REFERENCES
Abdel-Azeem, A.M. (1991). Effect of overgrazing on vegetation, microbes
and soil in Ismailia-desert habitat. Biological Diversity Symposium,
Madrid-Spain, pp. 241–246.
Abdel-Azeem, A.M. 2003. Ecological and taxonomical studies on
ascospore-producing fungi in Egypt. PhD Thesis, Faculty of Science,
Suez Canal University, Egypt.
Abdel-Azeem, A.M., 2009. Operation Wallacea in Egypt. I- A prelimi-
nary study on diversity of fungi in the world heritage site of Saint
Katherine, Egypt. Assiut Univ. J. Bot. 38 (1), 29–54.
Abdel-Azeem, A.M., Ibrahim, M.E., 2004. Diversity of terrophilous myco-
biota of Sinai. Egyptian J. Biol. 6, 21–31.
Abdel-Azeem, A.M., Rashad, H.M., 2013. Mycobiota of outdoor air
that can cause asthma: a case study from Lake Manzala, Egypt.
Mycosphere 4 (4), 1092–1104.
Abdel-Azeem, A.M., Abdel-Moneim, T.S., Ibrahim, M.E., Hassan,
M.A.A., Saleh, M.Y., 2007. Effect of long-term heavy metal contami-
nation on diversity of terricolous fungi and nematodes in Egypt- a case
study. Water Air Soil Pollut. 186 (1), 233–254.
Abdel-Azeem, A.M., El-Morsy, E.M., Nour El-Dein, M.M., Rashad, H.M.,
2015. Occurrence and diversity of mycobiota in heavy metal contami-
nated sediments of Mediterranean coastal lagoon El-Manzala, Egypt.
Mycosphere 6 (2), 228–240.
Abdel-Hafez, A.I.I., Mazen, M.B., Galal, A.A., 1989a. Keratinophilic and
cycloheximide resistant fungi in soils of Sinai Governorate, Egypt.
Cryptogam. Mycol. 10 (3), 265–275.
Abdel-Hafez, A.I.I., Mazen, M.B., Galal, A.A., 1989b. Some ecological
studies of osmophilic and halophilic soil fungi of Sinai Peninsula,
Egypt. J. Sohag Pure Appl. Sci. Bull. 5, 67–83.
Abdel-Hafez, A.I.I., Mazen, M.B., Galal, A.A., 1990. Glycophilic and cel-
lulose-decomposing fungi from soils of Sinai Peninsula, Egypt. Arab
Gulf J. Sci. Res. 8 (1), 153–168.
Abdel-Hafez, S., 1981. Halophilic fungi of desert soils in Saudi Arabia.
Mycopathologia 75, 75e80.
Abdel-Hafez, S.I., 1982a. Survey of microflora of desert soils in Saudi
Arabia. Mycopathologia 80, 3–8.
Abdel-Hafez, S.I., 1982b. Osmophilic fungi of desert soils in Saudi Arabia.
Mycopathologia 80, 9–14.
Abdel-Hafez, S.I., 1982c. Thermophilic and thermotolerant fungi of desert
soils in Saudi Arabia. Mycopathologia 80, 15–20.
Abdel-Hafez S.I.I. 1974. Ecological studies on Egyptian soil fungi, PhD
Thesis. Department of Botany, Faculty of Science, Assiut University,
Egypt.
Abdel-Hafez, S.I.I., 1994. Studies on soil mycoflora of desert soils in
Saudi Arabia. Mycopathologia 80, 3–8.
Abdel-Hafez, S.I.I., El-Maghraby, O.M.O., 1993. Thermophilic and ther-
motolerant fungi of Wadi-Bir-El-Ain soils. Eastern desert, Egypt.
Abhath Al-Yarmouk. Pure Sci. Eng. 2, 55–66.
Abdel-Hafez, S.I.I., Ismail, M.A., Hussein, N.A., Abdel-Hameed, N.A.,
2012. Fusaria and other fungi taxa associated with rhizosphere and
rhizoplane of lentil and sesame at different growth stages. Acta Mycol.
47 (1), 35–48.
Abdel-Hafez, S.I.I., Moharram, A.M., Abdel-Sater, M.A., 2000. Monthly
variations in the mycobiota of wheat fields in El-Kharga Oasis,
Western Desert, Egypt. Bull. Fac. Sci. Assiut Univ. 29 (2-D), 195–211.
Abdel-Hafez, S.I.I., Abdel-Kader, M.I.A., Abdel-Hafez, A.I.I., 1983.
Composition of the fungal flora of Syrian soils. Mycopathologia 81
(3), 161–166.
Abdel-Kader, M.I.A., Abdel-Hafez, A.I.I., Abdel-Hafez, S.I.I., 1983.
Composition of the fungal flora of Syrian soils. II Cellulose-
decomposing fungi. Mycopathologia 81, 167–171.
Abdel-Sater M.A. 1990. Studies on the mycoflora of the New Valley
area, Western Desert, Egypt. PhD Thesis, Faculty of Science, Assiut
University.
Abdel-Sater, M.A., 2000. Soil fungi of the New Valley area, Western
desert, Egypt. Bull. Fac. Sci. Assiut Univ. 29 (2-D), 255–271.
Abdullah, S.K., Al-Khesraji, T.O., Al-Edany, T.Y., 1986. Soil mycoflora of
the southern desert of Iraq. Sydowia 39, 8e16.
Abdullah, S.K., Al-Dossari, M.N., Al-Imara, F.J., 2010. Mycobiota of
surface sediments in marshes of southern Iraq. Marsh Bull. 5 (1),
14–26.
Abdulmoniem, M.A., Saadabi, A.M.A., 2006. On the fungal flora of Saudi
Arabian soils. Res. J. Microbiol. 1, 280–284.
Abou-Zeid, A.M., El-Fattah, R.I.A., 2007. Ecological Studies on the
Rhizosperic Fungi of some halophytic plants in Taif Governorate,
Saudi Arabia. World J. Agric. Sci. 3, 273–279.
Abramson, D., Sinha, R.N., Mills, J.T., 1987. Mycotoxin formation in
moist 2-row and 6-row barley during granary storage. Mycopathologia
97, 179–185.
Abrell, L., Borgeson, B., Crews, P., 1996. Chloro polyketides from the
cultured fungus (Aspergillus) separated from a marine sponge.
Tetrahedron Lett. 37, 2331–2334.
Abu Deraz, S.S. 2014. Isolation and characterization of microbiota inhab-
iting Al-Aqsa Mosque, Al-Quds, Palestine. Master thesis, Faculty of
Science, University of Suez Canal.
Adametz, L. 1886. Untersuchungen über die niederen Pilze der
Ackerkrume. Inaug. Diss., 1–78. Leipzig.
Adams, B.J., Bardgett, R.D., Ayres, E., Wall, D.H., Aislabie, J., Bamforth,
S., et al., 2006. Diversity and distribution of Victoria Land biota. Soil
Biol. Biochem. 38, 3003–3018.
Al-Doory, Y., Tolba, M.K., Al-Ani, A., 1959. On the fungal flora of Iraqi
soil.II: Central Iraq. Mycologia 51, 429–439.
Ali, M.I., 1977. Studies on the fungal flora of Saudi Arabia. 1-Wadi Hanif.
Bull. Fac. Sci. Riyadh Univ. 8, 7–20.
Ali, M.I., Abu-Zinada, A.H., El-Mashharawi, Z., 1977. On the fungal flora
of Saudi Arabia. 11-Seasonal fluctuations of fungi in the rhizosphere
of some plants. Bull. Fac. Sci., Riyadh Univ., 215–228.
Ali-Shtayeh, M.S., Jamous, R.M., 2000. Keratinophilic fungi and
related dermatophytes in polluted soil and water habitats. Revista
Iberoamericana de Micologia 17, 51–59.
Al-Subai, A.A.T. 1983. Soil fungi in state of Qatar. M.Sc. Thesis, Botany
Department, Faculty of Science, Qatar University, Qatar.
Alva, P., Mckenzie, E.H.C., Pointing, S.B., et al., 2002. Do seagrasses
harbour endophytes?. In: Hyde, K.D. (Ed.), Fungi in Marine
Environments, Fungal Diversity Research Series Hong Kong
University Press, Hong Kong.
Anderson, K., Morris, G., Kennedy, H., Croall, J., Michie, J., Richardson,
M., et al., 1996. Aspergillosis in immunocompromised paediatric
patients: associations with building hygiene, design, and indoor air.
Thorax 51, 256–261.
Andrews, S., Pitt, J.I., 1987. Further studies on the water relations of xero-
philic fungi, including some halophiles. J. Gen. Appl. Microbiol. 133,
233–238. http://dx.doi.org/10.1099/00221287-133-2-233.
Anke, H., Kolthoum, I., Zähner, H., Laatsch, H., 1980. Metabolic products
of microorganisms. The anthraquinones of Aspergillus glaucus group.
I. Occurrence,isolation, identification and antimicrobial activity. Arch.
Microbiol. 126, 223–230.
 Biodiversity of the Genus Aspergillus in Different Habitats Chapter | 1
Arenz, B.E., Blanchette, R.A., Farrell, R.L., 2014. Fungal diver-
sity in Antarctic soils. In: Cowan, D. (Ed.), Antarctic Terrestrial
Microbiology: Physical and Biological Properties of Antarctic Soils
Springer, Berlin, pp. 35–53.
Arif, I.A., Hashem, A.R., 1988. Soil analysis and mycoflora of Gizan City,
Saudi Arabia. Phyton 62, 109–113.
Arya, A., Shah, A.R., Sadasivan, S., 2001. Indoor aeromycoflora of
Baroda museum and deterioration of Egyptian mummy. Curr. Sci. 81,
793–799.
Baghdadi, V.Ch. (1968). De speciebus novis Penicilli Fr. et Aspergüli Fr. e
terris Syriae isolatis notula. - Novosti Sistematiki Vysshikh i nizshikh
Rastenii 1968, pp. 96–114.
Bai, Z.Q., Lin, X.P., Wang, Y.Z., Wang, J.F., Zhou, X.F., Yang, B., et al.,
2014. New phenyl derivatives from endophytic fungus Aspergillus
flavipes AIL8 derived of mangrove plant Acanthus ilicifolius.
Fitoterapia 95, 194–202.
Balbool, B.A., Abdel-Azeem, A.M., Khalil, W.F. El-Kazzaz, W.M.
(2013). Bioprospecting as a conservation tool: the genus Aspergillus
(Eurotium) in Egypt. Third International Congress on Fungal
Conservation, Akyaka, Mugla, Turkey, 11–15 November 2013. Abstract
book: 36.
Barakat, A., 1999. Incidence of halophilic and osmophoilic soil fungi and
glycerol biosynthesis by Eurotium amstelodami Mangin from Riyadh,
Saudi Arabia. Bull. Fac. Sci. Assiut Univ. 28 (2-D), 377–390.
Baranyi, N., Kocsubé́, S., Vágvölgyi, C., Varga, J., 2013. Current trends in
aflatoxin research. Acta Biologica Szegediensis 57 (2), 95–107.
Barkai-Golan, R., 2008. In: Barkai-Golan, R., Paster, N. (Eds.), Mycotoxins
in fruits and vegetables Academic Press, San Diego, pp. 115–151.
Battilani, P., Pietri, A., 2002. Ochratoxin A in grapes and wine. Euro. J.
Plant Pathol. 108, 639–643.
Bayman, P., Baker, J.L., Doster, M.A., Michailides, T.J., Mahoney, N.E.,
2002. Ochratoxin production by the Aspergillus ochraceus group and
Aspergillus alliaceus. Appl. Environ. Microbiol. 68, 2326–2329.
Behera, B.C., Mishra, R.R., Thatoi, H.N., 2012. Diversity of soil fungi
from mangroves of Mahanadi delta, Orissa, India. J. Microbiol.
Biotechnol. Res. 2, 375–378.
Belofsky, G.N., Jensen, P.R., Renner, M.K., et al., 1998. New cytotoxic
sesquiterpenoid nitrobenzoyl esters from a marine isolate of the fun-
gus Aspergillus versicolor. Tetrahedron 54, 1715–1724.
Besada, W.H., Yusef, H.M., 1968. On the mycoflora of UAR soil. Proc.
Egyp. Acad. Sci. 21, 103–109.
Betina, V., 1989. Mycotoxins – Chemical, Biological and Environmental
Aspects. Elsevier, Amsterdam.
Blanchette, R.A., 1995. Biodeterioration of archaeological wood. CAB
Biodeterioration Abstracts 9, 113–127.
Blanchette, R.A., 1998. A guide to wood deterioration caused by microor-
ganisms and insects. In: Dardes, K., Rotne, A. (Eds.), The Structural
Conservation of Panel Paintings Getty Conversion Institute, Los
Angeles, pp. 55–68.
Bonugli-Santos, R.C., Vasconcelos, M.R., Passarini, M.R.Z., Vieira,
G.A.L., Lopes, V.C.P., Mainardi, P.H., et al., 2015. Marine-derived
fungi: diversity of enzymes and biotechnological applications. Front.
Microbiol. http://dx.doi.org/10.3389/fmicb.2015.00269.
Borut, S., 1960. An ecological and physiological study on soil fungi of the
Northern Negev (Israel). Bull. Res. Coun. E. Israel 8, 65–80.
Boutaiba, S. 1997. Contribution à l’étude de la flore fongique du lac d’El
Goléa: taxonomie, écologie et production de metabolites. These de
Magister en Biologie, Universite Abou-Bekr Belkaid, Tlemcen,
Institut des Sciences de la Nature.
19
Bridge, P.D., Spooner, B.M., 2012. Non-lichenized Antarctic fungi: tran-
sient visitors or members of a cryptic ecosystem. Fungal Ecol 5,
381–394.
Brock, T.D., 1979. Ecology of saline lakes. In: Shilo, M. (Ed.), Strategies
of Microbial Life in Extreme Environments Dahlem Konferenzen,
Berlin, pp. 29–47.
Buchanan, J.R., Sommer, N.F., Fortlage, R.J., 1975. Aspergillus flavus
infection and aflatoxin production in fig fruits. Appl. Microbiol. 30,
238–241.
Butinar, L., Santos, S., Spencer-Martins, I., Oren, A., Gunde-Cimerman,
N., 2005a. Yeast diversity in hypersaline habitats. FEMS. Microbiol.
Lett. 244 (2), 229–234.
Butinar, L., Sonjak, S., Zalar, P., Plemenitaš, A., Gunde-Cimerman, N.,
2005b. Melanized halophilic fungi are eukaryotic members of micro-
bial communities in hypersaline waters of solar salterns. Bot. Mar. 48
(1), 73–79.
Butinar, L., Zalar, P., Frisvad, J.C., Gunde-Cimerman, N., 2005c. The
genus Eurotium—members of indigenous fungal community in hyper-
saline waters of salterns. FEMS Microbiol. Ecol. 51 (2), 155–166.
Butinar, L., Frisvad, J.C., Gunde-Cimerman, N., 2011. Hypersaline
waters- a potential source of foodborne toxigenic aspergilli and peni-
cillia. FEMS Microbiol. Ecol. 77, 186–199.
Callaghan, T.V., Björn, L.O., Chernov, Y., Chapin, T., Christensen, T.R.,
Huntley, B., et al., 2004. Biodiversity, distributions and adaptations
of Arctic species in the context of environmental change. Ambio 33,
404–417.
Cantrell, S.A., Casillas-Martinez, L., Molina, M., 2006. Characterization
of fungi from hypersaline environments of solar salterns using mor-
phological and molecular techniques. Mycol. Res. 110, 962–970.
Carbone, I., Kohn, L.M., 1999. A method for designing primer sets for
speciation studies in filamentous ascomycetes. Mycologia 91, 553–556.
Čavka, M., Glasnović, A., Janković, I., Šikanjić, P.R., Perić, B., Brkljačić, B.,
et al., 2010. Microbiological analysis of a mummy from the archeo-
logical museum in Zagreb. Coll. Antropol. 34, 803–805.
Christensen, M., 1981. A synoptic key and evaluation of species in the
Aspergillus flavus group. Mycologia 73, 1056–1084.
Christensen, M., 1982. The Aspergillus ochraceous group: two new
species from western soils and a synoptic key. Mycologia 74 (2),
210–225.
Christensen, M., States, J.S., 1982. Aspergillus nidulans group: Aspergillus
navahoensis and a revised synoptic key. Mycologia 74, 226–235.
Christensen, M., Tuthill, D.E., 1985. Aspergillus: an overview. In: Samson,
R.A., Pitt, J.I. (Eds.), Advances in Pencillium and Aspergillus system-
atics Plenum Press, New York, NY, pp. 195–209.
Ciegler, A., 1972. Bioproduction of ochratoxin A and penicillic acid by
members of the Aspergillus ochraceus group. Canad. J. Microbiol. 18,
631–636.
Cole, R.J., 1984. Cyclopiazonic acid and related toxins. In: Betina, V.
(Ed.), Mycotoxins: Production, Isolation, Separation and Purification
Elsevier, Amsterdam, pp. 405–414.
Cole, R.J., Cox, R.H., 1981. Handbook of Toxic Fungal Metabolites.
Academic Press, New York, NY.
Conley, C.A., Ishkhanova, G., McKay, C.P., Cullings, K., 2006. A pre-
liminary survey of non-lichenized fungi cultured from the hyperarid
Atacama Desert of Chile. Astrobiology 6, 521–526.
Cruickshank, R.H., Pitt, J.I., 1990. Isoenzyme patterns in Aspergillus fia-
vus and closely related taxa. In: Samson, R.A., Pitt, J.I. (Eds.), Modern
Concepts in Penicillium and Aspergillus Classification Plenum Press,
New York and London, pp. 259–264.
20 SECTION | I Biology and Biodiversity
Cui, L., Morris, A., Ghedin, E., 2013. The human mycobiome in health
and disease. Genome Med. 5, 63. <http://genomemedicine.com/
content/5/7/63>.
Cuijpers, C.E.J., Swaen, G.M.H., Wesseling, G., Sturmans, F., Wouters,
E.F.M., 1995. Adverse effects of the indoor environment on respira-
tory health in primary school children. Environ. Res. 68, 11–23.
Darwish, S.S., El Hadidi, N., Mansour, M., 2013. The effect of fungal
decay on Ficus sycomorus wood. Int. J. Conserv. Sci. 4 (3), 271–282.
Davis, N.D., 1981. Sterigmatocystin and other mycotoxins produced by
Aspergillus species. J. Food. Prot. 44, 711–714.
Dendouga, W., Boureghda, H., Belhamra, M., 2015. Edaphic factors
affecting distribution of soil fungi in three chotts located in Algeria
desert. Courrier du Savoir 19, 147–152.
Devi, L.S., Joshi, S.R., 2012. Antimicrobial and synergistic effects of silver
nanoparticles synthesized using soil fungi of high altitudes of eastern
Himalaya. Mycobiology (40), 27–34.
de Vries, R.P., Visser, J., 2001. Aspergillus enzymes involved in degrada-
tion of plant cell wall polysaccharides. Microbiol. Mol. Biol. Rev. 65,
497–522.
Ding, B., Yin, Y., Zhang, F., Li, Z., 2011. Recovery and phylogenetic diver-
sity of culturable fungi associated with marine sponges Clathrina lute-
oculcitella and Holoxea sp. in the South China Sea. Mar. Biotechnol.
13, 713–721.
Dörfelt, H., Schmidt, A.R., 2005. A fossil Aspergillus from Baltic amber.
Mycol. Res. 109, 956–960.
Dorner, J.W., Cole, R.J., Diener, U.L., 1984. The relationship of Aspergillus
flavus and Aspergillus parasiticus with reference to production of afla-
toxins and cyclopiazonic acid. Mycopathologia 87, 13–15.
Doster, M.A., Michailides, T.J., Morgan, D.P., 1996. Aspergillus species
and mycotoxins in figs from Californian orchards. Plant Dis. 80,
484–489.
Durley, R.C., MacMillan, J., Simpson, T.J., et al., 1975. Fungal products.
XIII Xanthomegnin, viomellein, rubrosulphin and viopurpurin, pig-
ments from Aspergillus sulphureus and Aspergillus melleus. J. Chem.
Perkin Trans. 1, 163–169.
Ein-Gil, N., Ilan, M., Carmeli, S., Smith, G.W., Pawlik, J.R., Yarden, O.,
2009. Presence of Aspergillus sydowii, a pathogen of gorgonian sea-
fans in the marine sponge Spongia obscura. ISME J. 3, 752–755.
http://dx.doi.org/10.1038/ismej.2009.18.
El-Buni, A.M., Rattan, S.S., 1981. Check List of Libyan Fungi. Department
of Botany, Al Faateh University, Tripoli, 169 pages.
El-Dohlob, S.M. F.F. Migahed (1985): Seed Borne and Rhizosphere Fungi
of Four Varieties of Crop Plants. 2nd Agric. Conf. Bot. Sci. 21–23 Sept.
El-Said, A.H.M., 1994. Studies on soil mycoflora of Bahreen. Microbiol.
Res. 149, 263–269.
El-Said, A.H.M., Saleem, A., 2008. Ecological and physiological studies
on soil fungi at western region, Libya. Mycobiology 36, 109.
Fathi, S.M., El-Husseini, T.M., Abu-Zinada, A.H., 1975. Seasonal varia-
tions of soil microflora and their activities in Riyadh region, Saudi
Arabia. Bull. Fac. Sci., Riyadh Univ. 7, 17–30.
Findley, K., Oh, J., Yang, J., Conian, S., Deming, C., et al., 2013.
Topographic diversity of fungal and bacterial communities in human
skin. Nature <http://dx.doi.org/10.1038/nature12171f> (online 22
May 2013).
Flannigan, B., Pearce, A.R., 1994. Aspergillus spoilage: spoilage of cere-
als and cereal products by the hazardous species Aspergillus clava-
tus. In: Powell, K.A., Renwick, A., Peberdy, J.F. (Eds.), The Genus
Aspergillus from Taxonomy and Genetics to Industrial Application
Plenum Press, New York, NY, pp. 55–62.
Flannigan, B., McCabe, E.M., McGarry, F., 1991. Allergenic and toxi-
genic micro-organisms in houses. J. Appl. Bacteriol. Sympos. 70,
61S–73S.
Microorganisms in home and indoor workFlannigan, B., Samson, R.A.,
Miller, J.D. (Eds.), 2011. Environments: Diversity, Health Impacts,
Investigation and Control, second ed. CRC Press, Boca Raton.
Fraga, M.E., Santana, D.M.N., Gatti, M.J., Direito, G.M., Cavaglieri, L.R.,
Rosa, C.A.R., 2008. Characterization of Aspergillus species based on
fatty acid profiles. Mem. Ist. Oswaldo Cruz 103 (6), 540–544.
Frisvad, J.C., 1989. The use of high-performance liquid chromatography
and diode array detection in fungal chemotaxonomy based on profiles
of secondary metabolites. Bot. J. Linnean Soc. 99, 81–95.
Frisvad, J.C., 2008. Fungi in cold ecosystems. In: Margesin, R., Schinner,
F., Marx, J.-C., Gerday, C. (Eds.), Psychrophiles: from Biodiversity to
Biotechnology Springer, Berlin, pp. 137–156.
Frisvad, J.C., Frank, J.M., Houbraken, J.A.M.P., Kuijpers, A.F.A., Samson,
R.A., 2004. New ochratoxin A producing species of Aspergillus sec-
tion Circumdati. Stud. Mycol. 50, 23–43.
Frisvad, J.C., Andersen, B., Thrane, U., 2007. The use of secondary metab-
olite profiling in chemotaxonomy of filamentous fungi. Mycol. Res.
http://dx.doi.org/10.1016/j.mycres.2007.08.018.
Frisvad, J.C., Thrane, U., Filtenborg, O., 1998. Role and use of secondary
metabolites in fungal taxonomy. In: Frisvad, J.C., Bridge, P.D., Arora,
D.K. (Eds.), Chemical Fungal Taxonomy Marcel Dekker, New York,
NY, pp. 289–319.
Frisvad, J.C., Larsen, T.O., Thrane, U., 2011. Fumonisin and ochratoxin
production in industrial Aspergillus niger strains. PLoS ONE 6,
e23496.
Fröhlich-Nowoisky, J., Burrows, S.M., Xie, Z., Engling, G., Solomon,
P.A., Fraser, M.P., et al., 2012. Biogeography in the air: fungal diver-
sity over land and oceans. Biogeosciences 9, 1125–1136. http://dx.doi.
org/10.5194/bg-9-1125-2012.
Galagan, J.E., Calvo, S.E., Cuomo, C., Ma, L.J., Wortman, J.R.,
Batzoglou, S., et al., 2005. Sequencing of Aspergillus nidulans and
comparative analysis with A. fumigatus and A. oryzae. Nature 438,
1105–1115.
Gallagher, R.T., Richard, J.L., Stahr, H.M., Cole, R.J., 1978. Cyclopiazonic
acid production by aflatoxigenic and non-aflatoxigenic strains of
Aspergillus flavus. Mycopathologia 66, 31–36.
Gams, W., Christensen, M., Onions, A.H.S., Pitt, J.I., Samson, R.A., 1985.
Infrageneric taxa of Aspergillus. In: Samson, R.A., Pitt, J.I. (Eds.),
Advances in Penicillium and Aspergillus Systematics Plenum Press,
New York, NY, pp. 55–64.
Gao, Z., Li, B., Zheng, C., Wang, G., 2008. Molecular detection of fun-
gal communities in the Hawaiian marine sponges Suberites zeteki and
Mycale armata. Appl. Environ. Microbiol. 74, 6091–6101.
Geiser, D.M., Taylor, J.W., Ritchie, K.B., Smith, G.W., 1998. Cause of sea
fan death in the West Indies. Nature 394, 137–138.
Ghannoum, M.A., Jurevic, R.J., Mukherjee, P.K., Cui, F., Sikaroodi, M.,
Naqvi, A., et al., 2010. Characterization of the oral fungal microbiome
(mycobiome) in healthy individuals. PLoS Pathog. 6, e1000713.
Gill-Carey, D., 1949. The nature of some antibiotics from aspergilli. Brit.
J. Exp. Path 30, 123.
Giridhar, P., Reddy, S.M., 2001. Incidence of mycotoxigenic fungi on rai-
sins. Adv. Plant Sci. 14, 291–294.
Giusiano, G., Piontelli, E., Mangiaterra, M., Sosa, M.A., 2002. Distribución
altitudinal de hongos queratinófilos, epífitos y endófitos en suelos
semiáridos del noroeste argentino (Prov. De Jujuy, 23°l.S Y 66°l.W).
Boletín Micológico 17, 51–62.
 Biodiversity of the Genus Aspergillus in Different Habitats Chapter | 1
Glass, N.L., Donaldson, G.C., 1995. Development of primer sets designed
for use with the PCR to amplify conserved genes from filamentous
ascomycetes. Appl. Environ. Microbiol. 61, 1323–1330.
Glassbrook, N.J. 2008. Biochemical markers for the detection and clas-
sification of Aspergillus. PhD Thesis, Cardiff University.
Google Scholar, <https://scholar.google.com.eg/> (Accessed 17. 07.
2015).
Gorst-Allman, C.P., Steyn, P.S., 1979. Screening methods for the detection
of thirteen common mycotoxins. J. Chromat. 175, 325–331.
Grishkan, I., Nevo, E., 2010. Spatiotemporal distribution of soil micro-
fungi in the Makhtesh Ramon area, central Negev desert, Israel.
Fungal Ecol. 3, 326e337.
Grishkan, I., Rong-Liang, J., Kidron, G.J., Xin-Rong, L., 2015. Cultivable
microfungal communities inhabiting biological soil crusts in the
Tengger Desert, China. Pedosphere 25 (3), 351–363.
Guarro, J., Gene, J., Stchigel, A.M., 1999. Developments in fungal tax-
onomy. Clin. Microbiol. Rev. 12, 454–500.
Guatam, A.K., 2014. Diversity of fungal endophytes in some medicinal
plants of Himachal Pradesh, India. Arch. Phytopathol. Plant Protect.
47 (5), 537–544.
Guiraud, P., Steiman, R., Seigle-Murandi, F., Sage, L., 1995. Mycoflora of
soil around the Dead Sea II—Deuteromycetes (except Aspergillus and
Penicillium). Syst. Appl. Microbiol. 18, 318–322.
Gunde-Cimerman, N., Zalar, P., de Hoog, S., Plemenitaš, A., 2000.
Hypersaline waters in salterns –natural ecological niches for halo-
philic black yeasts. FEMS Microbiol. Ecol. 32 (3), 235–240.
Gunde-Cimerman, N., Sonjak, S., Zalar, P., Frisvad, J.C., Diderichsen, B.,
Plemenitaš, A., 2003. Extremophilic fungi in Arctic ice: a relation-
ship between adaptation to low temperature and water activity. Phys.
Chem. Earth Pt. B. 28, 1273–1278.
Gunde-Cimerman, N., Oren, A., Plemenitaš, A., Butinar, L., Sonjak, S.,
Turk, M., et al., 2005. Halotolerant and halophilic fungi from coastal
environments in the Arctics. In: Seckbach, J. (Ed.), Adaptation to life
at High Salt Concentrations in Archaea, Bacteria, and Eukarya, vol 9,
Cellular Origin, Life in Extreme Habitats and Astrobiology Springer,
Netherlands, pp. 397–423.
Hafez, W.A. 2012. Comparative ecological studies on soil and rhizospheric
fungi of maize and wheat plants in different areas in Minia Governorate
Egypt. M.S. Thesis, Faculty of Science, El-Minina University.
Halwagy, R., Moustafa, A.F., Kamel, S.M., 1982. Ecology of the
soil mycoflora in the desert soil of Kuwait. J. Arid Environ. 5,
109–125.
Hashem, A.R., 1991. Studies on the fungal flora of Saudi Arabian soil.
Crypt. Bot. 2/3, 179–182.
Hashem, A.R., 1995. Soil analysis and mycoflora of the Jubail industrial
city in Saudi Arabia. J. Univ. Kuwait (Sci) 22, 231–237.
Hatakka, A. and Hammel, K.E. (2010). Fungal biodegradation of ligno-
celluloses, In: Esser, K. (series Ed.), The Mycota, A Comprehensive
Treatise on Fungi as Experimental Systems for Basic and Applied
Resarch. In: Hofrichter, M. (vol. Ed.), Industrial Applications, second
ed., vol. 10. Springer Berlin Heidelberg, pp. 319–340.
Hesseltine, C.W., Vandegraft, E.E., Fennell, I., et al., 1972. Aspergilli as
ochratoxin producers. Mycologia 64, 539–550.
Hill, R.A., Blankenship, P.D., Cole, R.J., Sanders, T.H., 1983. Effects of
soil moisture and temperature on preharvest invasion of peanuts by the
Aspergillus flavus group and subsequent aflatoxin development. Appl.
Environ. Microbiol. 45, 628–633.
Hogarth, P.J., 2007. The Biology of Mangroves and Seagrasses. Oxford
University Press, Oxford, 272 pp.
21
Höller, U., Wrigh, A.D., Matthee, G.F., et al., 2000. Fungi from marine
sponges: diversity, biological activity and secondary metabolites.
Mycol. Res. 104, 1354–1365.
Hong, S.B., Go, S.J., Shin, H.D., Frisvad, J.C., Samson, R.A., 2005.
Polyphasic taxonomy of Aspergillus fumigatus and related species.
Mycologia 97, 1316–1329.
Horn, B.W., 2003. Ecology and population biology of aflatoxigenic fungi
in soil. J. Toxicol.—Toxin Rev. 22, 351–379.
Horn, B.W., Dorner, J.W., 2002. Effect of competition and adverse culture
conditions on aflatoxin production by Aspergillus flavus through suc-
cessive generations. Mycologia 94, 741–751.
Horner, W.E., Helbling, A., Salvaggio, J.E., Lehrer, S.B., 1995. Fungal
allergens. Clin. Microbiol. Rev. 8, 161–179.
Horré, R., Symoens, F., Delhaes, L., Bouchara, J.-P., 2010. Fungal respira-
tory infections in cystic fibrosis: a growing problem. Med. Mycol. 48,
S1–S3. http://dx.doi.org/10.3109/13693786.2010.529304.
Houbraken, J., Due, M., Varga, J., et al., 2007. Polyphasic taxonomy of
Aspergillus section Usti. Stud. Mycol. 59, 107–128.
Hu, F.B., Persky, V., Flay, B.R., Richardson, J., 1997. An epidemiological
study of asthma prevalence and related factors among young adult. Br.
Med. J. 34, 67–76.
Hubka, V., Kolarik, M., 2012. β-Tubulin paralogue tubC is frequently
misidentified as the benA gene in Aspergillus section Nigri taxonomy:
primer specificity testing and taxonomic consequences. Persoonia 29.
http://dx.doi.org/10.3767/003158512X658123.
Hubka, V., Kolarik, M., Alena Kubátova, A., Peterson, S.W., 2013.
Taxonomic revision of Eurotium and transfer of species to Aspergillus.
Mycologia 105 (4), 912–937.
Huffnagle, G.B., Noverr, M.C., 2013. The emerging world of the fungal
microbiome. Trends Microbiol. 21, 334–341. Available from: http://
dx.doi.org/10.1016/j.tim.2013.04.002.
Hyde, K.D., Jones, E.B.G., Leaño, E., Pointing, S.B., Poonyth, A.D.,
Vrijmoed, L.L.P., 1998. Role of fungi in marine ecosystems. Biodivers.
Conserv. 7, 1147–1161.
Iamanaka, B.T., Taniwaki, M.H., Menezes, H.C., et al., 2005. Incidence of
toxigenic fungi and ochratoxin A in dried fruits sold in Brazil. Food
Addit. Contam. 22, 1258–1263.
Imran, Z.K., Al Rubaiy, A.A., 2015. Molecular ecological typing of wild
type Aspergillus terreus from arid soils and screening of lovastatin
production. Afr. J. Microbiol. Res. 9 (8), 534–542.
Irbe, I., Andersone, I., Andersons, B., 2009. Diversity and distribution of
wood decay fungi and wood discoloring fungi in buildings in Latvia.
LLU Raksti 23 (318), 91–102.
Ismail, A.L.S., Abdullah, S.K., 1977. Studies on the soil fungi of Iraq.
Proc. Indian Acad. Sci. 86, 151–154.
Ivarson, K.C., 1965. The microbiology of some permafrost soils in the
McKenzie Valley, N.W.T. Arctic 18, 256–260.
Jabra-Rizk, M.A., Ferreira, S.M., Sabet, M., Falkler, W.A., Merz, W.G.,
Meiller, T.F., 2001. Recovery of Candida dubliniensis and other yeasts
from human immunodeficiency virus associated periodontal lesions.
J. Clin. Microbiol. 39, 4520–4522.
Jaime-Garcia, R., Cotty, P.J., 2006. Spatial relationships of soil texture
and crop rotation to Aspergillus flavus community structure in South
Texas. Phytopathology 96, 599–607.
Jaime-Garcia, R., Cotty, P.J., 2010. Crop rotation and soil temperature
influence the community structure of Aspergillus flavus in soil. Soil
Biol. Biochem. 42, 1842–1847.
Jeewon, R., Hyde, K.D., 2007. Diversity and detection of fungi from
environmental samples: traditional versus molecular approaches.
22 SECTION | I Biology and Biodiversity
In: Varma, A., Oelmuller, R. (Eds.), Advanced Techniques in Soil
Microbiology. Soil Biology Series Springer-Verlag Press.
Junior, P.R.J., Yamamoto, A.C.A., Amadio, J.V.R., Martins, E.R., Leal,
F.A., et al., 2012. Trichocomaceae: biodiversity of Aspergillus spp
and Penicillium spp residing in libraries. J. Infect. Dev. Ctries 6 (10),
734–743.
Jurjević, Ž., Peterson, S.W., Horn, B.W., 2012. Aspergillus section
Versicolores: nine new species and multilocus DNA sequence based
phylogeny. IMA Fungus 3, 59–79.
Karlshøj, K., Nielsen, P.V., Larsen, T.O., 2007. Fungal volatiles: biomark-
ers of good and bad food quality. In: Dijksterhuis, J., Samson, R.A.
(Eds.), Food Mycology. A multifaceted Approach to Fungi and Food
CRC Press, Boca Raton, pp. 279–302.
Kathiresan, K., Bingham, B.L., 2001. Biology of mangroves and man-
grove ecosystems. Adv. Mar. Biol. 40, 81–251.
Kelecom, A., 2002. Secondary metabolites from marine microorganisms.
An. Acad. Bras. Cienc. 74, 151–170.
Khalil, A.M.A., El-sheikh, H.H., Sultan, M.H., 2013. Distribution of fungi
in mangrove soil of coastal areas at Nabq and Ras Mohammed protec-
torates. Int. J. Curr. Microbiol. App. Sci. 2 (12), 264–274.
Khosravi, A.R., Arash, C.N., Hojjatollah, S., 2012. Protein profiles of
Aspergillus species isolated from the tea gardens and factories air in
northern Iran. Jundishapour J. Microbiol. 6 (1), 4–10.
Kim, K.W., Sugawara, F., Yoshida, S., et al., 1993. Structure of malformin
A, a phytotoxic metabolite produced by Aspergillus niger. Biosci.
Biotech. Biochem. 57, 240–243.
Kirk, J.L., Beaudette, L.A., Hart, M., Moutoglis, P., Klironomos, J.N., Lee,
H., 2004. Methods of studying soil microbial diversity. J. Microbiol.
Methods. 58, 169–188.
Klich, M.A., 2002a. Biogeography of Aspergillus species in soil and litter.
Mycologia 94 (1), 21–27.
Klich, M.A., 2002b. Identification of Common Aspergillus Species.
Centraalbureau voor Schimmelcultures, Utrecht.
Klich, M.A., Pitt, J.I., 1988. A Laboratory Guide to Common Aspergillus
Species and Their Teleomorphs. CSIRO Division of Food Processing,
North Ryde, NWS.
Klich, M.A., Mullaney, E.J., Daly, C.B., Cary, J.W., 2000. Molecular and
physiological aspects of aflatoxin and Sterigmatocystin biosynthe-
sis by Aspergillus tamarii and A. ochraceoroseus. Appl. Microbiol.
Biotechnol. 53, 605–609.
Kohlmeyer, J., Kohlmeyer, E., 1979. Marine Mycology The Higher Fungi.
Academic Press, New York, NY.
König, G.M., Kehraus, S., Seiber, S.F., Abdel-Lateff, A., Müller, D.,
2006. Natural products from marine organisms and their associated
microbes. Chem. Biochem. 7, 229–238.
Kozakiewicz, Z., 1989. Aspergillus species on stored products. Myc.
Papers 161, 1–188.
Kredics, L., Hatvani, L., Naeimi, S., et al., 2014. Biodiversity of the
genus Hypocrea/Trichoderma in different habitats. In: Gupta, V.G.,
Schmoll, M., Herrera-Estrella, A. (Eds.), Biotechnology and Biology
of Trichoderma Elsevier, pp. 3–24.
Krishnan, A., Alias, S.A., Michael Wong, C.V.L., Pang, K.L., Convey, P.,
2011. Extracellular hydrolase enzyme production by soil fungi from
King George Island, Antarctica. Polar Biol. 4, 1535–1542.
Krnjaja, V., Stojanovic, L.J., Tomic, Z., Nesic, Z., 2008. The presence of
potentially toxigenic fungi in dairy cattle feed with focus on species of
genus Аspergillus. J. Mountain Agric. Balkans 11, 621–630.
Kubanek, J., Jensen, P.R., Keifer, P.A., Sullards, M.C., Collins, D.O.,
Fenical, W., 2003. Seaweed resistance to microbial attack: a targeted
chemical defense against marine fungi. Proc. Nat. Acad. Sci. USA
100, 6916–6921.
Kuraishi, H., Itoh, M., Katayama, Y., Ito, T., Hasegawa, A., Sugiyama,
J., 2000. Ubiquinone systems in fungi. V. Distribution and taxo-
nomic implications of ubiquinones in Eurotiales, Onygenales and the
related plectomycete genera, except for Aspergillus, Paecilomyces,
Penicillium, and their related teleomorphs. Antonie Van Leeuwenhoek.
77 (2), 179–186.
Kurakov, A.V., Somova, N.G., Ivanovskii, R.N., 1999. Micromycetes pop-
ulating limestone and red brick surfaces of the Novodevichii Convent
masonry. Microbiologia 68, 232–241.
Kuriashi, H., Itoh, M., Tsuzaki, N., Katayama, Y., Yokoyama, T., Sugiyama,
J., 1990. The ubiquinone system as a taxonomic aid in Aspergillus and
its teleomorphs. In: Samson, R.A., Pitt, J.I. (Eds.), Modern Concepts
in Penicillium and Aspergillus Classification Plenum Press, New York,
NY, pp. 407–421.
Kurtzman, C.P., Horn, H.B., Hesseltine, C.W., 1987. Aspergillus nomius:
a new aflatoxin-producing species related to Aspergillus flavus and
Aspergillus tamarii. J. Microbiol. 12, 85–87.
Kurzeja, K.C., Gabber, E.D., 1973. A genetic study of electrophoreti-
cally variant extracellular amylolytic enzymes of wild-type strains of
Aspergillus nidulans. Can. J. Genet. Cytol. 15, 275–287.
Kusari, S., Lamshöft, M., Spiteller, M., 2009. Aspergillus fumiga-
tus Fresenius, an endophytic fungus from Juniperus communis L.
Horstmann as a novel source of the anticancer pro-drug deoxypodo-
phyllotoxin. J. Appl. Microbiol. 107, 1019–1030.
Lam, C., Stang, A., Harder, T., 2008. Planktonic bacteria and fungi are
selectively eliminated by exposure to marine macroalgae in close
proximity. FEMS Microbiol. Ecol. 63, 283–291.
Larsen, T.O., Smedsgaard, J., Nielsen, K.F., Hansen, M.E., Frisvad, J.C.,
2005. Phenotypic taxonomy and metabolite profiling in microbial
drug discovery. Nat. Prod. Rep. 22, 672–693.
Lechevalier, H., Lechevalier, M.P., 1988. Chemotaxonomic use of lipids
an overview In: Ratledge, C. Wilkison, S.G. (Eds.), Microbiol. Lipids,
Vol. I Academic Press, London, pp. 869–902.
Lee, O.O., Wang, Y., Yang, J., Lafi, F.F., Al-Suwailem, A., Qian, P.Y.,
2010. Pyrosequencing reveals highly diverse and species-specific
microbial communities in sponges from the Red Sea. ISME J. 5,
650–664.
Lee, L.S., Bennett, J.W., Cucullu, A.F., Stanley, J.B., 1975. Synthesis of
versicolorin A by a mutant of Aspergillus parasiticus deficient in afla-
toxin production. J. Agric. Food Chem. 23, 1132–1134.
Lee, S.M., Li, X.F., Jiang, H., Cheng, J.G., Seong, S., Choi, H.D., et al.,
2003. Terreusinone, a novel UV-A protecting dipyrroloquinone from
marine algicolous fungus Aspergillus terreus. Tetrahedron Lett. 44,
7707–7710.
Leila, S., Azar, S., Alireza, K., Mansour, B., Amir, B., 2010. Determining
protein patterns for three fungus species Aspergillus fumigatus, Asp.
flavus and Asp. niger, obtained from outdoor air in Iran. Global
Veterinaria 4 (2), 130–134.
Lević, J., Gošić-Dondo, S., Ivanović, D., Stanković, S., Krnjaja, V.,
Boćarov-Stancić, A., et al., 2013. An outbreak of Aspergillus species
in response to environmental conditions in Serbia. Pestic. Phytomed.
(Belgrade) 28 (3), 167–179.
Li, Q., Wang, G., 2009. Diversity of fungal isolates from three Hawaiian
marine sponges. Microbiol. Res. 164, 233–241.
Li, W.C., Zhou, J., Guo, S.Y., Guo, L.D., 2007. Endophytic fungi asso-
ciated with lichens in Baihua mountain of Beijing, China. Fungal
Divers. 25, 69–80.
 Biodiversity of the Genus Aspergillus in Different Habitats Chapter | 1
Li, X.J., Zhang, Q., Zhang, A.L., Gao, J.M., 2012. Metabolites from
Aspergillus fumigatus, an endophytic fungus associated with Melia
azedarach, and their antifungal, antifeedant, and toxic activities.
J. Agr. Food Chem. 60, 3424–3431.
Lin, A., Lu, X., Fang, Y., et al., 2008. Two new 5-Hydroxy-2-pyrone
derivatives isolated from a marine-derived fungus Aspergillus flavus.
J. Antibiot. 61, 245–249.
Lin, W., Brauers, G., Ebel, R., Wray, V., Berg, A., Sudarsono, P.P., 2003.
Novel chromone derivatives from fungus Aspergillus versicolor iso-
lated from the marine sponge Xestospongia exigua. J. Nat. Prod. 66,
57–61.
Liu, W., Li, C., Zhu, P., Yang, J., Cheng, K., 2010. Phylogenetic diversity
of culturable fungi associated with two marine sponges: Haliclona
simulans and Gelliodes carnosa, collected from the Hainan Island
coastal waters of the South China Sea. Fungal Divers. 42, 1–15.
Londero, A.T., Guadalupe-Cortés, J.M., 1990. Aspergiloses Pulmonares.
J. Pneumologia 16, 78–90.
Lopez-Diaz, T.M., Flannigan, B., 1997. Production of patulin and cytocha-
lasin E by Aspergillus clavatus during malting of barley and wheat.
Int. J. Food. Microbiol. 35, 129–136.
Madavasamy, S., Pannerselvam, A., 2012. Isolation, identification of fungi
from Avecinnia marina Muthupet Mangroves Thiruvarur Dt. Asian J.
Plant Sci. Res. 2 (4), 452–459.
Magnoli, C., Astoreca, A., Ponsone, L., et al., 2004. Survey of mycoflora
and ochratoxin A in dried vine fruits from Argentina markets. Lett.
Appl. Mycobiol 39, 326–331.
Mahmoud, S.A.Z., Abou El-Fadle, M., El-Mofty, M., 1964. Studied on the
rhizosphere microflora of a desert plants. Folia. Microbiol. (Praha).
9, 1–8.
Manoharachary, C., Kunwar, I.K., Tilak, K.V., 2013. Diversity and charac-
terization of fungi and its relevance. Indian Phytopath. 66 (1), 10–13.
Mansour, A.M.A., 2010. Contribution to knowledge of some soil fungi in
Eastern region, in Libya. J Prod. Dev. (Agricultural Research) 15 (3), 10.
Marguet, C., Favennec, L., Matray, O., Bertout, S., Giraud, S., Couderc,
L., et al., 2012. Clinical and microbiological efficacy of micafungin
on Geosmithia argillacea infection in a cystic fibrosis patient. Med.
Mycol. Case Rep 1, 79–81. Available from: http://dx.doi.org/10.1016/j.
mmcr.2012.08.004.
Marín, S., Ramos, A.J., Sanchis, V., 2012. Modeling Aspergillus flavus
growth and aflatoxins production in pistachio nuts. Food Microbiol.
32, 378–388. http://dx.doi.org/10.1016/j.fm.2012.07.018.
Masic, Z., Bocarov-Stancic, A., Sinovec, Z., Dilas, S., Adamovic, M.,
(2003) Mycotoxin in feed for animals in the Republic of Serbia.
10th Symposium Food Technology for Animal Safety and Quality,
Vrnjačka Banja, Serbia and Montenegro. Book of Prooceedings.
Matsuda, H., Kohno, S., Maesaki, S., Yamada, H., Koga, H., Tamura,
M., et al., 1992. Application of ubiquinone systems and electropho-
retic comparison of enzymes to identification of clinical isolates of
Aspergillus fumigatus and several other species of Aspergillus. J. Clin.
Microbiol. 30, 1999–2005.
Mazen, M.B., Shaban, G.M., 1983. Air-borne fungi of wheat fields in
Egypt. Qatar Univ. Sci. Bull. 3, 131–139.
McClenny, N., 2005. Laboratory detection and identification of Aspergillus
species by microscopic observation and culture: the traditional
approach. J. Med. Vet. Mycol. 43 (Suppl. 1), S125–S128.
McGinnis, Mr, 2007. Indoor mould development and dispersal. Med.
Mycol. 45 (1), 1–9.
Medina, A., Mateo, R., López-Ocaná, L., et al., 2005. Study of Spanish
grape mycobiota and ochratoxin A production by isolates of
23
Aspergillus tubingensis and other members of Aspergillus section
Nigri. Appl. Environ. Microbiol. 71, 4696–4702.
Mehl, H.L., Cotty, P.J., 2013. Influence of plant host species on intraspe-
cific competition during infection by Aspergillus flavus. Plant Pathol.
62, 1310–1318.
Menezes, C.B.A., Bonugli-Santos, R.C., Miqueletto, P.B., Passarini,
M.R.Z., Silva, C.H.D., Justo, M.R., et al., 2010. Microbial diversity
associated with algae, ascidians and sponges from the north coast of
Sao Paulo state, Brazil. Microbiol. Res. 165, 466–482. Available from:
http://dx.doi.org/10.1016/j.micres.2009.09.005.
Micales, J.A., Bonde, M.R., Peterson, G.L., 1992. Isozyme analysis in fun-
gal taxonomy and molecular genetics. In: Arora, D.K., Elander, R.P.,
Mukerji, K.G. (Eds.), Handbook of Applied Mycology, vol. 4, Fungal
Biotechnology Dekker, New York, NY, pp. 57–79.
Mislivec, P.B., Dieter, C.T., Bruce, V.R., 1975. Effect of temperature
and relative humidity on spore germination of mycotoxic species of
Aspergillus and Penicillium. Mycologia 67, 1187–1189.
Mitterdorfer, G., Mayer, H.K., Kneifel, W., Viernstein, H., 2002.
Clustering of Saccharomyces boulardii strains within the species S.
cerevisiae using molecular typing techniques. J. Appl. Microbiol. 93
(4), 521–530.
Montasir, A.H., Mostafa, M.A., Elwan, S.H., 1956a. Development of soil
microflora under Zygophyllum album L. and Zygophyllum coccineum
L. Ain Shams Sci. Bull. 1, 9–22.
Montasir, A.H., Mostafa, M.A., Elwan, S.H., 1956b. Development of soil
microflora in relation to vegetation along a transect line at yellow hills,
North Cairo. Ain Shams Sci. Bull. 1, 23–32.
Moss, M.O., 1977. Aspergillus mycotoxins. In: Smith, J.E., Patlman, J.A.
(Eds.), Genetics and Physiology of Aspergillus Academic Press, New
York and London, pp. 499–524.
Moubasher, A.H., 1993. Soil Fungi of Qatar and Other Arab Countries.
The Scientific and Applied Research Centre, University of Qatar.
Moubasher, A.H., Abdel-Hafez, S.I.I., 1978. Studies on the mycoflora of
Egyptian soils. Mycopathologia 63 (1), 3–10.
Moubasher, A.H., El-Dohlob, S.M., 1970. Seasonal fluctuation of Egyptian
soil fungi. Trans. Brit. Mycol. Soc. 54, 45–51.
Moubasher, A.H., Moustafa, A.F., 1970. A survey of Egyptian soil fungi
with special reference to Aspergillus, Penicillium and Penicillium
related genera. Trans. Brit. Mycol. Soc. 54, 35–44.
Moubasher, A.H., Moustafa, A.F., 1972. Aspergillus aegyptiacus sp. nov.
Egypt. J. Bot. 15, 153–154.
Moubasher, A.H., Abdel-Hafez, S.I.I., El-Maghraby, O.M.O., 1985.
Studies on soil mycoflora of Wadi Bir- El- Ain, Eastern Desert, Egypt.
Cryptogamie Mycologie 6, 129–143.
Moubasher, A.H., Abdel-Hafez, S.I.I., El-Maghraby, O.M.O.,
1988. Seasonal fluctuations of soil and air borne fungi of Wadi
Bir- El-Ain in Eastern Desert of Egypt. Nat. Monspel. Ser. Bot. 52,
57–70.
Moubasher, A.H., Abdel-Hafez, S.I.I., Bagy, M.M.K., Abdel-Sater, M.A.,
1990. Halophilic and halotolerant fungi in cultivated, desert and salt
marsh soils from Egypt. Acta Mycologica 27, 65–81.
Mouchacca, J., 1971. Pseudeurotium desertorum sp. nov. Revue de
Mycologie 36, 123–127.
Mouchacca, J., 1973a. Deux Alternaria des sols arides d’Egypte: A. chla-
mydospora sp. nov. et A. phragmospora van Emden. Mycopathol.
Mycol. Appl. 50, 217–225.
Mouchacca, J., 1973b. Les Thielavia des sols arides: espèces nouvelles et
analyse générique. Bulletin de la Société Mycologique de France 89,
295–311.
24 SECTION | I Biology and Biodiversity
Mouchacca, J., 1977. Sur un nouveau Discomycetes Ascobolus egyp-
tiacus Travaux dédiès à G. Viennot-Bourgin. Société Francaise de
Phytopathologoie, Paris.236–267
Mouchacca J., 1982. Etude analytique de la mycoflore de quelques sols
de régions arides de l’Egypte. Thèse de Doctorat d’Etat, Muséum
National d’Histoire Naturelle et Université Pierre et Marie Curie
(Paris VI), 247 pp., 90 tabs., 30 figs.
Mouchaca, J., 1985. Les champignons. In: Balout, D.L., Roubet, C. (Eds.),
La momie de Ramses II Editions Recherches sur les Civilisations,
Paris, pp. 119–152.
Mouchacca, J., 1995. Check-list of novel fungi from the Middle East
described mainly from soil since 1930. Sydowia 47, 240–257.
Mouchacca, J., Joly, P., 1974. Etude de la mycoflore des sols arides de
l’Egypte. I. Le genre Penicillium. Revue d’Ecologie et de Biologie du
Sol 11, 67–88.
Mouchacca, J., Joly, P., 1976. Etude de la mycoflore des sols arides de
l’Egypte. II. Le genre Aspergillus. Revue d’Ecologie et de Biologie
du Sol 13, 293–313.
Mouchacca, J., Nicot, J., 1973. Les Fusariella des sols arides. Revue de
Mycologie 37, 168–182.
Moustafa, A.F., 1975. Osmophilous fungi in the salt marshes of Kuwait.
Can. J. Microbiol. 21, 1573–1580.
Mustafa, A.I., Abdel-Azeem, A.M. Salem, F.M. (2013) Surveying and
exploitation of some taxa for extracellular biosynthesis of silver
nanoparticles. Third International Congress on Fungal Conservation,
Akyaka, Mugla, Turkey, pp. 11–15 November 2013. Abstract book:
44.
Muthomi, J.W., Mureithi, B.K., Chemining’wa, G.N., Gathumbi, J.K.,
Mutitu, E.W., 2012. Aspergillus species and Aflatoxin B1 in soil,
maize grain and flour samples from semi-arid and humid regions of
Kenya. Int. J. AgriSci. 2 (1), 22–34.
Myers, N., Mittermeier, A., Mittermeier, C.G., da Fonseca, A.B., Kent, I.,
2000. Biodiversity hotspots for conservation priorities. Nature 403,
853–858.
Naguib, A.I., Mouchacca, J., 1970–1971. The mycoflora of Egyptian
desert soils. Bulletin de l’Institut d’Egypte 52, 37–61.
Naim, M.S., 1967a. Contribution to the knowledge of soil fungi in Libya.
I. Rhizosphere and soil fungi of Artemisia herba alba in Tripoli.
Mycopath. Mycol. Appl. 31, 296–299.
Naim, M.S., 1967b. Contribution to the knowledge of soil fungi in Libya.
II. Fungus flora under Citrus trees in Libya. Mycopath. Mycol. Appl.
31, 300–304.
Nassar, M.S.M., 1998. Soil mycoflora of Wadi Abu-Subayrah at Aswan
region at Eastern Desert of Egypt. Egypt. J. Bot. 38 (1-2), 21–46.
Nasuno, S., 1971. Polyacrylamide gel disc electrophoresis of alkaline pro-
teinases from Aspergillus species. Agric. Biol. Chem. 35, 1147–1150.
Nasuno, S., 1972a. Differentiation of Aspergillus sojae from Aspergillus
oryzae by polyacrylamide gel disc electrophoresis. J. Gen. Microbiol.
71, 29–33.
Nasuno, S., 1972b. Electrophoretic studies of alkaline proteinases from
strains of Aspergillus flavus group. Agric. Biol. Chem. 36, 684–689.
Nasuno, S., 1974. Further evidence on differentiations of Aspergillus sojae
from Aspergillus oryzae by electrophoretic patterns of cellulase, pec-
tin-lyase, and acid proteinase. Can. J. Microbiol. 20, 413–416.
Nealson, K.H., Garber, E.D., 1967. An electrophoretic survey of esterases,
phosphatases and leucine aminopeptidases in mycelial extracts of
Aspergillus. Mycologia 59, 330–336.
Nemec, T., Jernec, K., Cimerman, A., 1997. Sterols and fatty acids of dif-
ferent Aspergillus species. FEMS Microbiol. Lett. 149, 201–205.
Nielsen, K.F., Snmedsgaard, J., Larsen, T.O., Lund, F., Thrane, U., Frisvad,
J.C., 2004. Chemical identification of fungi – metabolite profiling
and metabolomics. In: Arora, D.K. (Ed.), Fungal Biotechnology in
Agricultural, Food and Environmental Application Marcel Dekker,
New York, NY, pp. 19–35.
Nierman, W.H., Pain, A., Anderson, M.J., Wortman, J.R., Kim, H.S.,
Arroya, J., et al., 2005. Genomic sequence of the pathogenic and
allergenic filamentous fungus Aspergillus fumigatus. Nature 438,
1151–1156.
Nilsson, T., Daniel, G., Kirk, K.T., Obst, J.R., 1989. Chemistry and micros-
copy of wood decay by some higher ascomycetes. Holzforschung 43,
11–18.
Okuda, T., Klich, M.A., Seifert, K.A., et al., 2000. Media and incubation
effect on morphological characteristics of Penicillium and Aspergillus.
In: Samson, R.A., Pitt, J.I. (Eds.), Integration of Modern Taxonomic
Methods for Penicillium and Aspergillus Classification Harwood
Academic Publishers, Amsterdam, pp. 83–99.
Oren, A., 2002. Halophilic Microorganisms and Their Environments.
Kluwer Academic Publishers, Dordrecht.
Ozerskaya, S., Kochkina, G., Ivanushkina, N., Gilichinsky, D.A., 2009.
Fungi in permafrost In: Margesin, R. (Ed.), Permafrost Soils. Soil
Biology, Vol. 16 Springer, pp. 85–95.
Palenicia, E.R., 2012. Eendophytic associations of species in the
Aspergillus section NIGRI with maize (Zea mays) and peanut (Arachis
hypogea) hosts, and their mycotoxins, PhD Thesis. Graduate Faculty
of The University of Georgia, Athens.
Pathan, A.A.K., Bhadra, B., Begum, Z., Shivaji, S., 2009. Diversity of
yeasts from puddles in the vicinity of Midre Lovénbreen glacier, Arctic
and bioprospecting for enzymes and fatty acids. Curr. Microbiol. 60,
307–314.
Paz, Z., Komon-Zelazowska, M., Druzhinina, I.S., et al., 2010. Diversity
and potential antifungal properties of fungi associated with a
Mediterranean sponge. Fungal Divers 42, 17–26.
Pel, H.J., de Winde, J.H., Archer, D.B., Dyer, P.S., Hofmann, G., Schaap,
P.J., et al., 2007. Genome sequencing and analysis of the versatile cell
factory Aspergillus niger CBS 513.88. Nat. Biotechnol. 25, 221–231.
Perrone, G., Mulbe, G., Antonia, S., et al., 2006. Ochratoxin A production
and amplified fragment length polymorphism analysis of Aspergillus
carbonarius, Aspergillus tubingensis and Aspergillus niger strains
isolated from grapes in Italy. Appl. Environ. Microbiol. 72, 680–685.
Peterson, S.W., 2008. Phylogenetic analyses of Aspergillus species using
DNA sequences from four loci. Mycologia 100, 205–226.
Petrini, O., 1991. Fungal endophytes of tree leaves. In: Fokkema, N.J.,
van den Heuvel, J. (Eds.), Microbial ecology of the leaves Cambridge
University Press, Cambridge, pp. 185–187.
Pettersson, O., Leong, S.-l L., 2011. Fungal Xerophiles (Osmophiles). eLS
John Wiley & Sons Ltd, Chichester.
Pimentel, M., Lembo, A., Chey, W., et al., 2011. Rifaximin therapy for
patients with irritable bowel syndrome without constipation. N. Eng.
J. Med. 364, 22–32.
Piñar, G., Piombino-Mascali, D., Maixner, F., Zink, A., Sterflinger,
K., 2013. Microbial survey of the mummies from the Capuchin
Catacombs of Palermo, Italy: biodeterioration risk and contamination
of the indoor air. FEMS Microbiol. Ecol. 86, 341–356. Available from:
http://dx.doi.org/10.1111/1574-6941.12165.
Piontelli, E., Toro, M., Giusiano, G., Vivar, V., 2002. Distribución altitudi-
nal de hongos queratinófilos, epífitos y endíofitos en suelos desérticos
del norte chileno (II Región, 23°L.S Y 68°L.W). Boletín Micológico
17, 33e49.
 Biodiversity of the Genus Aspergillus in Different Habitats Chapter | 1
Pitt, J.I., 1985. Nomenclatorial and taxonomic problems in the genus
Eurotium. In: Samson, R.A., Pitt, J.I. (Eds.), Integration of Modern
Taxonomic Methods for Penicillium and Aspergillus Classification
Harwood Academic Publishers, Amsterdam, pp. 383–396.
Pitt, J.I., Hocking, A.D., 2009. Fungi and Food Spoilage. Springer, US.
Proksch, P., Ebel, R., Edrada, R., et al., 2008. Sponge-associated fungi
and their bioactive compounds: the Suberites case. Bot. Mar. 51,
209–218.
Qiao, M.F., Ji, N.Y., Liu, X.H., Li, K., Zhu, Q.M., Xue, Q.Z., 2010.
Indoloditerpenes from an algicolous isolate of Aspergillus oryzae.
Bioorg. Med. Chem. Lett. 20, 5677–5680.
Raghukumar, C., et al., 1992. Endolithic fungi from deep sea calcare-
ous substrata: isolation and laboratory studies. In: Desai, B.N. (Ed.),
Oceanography of the Indian Ocean Oxford and IBH, pp. 3–9.
Raghunath, R., Radhakrishna, A., Angayarkanni, J., Palaniswamy,
M., 2012. Production and cytotoxicity studies of lovastatin from
Aspergillus niger PN2 an endophytic fungi isolated from Taxus bac-
cata. Int. J. Appl. Biol. Pharm. Technol. 3 (3), 342–351.
Rank, C., Nielsen, K.F., Larsen, T.O., Varga, J., Samson, R.A., Frisvad,
J.C., 2011. Distribution of sterigmatocystin in filamentous fungi.
Fungal Biol. 115, 406–420.
Raper, K.B., Fennell, D.I., 1965. The Genus Aspergillus. Williams &
Wilkins, Baltimore.
Rath, P.M., 2001. Phenotypic and genotypic characterization of reference
strains of the genus Aspergillus. Mycoses 44, 65–72.
Rayss, T., Borut, S., 1958. Contribution to the knowledge of soil fungi
in Israel. Mycopathol. Mycol. Applicata (Mycopathologia) 10,
142–174.
Reeve, J.N., Christner, B.C., Kvitko, B.H., Mosley-Thompson, E.,
Thompson, L.G. 2002. Life in glacial ice (Abstract). In: Rossi, M.,
Bartolucci, S., Ciaramella, M., Moracci, M., (Eds.), “Extremophiles
2002,” 4th International Congress on Extremophiles. 22–26 September
2002, Naples, Italy. pp. 27.
Research Gate. <http://www.researchgate.net/>(accessed 17.07.15).
Richard, J.L., Plattner, R.D., Mary, J., Liska, S.L., 1999. The occur-
rence of ochratoxin A in dust collected from a problem homehold.
Mycopathologia 146, 99–103.
Rodriguez, R., Redman, R., 2008. More than 400 million years of evolu-
tion and some plants still can’t make it on their own: plant stress toler-
ance via fungal symbiosis. J. Exp. Bot. 59 (5), 1109–1114.
Rodriguez, A., Dougall, T., Dodd, J.C., Clapp, J.P., 2001. The large subu-
nit ribosomal RNA genes of Entrophospora infrequens comprise
sequences related to two different glomelean families. New Phytol.
152, 159–167.
Rodrigues, P., Santos, C., Venâncio, A., Lima, N., 2011. Species identifi-
cation of Aspergillus section Flavi isolates from Portuguese almonds
using phenotypic, including MALDI-TOF ICMS, and molecular
approaches. J. Appl. Microbiol. 111, 877–892.
Rodriguez, R.J., White Jr, J.F., Arnold, A.E., Redman, R.S., 2009. Fungal
endophytes: diversity and functional roles. New Phytol. 182, 314–330.
Roussos, S., Zaoula, N., Salih, G., et al., 2006. Characterization of
filamentous fungi isolated from Moroccan olive and olive cake: toxi-
genic potential of Aspergillus strains. Molec. Nutr. Food Res. 50,
500–506.
Ruisi, S., Barreca, D., Selbmann, L., Zucconi, L., Onofri, S., 2007. Fungi
in Antarctica. Rev. Environ. Sci. Biotechnol. 6, 127–141.
Sage, L., Krivobok, S., Delbos, E., et al., 2002. Fungal flora and ochratoxin
A production in grapes and musts from France. J. Agric. Food Chem.
50, 1306–1311.
25
Sage, L., Garon, D., Seigle-Murandi, F., 2004. Fungal microflora and
ochratoxin. A risk in French vineyards. J. Agric. Food. Chem. 52,
5764–5768.
Saito, M., Kusumoto, K., Kawasumi, T., 1991. A simple method for identi-
fication of Aspergillus flavus and Aspergillus parasiticus by slab poly-
acrylamide gel electrophorosesis of alkaline proteinases. Rep. Natl.
Food. Res., Inst. 55, 49–51.
Salama, A.M., Elbatanoni, K., Ali, M.I., 1971. Studies on the fungal flora
of Egyptian soils. I. Western Mediterranean coast and Libyan Desert.
United Arab Republic J. Bot. 14 (1), 99–114.
Salem, F.M., Abdel-Azeem, A.M., 2014. Screening of anticancer metab-
olites produced by endophytic fungi. LAP LAMBERT Academic
Publishing., ISBN 978-3-659-53697-7.
Salonen, J.H., Richardson, M.D., Gallacher, K., Issakainen, J., Helenius,
H., Lehtonen, O.P., et al., 2000. Fungal colonization of haematological
patients receiving cytotoxic chemotherapy: emergence of azole-resist-
ant Saccharomyces cerevisiae. J. Hosp. Infect. 45, 293–301.
Samaniego-Gaxiola, J.A., Chew-Madinaveitia, Y., 2007. Diversidad de
géneros de hongos en suelo en tres campos con diferente condiciones
agrícola en la Laguna, México. Revista Mexicana de Biodiversidad
78, 383e390.
Samson, R., Gams, W., 1985. Typification of the species of Aspergillus and
associated teleomorphs. In: Samson, R.A., Pitt, J.I. (Eds.), Advances
in Penicillium and Aspergillus Systematics Plenum Press, New York,
NY, pp. 31–54.
Samson, R., Pitt, J.I. (Eds.), 2000. Integration of Modern Taxonomic
Methods for Penicillium and Aspergillus Classification Harwood
Academic Publishers, Reading, United Kingdom. 510 p.
Samson, R.A., Mouchacca, J., 1974. Some interesting species of
Emericella and Aspergillus from Egyptian desert soil. Antonie van
Leeuwenhoek 40, 121–131.
Samson, R.A., Mouchacca, J., 1975. Additional notes on species of
Aspergillus, Eurotium and Emericella from Egyptian desert soil.
Antonie van Leeuwenhoek 41, 343–351.
Samson, R.A., Houbraken, J.A.M.P., Kuijpers, A.F.A., Frank, J.M.,
Frisvad, J.C., 2004. New ochratoxin A or sclerotium producing
species of Aspergillus section Nigri. Studies in Mycology 50,
45–61.
Samson, R.A., Varga, J., Witiak, S.M., et al., 2007. The species concept in
Aspergillus: recommendations of an international panel. Stud. Mycol.
59, 71–73.
Samson, R.A., Houbraken, J., Thrane, U., et al., 2010. Food and Indoor
Fungi. CBS KNAW Biodiversity Center, Utrecht.
Samson, R.A., Visagie, C.M., Houbraken, J., Hong, S.-B., Hubka, V.,
et al., 2014. Phylogeny, identification and nomenclature of the genus
Aspergillus. Stud. Mycol. 78, 141–173.
Saric, L.C., Skrinjar, M.M., 2008. Share of aflatoxigenic molds from gen-
era Aspergillus and Penicillium in mycopopulations isolated from
spices for meat processing industry. Matica Srpska Proceedings for
Natural Sciences 114, 115–122.
Säwström, C., Mumford, P., Marshall, W., Hodson, A., Laybourn-Parry,
J., 2002. The microbial communities and primary productivity of
cryoconite holes in an Arctic glacier (Svalbard 79°N). Polar Biol. 25,
591–596.
Schoch, C.L., Seifert, K.A., Huhndorf, S., Robert, V., Spouge, J.L.,
Levesque, C.A., et al., 2012. Fungal Barcoding Consortium; Fungal
Barcoding Consortium Author List. Nuclear ribosomal internal tran-
scribed spacer (ITS) region as a universal DNA barcode marker for
Fungi. Proc. Natl. Acad. Sci. USA 109 (16), 6241–6246.
26 SECTION | I Biology and Biodiversity
Schulz, B., Boyle, C., Drager, S., et al., 2002. Endophytic fungi; a source
of novel biologically active secondary metabolites. Mycol. Res. 106,
996–1004.
Schuster, G.S., 1999. Oral flora and pathogenic organisms. Infect. Dis.
Clin. North Am. 13, 757–774.
Scudamore, K.A., Atkin, P.M., Buckle, A.E., 1986. Natural occurrence
of the naphtoquinone mycotoxins, xanthomegnin, viomellein and
vioxanthin in cereals and animal feedstuffs. J. Stored Prod. Res. 22,
81–84.
Seed, P.C., 2015. The human mycobiome. Cold Spring Harb. Perspect.
Med. Available from: http://dx.doi.org/10.1101/cshperspect.a019810.
Seifert, K.A., Lévesque, C.A., 2004. Phylogeny and molecular diagnosis
of mycotoxigenic fungi. Eur. J. Plant Pathol. 110, 449–471.
Semeniuk, G., Harshfield, G.S., Carlson, C.W., et al., 1971. Mycotoxins in
Aspergillus. Mycopath. Mycol. Appl 43, 137–152.
Serra, R., Abrunhosa, L., Kozakiewiez, Z., Venâncio, A., 2003. Black
Aspergillus species as ochratoxin A producers in Portuguese wine
grapes. Int. J. Food Microbiol. 88, 63–68.
Shearer, C., Descals, E., Kohlmeyer, B., Kohlmeyer, J., Marvanová, L.,
Padgett, D., et al., 2007. Fungal biodiversity in aquatic habitats.
Biodivers. Conserv 16 (1), 49–67.
Shehu, K., Bello, M.T., 2011. Effect of environmental factors on the
growth of Aspergillus species associated with stored millet grains in
Sokoto. Nigerian J. Basic Appl. Sci. 19 (2), 218–223.
Shelton, B.G., Kirkland, K.H., Flanders, W.D., Morris, G.K., 2002. Profiles
of airborne fungi in buildings and outdoor environments in the United
States. Appl. Environ. Microbiol. 68 (4), 1743–1753.
Silva, M.R.O., Almeida, A.C., Arruda, F.V.F., Gusmão, N., 2011.
Endophytic fungi from brazilian mangrove plant Laguncularia rac-
emosa (L.) Gaertn. (Combretaceae): their antimicrobial potential.
In: Méndez-Vilas, A. (Ed.), Science Against Microbial Pathogens:
Communicating Current Research And Technological Advances
Formatex, Badajoz, pp. 1260–1266.
Silva, T.L., Sousa, E., Pereira, P.T., Ferrão, A.M., Roseiro, C., 1998.
Cellular fatty acid profiles for the differentiation of Penicillium spe-
cies. FEMS Microbiol. Lett. 164, 303–310.
Šimonovičová, A., Lucia Kraková, L., Pangallo, D., Majorošová, M.,
Piecková, E., Bodoriková, S., et al., 2015. Fungi on mummified
human remains and in the indoor air in the Kuffner family crypt in
Sládkovičovo (Slovakia). Int. Biodeter. Biodegrad. 99, 157–164.
Singh, S.M., Singh, S.K., Yadav, L.S., Singh, P.N., Ravindra, R., 2012a.
Filamentous soil fungi from Ny-Alesund, Spitsbergen, and screening
for extracellular enzymes. Arctic 65, 45–55.
Singh, P., Raghukumar, C., Meea, R.M., Verma, P., Shiuche, Y., 2012b.
Fungal diversity in deep-sea sediments revealed by culture-dependent
and culture independent approaches. Fungal Ecol. 5, 543–553.
Sivakumar, T., Ravikumar, M., Sivakumar, N., 2006. Abundance of
mangrove fungi along the east coast of Tamil Nadu, India. Asian J.
Microbiol. Biotech. Env. Sci. 18 (3), 589–594.
Sizova, T.P., Baghdadi, V.Kh, Gorlenko, M.V., 1967. Mycoflora
of mukhafez of Damascus and Es-Suveida (Syria). Mikologia
Fitopatdogii 1, 286–293.
Sommer, N.F., Buchanan, J.R., Fortlage, R.J., 1976. Aflatoxin and sterig-
matocystin contamination of pistachio nuts in orchards. Appl. Environ.
Microbiol. 32, 64–67.
Spalding, M., Blasco, F., Field, C., 1997. World Mangrove Atlas. The
International Society for Mangrove Ecosystems, Okinawa, 178 pp.
Spiering, M.J., Greer, D.H., Schmid, J., 2006. Effects of the fungal endo-
phyte, Neotyphodium lolii, on net photosynthesis and growth rates of
perennial ryegrass (Lolium perenne) are independent of in plant endo-
phyte concentration. Ann. Bot. 98 (2), 379–387.
Stack, M.E., Mislivec, P.B., 1978. Production of xanthomegnin and viomel-
lein by isolates of Aspergillus ochraceus, Penicillium cyclopium and
Penicillium viridicatum. Appl. Environ. Microbiol. 36, 552–554.
Stahl, P.D., Klug, M.J., 1996. Characterization and differentiation of
filamentous fungi based on fatty acid composition. Appl. Environ.
Microbiol. 62, 4136–4146.
Steiman, R., Guiraud, P., Sage, L., Siegle-Murandi, F., Lafond, J.L., 1995.
Mycoflora of soil around the Dead Sea I—Ascomycetes (including
Aspergillus and Penicillium), Basidiomycetes, Zygomycetes. Syst.
Appl. Microbiol. 18, 310–317.
Strobel, G., Daisy, B., 2003. Bioprospecting for microbial endophytes and
their natural products. Microbiol. Mol. Biol. Rev. 67 (04), 491–502.
Strobel, G.A., Knighton, B., Ren, Y., et al., 2008. The production of
mycodiesel hydrocarbons and their derivatives by the endophytic
fungus Gliocladium roseum (NRRL 50072). Microbiology 154 (11),
3319–3328.
Sugiyama, J., Yamatoya, T., 1990. Electrophoretic comparison of enzymes
as a chemotaxonomic aid among Aspergillus taxa (1). Aspergillus
sects. Ornati and Cremei. In: Samson, R.A., Pitt, J.I. (Eds.), Modern
Concepts of Penicillium and Aspergillus Classification Plenum Press,
New York and London, pp. 385–394.
Sugiyama, J., Rahayu, E.S., Chang, J., Oyaizu, H., 1991. Chemotaxonomy
of Aspergillus and associated teleomorphs. Jpn. J. Med. Mycol. 32
(suppl 2), 39–60. 31.
Suryanarayanan, T.S., 2012. Fungal endosymbionts of seaweeds. In:
Raghukumar, C. (Ed.), Biology of Marine Fungi Springer, Berlin, pp.
53–69.
Suryanarayanan, T.S., Thirunavukkarasu, N., Hariharan, G.N., Balaji, P.,
2005. Occurrence of non-obligate microfungi inside lichen thalli.
Sydowia 57, 120–130.
Suryanarayanan, T.S., Venkatachalam, A., Thirunavukkarasu, N., et al.,
2010. Internal mycobiota of marine macroalgae from the Tamilnadu
coast: distribution, diversity and biotechnological potential. Bot. Mar.
53, 457–468.
Tang, Y., Lian, B., Dong, H., Liu, D., Hou, W., 2012. Endolithic bacte-
rial communities in dolomite and limestone rocks from the Nanjiang
Canyon in Guizhou Karst area (China). Geomicrobiol. J. 29, 213–225.
Tariq, M., Dawar, S., Mehdi, F.S., 2008. Studies on the rhizosphere myco-
flora of mangroves. Turkish J. Bot. 32, 97–101.
Taylor, T.N., Krings, M., Taylor, E.L., 2015. Fungal diversity in the fossil
record. In: McLaughlin, D.J., Spatafora, J.W. (Eds.), The Mycota, vol.
VII part B, 2nd ed. Systematics and Evolution Springer Verlag, Berlin,
Heidelberg, pp. 259–278.
Tedersoo, L., Bahram, M., Põlme, S., Kõljalg, U., Yorou, N.S., Wijesundera,
R., et al., 2014. Fungal biogeography. Global diversity and geography
of soil fungi. Science 346 (6213), 1256688. Available from: http://
dx.doi.org/10.1126/science.1256688.
Thirunavukkarasu, N., Suryanarayanan, T.S., Girivasan, K.P.,
Venkatachalam, A., Geetha, V., Ravishankar, J.P., et al., 2012. Fungal
symbionts of marine sponges from Rameswaram, southern India: spe-
cies composition and bioactive metabolites. Fungal Divers. 55, 37–46.
Thom, C., Church, M.B., 1926. The Aspergilli. Williams & Wilkins,
Baltimore, MD.
Thom, C., Raper, K.B., 1945. Manual of the Aspergilli. Williams &
Wilkins, Baltimore, MD.
Thomas, G.M., Poinar Jr, G.O., 1988. A fossil Aspergillus from Eocene
Dominican amber. J. Paleontol. 62, 141–143.
 Biodiversity of the Genus Aspergillus in Different Habitats Chapter | 1
Tolba, M.K., Al-Doory, Y., Al-Wahhab, M.H. 1957. On the fungal flora
of Iraqi soils 1. Baghdad area. Pro. 3rd Arab Sci. Cong., Beirut, 198–214.
Tomita, T., 2003. Amylin in pancreatic islets and pancreatic endocrine neo-
plasms. Pathol. Int. 53 (9), 591–595.
Tresner, H.D., Hayes, J.A., 1971. Sodium chloride tolerance of terrestrial
fungi. Appl. Microbiol. 22 (2), 210–213.
Tripathi, M., Joshi, Y., 2015. Endolichenic fungi in Kumaun Himalaya: a
case study. In: Upreti, D.K. (Eds.), Recent Advances in Lichenology.
Springer, India, pp. 111–120.
Tripathi, M., Gupta, R.C., Joshi, Y., 2014a. Spegazzinia tessarthra iso-
lated as a true endophyte from lichen Heterodermia flabellata. Indian
Phytopathol. 67 (1), 109–110.
Tripathi, M., Gupta, R.C., Joshi, Y., 2014b. Physcia dilatata Nyl.
(lichenized fungi, Physciaceae); a new host of Bipolaris australiensis
(M.B. Ellis) Tsuda and Ueyama from Kumaun Himalaya, India. Proc.
Nat. Acad. Sci. Lett. 37 (5), 477–479.
Tripathi, M., Joshi, Y., Gupta, R.C., 2014c. Assessment of endolichenic
fungal diversity in some forests of Kumaun Himalaya. Curr. Sci. 107
(5), 745–748.
Trüper, H.G., Galinski, E.A., 1986. Concentrated brines as habitats for
microorganisms. Experientia 42 (11–12), 1182–1187.
Turner, W.B., Aldridge, D.C., 1983. Fungal Metabolites II. Academic
Press, London.
Underhill, D.M., Iliev, I.D., 2014. The mycobiota: interactions between
commensal fungi and the host immune system. Nat. Rev. Immunol.
14, 405–416. Available from: http://dx.doi.org/10.1038/nri3684.
Urairuj, C., Khanongnuch, C., Lumyong, S., 2003. Ligninolytic
enzymes from tropical endophytic Xylariaceae. Fungal Divers. 13,
209–219.
van Woerden, H.C., Gregory, C., Brown, R., Marchesi, J.R., Hoogendoorn,
B., Matthews, I.P., 2013. Differences in fungi present in induced spu-
tum samples from asthma patients and non-atopic controls: a commu-
nity based case control study. BMC Infect. Dis. 13, 69. Available from:
http://dx.doi.org/10.1186/1471-2334-13-69.
Varga, J., Samson, R.A. (Eds.), 2008. Aspergillus in the Genomic Era
Wageningen Academic Pubs., Wageningen, Gelderland.
Varga, J., Kevei, E., Rinyu, E., Teren, J., Kozakiewicz, Z., 1996. Ochratoxin
production by Aspergillus species. Appl. Environ. Microbiol. 62,
4461–4464.
Varga, J., Juhász, A., Kevei, F., Kozakiewicz, Z., 2004. Molecular diversity
of agriculturally important Aspergillus species. Eur. J. Plant Pathol.
110, 627–640.
Varga, J., Tóth, B., Kocsubé, S., Farkas, B., Szakács, G., Téren, J., et al.,
2005. Evolutionary relationships among Aspergillus terreus isolates
and their relatives. Antonie Van Leeuwenhoek 88, 141–150.
Varga, J., Frisvad, J.C., Samson, R.A., 2007a. Polyphasic taxonomy of
Aspergillus section Candidi based on molecular, morphological and
physiological data. Stud. Mycol. 59, 75–88.
Varga, J., Due, M., Frisvad, J.C., Samson, R.A., 2007b. Taxonomic revi-
sion of Aspergillus section Clavati based on molecular, morphological
and physiological data. Stud. Mycol. 59, 89–106.
Varga, J., Frisvad, J.C., Samson, R.A., 2009. A reappraisal of fungi produc-
ing aflatoxin. World Mycotoxin J. 2, 263–277.
Varga, J., Frisvad, J.C., Samson, R.A., 2011. Two new aflatoxin producing
species, and an overview of Aspergillus section Flavi. Stud. Mycol.
69, 57–80.
Varoglu, M., Crews, P., 2000. Biosynthetically diverse compounds from
a saltwater culture of sponge-derived Aspergillus niger. J. Nat. Prod.
63, 41–43.
27
Vega, F.E., Posada, F., Aime, M.C., et al., 2008. Entomopathogenic fungal
endophytes. Biol. Control 46, 72–82.
Verma, A., Johri, B.N., Prakash, A., 2014. Antagonistic evaluation of
bioactive metabolite from endophytic fungus, Aspergillus flavipes
KF671231. J. Mycol. Article ID 371218, 5 pages. Available from:
http://dx.doi.org/10.1155/2014/371218.
Verma, V.C., Kharwar, R.N., Gange, A.C., 2010. Biosynthesis of anti-
microbial silver nanoparticles by the endophytic fungus Aspergillus
clavatus. Nanomedicine 5 (1), 33–40.
Vesonder, R.F., Lambert, R., Wicklow, D.T., Biehl, M.L., 1988. Eurotium
spp. and echinulin in feed refused by swine. Appl. Environ. Microbiol.
54, 830–831.
Visagie, C.M., Hirooka, Y., Tanney, J.B., et al., 2014. Aspergillus,
Penicillium and Talaromyces isolated from in house dust samples col-
lected around the world. Stud. Mycol. 78, 63–139.
Volz, P.A., Ellanskaya, I.A., Wasser, S.P., Nevo, E., Grishkan, I., 2001.
Soil microfungi of Israel. Biodiversity of Cyanoprocaryotes,
Algae and Fungi of Israel. In: Subramanian, C.V., Wasser, S.P. (Eds.),
A.R.A. Gantner Verlag K.-G., Ruggell. Fifty-two photographic plates.
546 pp.
Watanabe, T., 2002. Pictorial Atlas of Soil and Seed Fungi, Morphologies
of Cultured Fungi and Key to Species, second ed. CRC Press.
Welch, D., 1991. Applications of cellular fatty acid analysis. Clin.
Microbiol. Rev. 4, 422–438.
Wessels, J.G.H., 2005. Fungal physiology Encyclopedia of Life Sciences
(ELS). John Wiley & Sons, Chichester, http://www.els.net. Available
from: http://dx.doi.org/10.1038/npg.els.0004305.
White, T.J., Bruns, T., Lee, S., et al., 1990. Amplification and direct
sequencing of fungal ribosomal RNA genes for phylogenetics. In:
Innis, M.A., Gelfand, D.H., Shinsky, T.J., White, T.J. (Eds.), PCR pro-
tocols: A Guide To Methods and Applications Academic Press Inc,
New York, NY, pp. 315–322.
Wicklow, D.T., Cole, R.J., 1982. Tremorgenic indole metabolites and afla-
toxins in sclerotia of Aspergillus flavus: an evolutionary perspective.
Can. J. Bot. 60, 525–528.
Wiese, J., Ohlendorf, B., Bl·mel, M., Schmaljohann, R., Imhoff, J.F., 2011.
Phylogenetic identification of fungi isolated from the marine sponge
Tethya aurantium and identification of their secondary metabolites.
Mar. Drugs 9, 561–585.
Wildman, H.G., 2003. The rise and fall of natural products screening for
drug discovery. Fungal Divers. 13, 221–231.
Williams, D.W., Lewis, M.A.O., 2006. Isolation and identification of
Candida from the oral cavity. Oral Dis. 6 (1), 3–11.
Yaguchi, T., Someya, A., Udagawa, S., 1996. A reappraisal of intrage-
neric classification of Talaromyces based on the ubiquinone systems.
Mycoscience 37, 55–60.
Yamada, Y., Sugihara, K., van Eijk, G.W., Roeijmans, H.J., de Hoog, G.S.,
1989. Coenzyme Q systems in ascomycetous black yeasts. Antonie
Van Leeuwenhoek 6, 349–356.
Yamatoya, T., Sugiyama, J., Kuraishi, H., 1990. Electrophoretic compar-
ison of enzymes as a chemotaxonomic aid among Aspergillus taxa
(2). Aspergillus sect. Flavi. In: Samson, R.A., Pitt, J.I. (Eds.), Modern
Concepts in Penicillium and Aspergillus Classification Plenum Press,
New York, NY, pp. 395–406.
Youssef, Y.A., 1974. On the fungal flora of Libyan soils. Arch. Microbiol.
99, 167–171.
Yu, Z., Zhang, B., Sun, W., Zhang, F., Li, Z., 2012. Phylogenetically
diverse endozoic fungi in the South China Sea sponges and their
potential in synthesizing bioactive natural products suggested by PKS
28 SECTION | I Biology and Biodiversity
gene and cytotoxic activity analysis. Fungal Divers. Available from:
http://dx.doi.org/10.1007/s13225-012-0192-7.
Zalar, P., Frisvad, J.C., Gunde-Cimerman, N., Varga, J., Samson, R.A.,
2008. Four new species of Emericella from the Mediterranean region
of Europe. Mycologia 100 (5), 779–795.
Zelles, L., 1999. Fatty acid patterns of phospholipids and lipopolysac-
charides in the characterization of microbial communities in soil: a
review. Biol. Fertil. Soils 5, 111–129.
Zhang, X.-y, Tang, G.-l, Xu, X.-y, Nong, X.-h, Qi, S.-H., 2014. Insights
into deep-sea sediment fungal communities from the East Indian
Ocean using targeted environmental sequencing combined with tradi-
tional cultivation. PLoS ONE 9 (10), e109118. Available from: http://
dx.doi.org/10.1371/journal.pone.0109118.
Zhang, Y., Li, X.M., Proksch, P., 2007a. Ergosterimide, a new natu-
ral Diels–Alder adduct of a steroid and maleimide in the fungus
Aspergillus niger. Steroids 72, 723–727.
Zhang, Y., Li, X.M., Wang, C.Y., et al., 2007b. A new naphthoquinon-
eimine derivative from the marine algal-derived endophytic fungus
Aspergillus niger EN-13. Chin. Chem. Lett. 18, 951–953.
Zhang, Y., Wang, S., Li, X.M., Cui, C.M., Feng, C., Wang, B.G., 2007c.
New sphingolipids with a previously unreported 9-methyl-C20- sphin-
gosine moiety from a marine algous endophytic fungus Aspergillus
niger EN-13. Lipids 42, 759–764.
Zhang, Y., Han, T., Ming, Q., Wu, L., Rahman, K., Qin, L., 2012a. Alkaloids
produced by endophytic fungi: a review. Nat. Prod. Commun. 7 (7),
963–968.
Zhang, Y., Li, X.M., Wang, B.G., 2012b. Anthraquinone derivatives pro-
duced by marine-derived fungus Aspergillus versicolor EN-7. Biosci.
Biotechnol. Biochem. 76, 1774–1776.
Zhao, K., Ping, W., Li, Q., Hao, S., Zhao, L., Gao, T.D., 2009. Aspergillus
niger var. taxi, a new species variant of taxol producing fungus iso-
lated from Taxus cuspidata in China. J. Appl. Microbiol., 1364–5072.
Zhou, K., Zhang, X., Zhang, F., Li, Z., 2011. Phylogenetically diverse cul-
tivable fungal community and polyketide synthase (PKS), non-riboso-
mal peptide synthase (NRPS) genes associated with the South China
Sea sponges. Microb. Ecol. 62, 644–654.
Zidan, Y., Handoussa, T., Hosni, H., El Hadidi, N.M.N., 2006. The con-
servation of a wooden Graeco- Roman coffin box, e-Preservation.
Science 3, 27–33.
Zuccaro, A., Summerbell, R.C., Gams, W., Schroers, H.J., Mitchell, J.I.,
2004. A new Acremonium species associated with Fucus spp., and its
affinity with a phylogenetically distinct marine Emericellopsis clade.
Stud. Mycol. 50, 283–297.
Chapter 2
Understanding the Diversity of Aspergillus
by Next-Generation Sequencing
Md. Shamim1,2, M. Kumar2, Ravi Ranjan Kumar2, P. Pandey1, D. Srivastava1, D. Kumar1, N.A. Khan1,
Ranjeet Ranjan Kumar3 and K.N. Singh1
1
N.D. University of Agriculture and Technology, Faizabad, UP, India, 2Bihar Agricultural University, Sabour Bhagalpur, Bihar, India,
3
Division of Biochemistry, Indian Agricultural Research Institute, New Delhi, India
INTRODUCTION
Aspergillus is a varied genus of important fungus with high
economic and social impact on humans and agriculture.
Aspergillus species are found worldwide in a variety of
habitats and they are responsible for spoiling different food
materials, producing mycotoxins, and are frequently reported
as human and animal pathogens (Samson et al., 2007). The
Aspergillus filamentous fungal genus consists of more than
250 saprophytic species (Geiser et al., 2007). Some species
of Aspergillus, such as Aspergillus niger, Aspergillus terreus,
and Aspergillus oryzae, are commonly exploited commer-
cially for the production of several enzymes, pharmaceuticals,
and traditional Asian foods and beverages. There are several
other Aspergillus capable of colonizing and infecting immune-
compromised individuals (Baker and Bennett, 2008). The
filamentous fungal genus Aspergillus contains approximately
250 species and spans over 200 million years of evolutionary
history (Geiser et al., 2007). Several species in the genus can
cause a range of frequently deadly diseases, which are col-
lectively known as aspergillosi (Denning, 1998; Latge, 1999).
The genus Aspergillus represents groups of a very large num-
ber of asexual fungi (Fungi Imperfecti or Deuteromycetes)
that are found in a broad range of habitats. Separation of indi-
vidual species into various groups or sections was originally
based on overlapping morphological or physiological charac-
teristics (Raper and Fennell, 1965; Chang and Ehrlich, 2010).
The genus Aspergillus, whose members include toxin-produc-
ing pathogens (Aspergillus flavus and Aspergillus fumigatus)
and pharmaceutical-producing species (Aspergillus nidulans
and A. terreus), is renowned for prodigious metabolite produc-
tion and serves as the model for natural-product exploration.
Species of the genus Aspergillus are of great economic impor-
tance because of their biochemical properties of producing
enzymes that are used in industry. However, certain species
can produce secondary metabolites, called mycotoxins, which
New and Future Developments in Microbial Biotechnology and Bioengineering. DOI: http://dx.doi.org/10.1016/B978-0-444-63505-1.00002-6
© 2016 Elsevier B.V. All rights reserved.
are highly hazardous to human and animal health. Aspergillus
species are more common in warm climates and several ther-
motolerant species exist (Pitt, 1994; Klich, 2002; Domsch
et al., 2007). The use of nontoxigenic strains for the bio-
logical control of toxigenic ones has already been suggested
(Egel et al., 1994). The nontoxigenic varieties would be arti-
ficially disseminated in nature to compete with the toxigenic
strains, driving them out of their ecological niches (Trail et al.,
1995). Complexes of pathogenic and opportunistic species of
Aspergillus can colonize and induce disease symptoms in vari-
ous plants and plant products, and produce toxic secondary
metabolites (mycotoxins) in the infected tissue.
Population studies of A. flavus centered on its genetic
variability are indeed required. Therefore it will be important
to better understand the genetic diversity within this fungal
group and the critical factors for retention or loss of charac-
teristics such as toxigenic capacity and virulence to plants.
The genus Aspergillus is one of the most important fila-
mentous fungal genera. Aspergillus species are used in the
fermentation industry, but they are also responsible for vari-
ous plant and food secondary rots, with the consequence of
possible accumulation of mycotoxins (Perrone et al., 2007).
The aflatoxin-producing A. flavus and Aspergillus parasiti-
cus, and ochratoxinogenic A. niger, Aspergillus ochraceus,
and Aspergillus carbonarius species are frequently encoun-
tered in agricultural products (Cotty et al., 1994). Studies on
the biodiversity of toxigenic Aspergillus species are useful
to clarify molecular, ecological, and biochemical character-
istics of the different species in relation to their different
adaptation to environmental and geographical conditions,
and to their potential toxigenicity. Aspergillus flavus is the
most common species associated with aflatoxin contamina-
tion of agricultural crops. Development of control strategies
against A. flavus and A. parasiticus, the major aflatoxin-
producing species, is dependent upon a basic understanding
of their diversity in agricultural ecosystems (Cotty, 1997).
29
30 SECTION | I Biology and Biodiversity
Recognizing the growing role of molecular meth-
ods in Aspergillus species identification, an international
Aspergillus working group (Balajee et al., 2007) proposed
the following recommendations: (1) the term “species com-
plex” as an alternative to “section,” (2) use of sequences
from the internal transcribed spacer (ITS) region for identi-
fication of Aspergillus isolates to the species complex level,
and (3) comparative sequence analyses of the β-tubulin
region for species identification within a complex. This
recommendation can be advantageous to clinical laborato-
ries that rely on comparative sequence analyses of the ITS
region (which are not variable enough for species identi-
fication within a section) and/or morphology for species
identification (where overlapping morphologies can hinder
resolution of species within the sections) as they can report
the identification of an unknown organism to species com-
plex, for instance, A. terreus complex. Thus, the term “com-
plex” in such an identification scheme would indicate the
placement of the isolate within a species complex but does
not identify the isolate to a species within the complex.
Aspergillus species are the important fungi representing
an essential functional component of terrestrial ecosystems
as pathogens, and are one of the most diverse groups of the
Eukarya. Studies on these ecological factors which support
the dynamics of Aspergillus communities are a challenge
because of this high taxonomic and ecological diversity.
Generally, polymerase chain reaction (PCR)-based molecu-
lar markers and sequencing of ribosomal DNA have been
successful in characterization and identification of the
abundance of Aspergillus species and have provided insight
into the Aspergillus communities (Midorikawa et al., 2008).
Genetic variations were also detected in a large number of
Aspergillus species representing a broad range of biologi-
cal material and this helped understand genetic mechanisms
of different diseases. Even though an increasing number
of genomes of different fungal species has already been
sequenced, the importance of technical developments in the
field of DNA analysis is clear for the future sequencing of
Aspergillus species. Aspergillus pathogen identification in
different infectious diseases relies mostly on routine cul-
tures and biochemical testing by means of semiautomated
platforms in the plant pathology. Genetic studies in differ-
ent Aspergillus, augmented by analysis of whole-genome
sequences, have revealed that most fungal SM biosynthetic
genes are found in compact clusters functioning as individ-
ual genetic loci (Keller et al., 2005).
There were several reports regarding the sequencing of
16S rRNA studies published in the 1990s which indicated
that this particular genes could be useful for pathogen dis-
covery and identification. The number of DNA sequencing
technologies used is currently high, even though some tech-
niques are more advantageous than others depending on the
application, and, therefore, a general ranking of the technol-
ogies may be incorporated or misleading. The advent of new
high-throughput DNA-sequencing technologies promises
to redefine how fungi and fungal communities, as well as
other groups of organisms, are studied in their natural envi-
ronment. The high technical nature and time-taking process
of Sanger DNA sequencing is shifting sequencing methods
toward widespread adoption of next-generation nucleic acid
sequencing for the identification of microbial pathogens.
The increasing use of molecular markers to identify fungi
and analyze fungal communities in a phylogenetic context
has initiated a boom in fungal ecology and phylogenetics
(Margulies et al., 2005).
In 2005 the first high-throughput sequencing platform
from 454 Life Sciences (Branford, CT, USA) was intro-
duced to the market (Margulies et al., 2005), and 3 years
later the first fungal ecology studies were published based
on this technology (Buee et al., 2009). The 454-sequenc-
ing technique is routinely used both for shotgun sequencing
of genomic DNA/cDNA and in-depth sequencing of PCR
amplicons (Jumpponen and Jones, 2009; Opik et al., 2009).
The simultaneous publication of three Aspergillus genome
manuscripts in Nature in December 2005 established
Aspergillus as the leading filamentous fungal genus for
comparative genomic studies (Galagan et al., 2005; Machida
et al., 2005; Nierman et al., 2005). Like most major genome
projects, these Aspergillus efforts were collaborations
between a large sequencing center and the respective com-
munity of scientists. For example, the Institute for Genome
Research (TIGR) worked with the Aspergillus fumigates
community. Aspergillus nidulans was sequenced at the
Broad Institute. Aspergillus oryzae was sequenced in Japan
at the National Institute of Advanced Industrial Science
and Technology. The Joint Genome Institute (JGI) of the
Department of Energy has released sequence data for a cit-
ric acid-producing strain of A. niger (Baker, 2006). TIGR,
now renamed the Venter Institute, is currently spearheading
a project on the A. flavus genome (Payne et al., 2006, 2007).
Aspergillus genomics was reviewed and the review pro-
vides URLs for major Aspergillus genome projects listing
genes, availability of other resources, links to relevant data
bases, and literature citations (Jones, 2007). Genome sizes
for sequenced species of Aspergillus range from approxi-
mately 29.3 MB for A. fumigatus to 37.1 MB for A. oryzae,
while the numbers of predicted genes vary from approxi-
mately 9926 for A. fumigates to approximately 12,071 for
A. oryzae (Machida et al., 2005; Nierman et al., 2005). The
genome size of an enzyme-producing strain of A. niger is of
intermediate size at 33.9 MB (Pel et al., 2007). Up-to-date
listings of Aspergillus genome projects are available at the
Genomes on Line Database (GOLD) at http://www.genome-
sonline.org/. All Aspergillus genomes sequenced so far have
eight chromosomes, ranging in size from 28 to 40 MB, and
appear to have similar characteristics, although karyotype
analyses suggest that natural populations of several of these
species harbor chromosomal variants (Geiser et al., 1996).
 Understanding the Diversity of Aspergillus by Next-Generation Sequencing Chapter | 2
Another major, and perhaps more complex, question whose
investigation has been dramatically enhanced by the avail-
ability of genomes is whether Aspergillus populations are
genetically differentiated, and the implication of population
structure for their lifestyles. There are several approaches
for the diversity assessments in Aspergillus, which are
discussed in the following sections.
METHODS FOR THE DIVERSITY
ASSESSMENTS OF DIFFERENT
ASPERGILLUS SPECIES
Morphological Characters
Morphology forms an important part of the species con-
cept of Aspergillus. Colony characters used for character-
izing species include colony growth rates, texture, degree of
sporulation, production of sclerotia or cleistothecia, colors
of mycelia, sporulation, soluble pigments, exudates, and
colony reverses, etc. Both sexual and asexual reproduction
occur in Aspergillus and the microscopic features of these
structures are important. Diagnostic conidiophores char-
acters include the shape of conidial heads, the presence or
absence of metulae between vesicle and phialides (ie, unise-
riate or biseriate), color of stipes, and the dimension, shape
and texture of stipes, vesicles, metulae (when present), phi-
alides, conidia, and Hulle cells (when present). The same
applies for cleistothecia, asci, and ascospores. For cleisto-
thecia, the development of ascomata and the way their walls
are produced is also an important character. Ascospore sizes
and morphology, particularly the often diagnostic ornamen-
tation (roughening, rims, wings, furrows, etc.), are impor-
tant for identifying species. Media, inoculation technique,
and incubation conditions affect morphological characters
of Aspergillus (Okuda et al., 2000). The classification of this
section was traditionally based on morphological identifica-
tion (Dalcero et al., 2002; Chulze et al., 2006), which is very
difficult and can lead to misidentification, especially within
the A. niger species aggregate (a group of morphologically
indistinguishable species). This method was then integrated
with physiological characters, that is, extrolite production
data (Frisvad et al., 2007), that often provide similar results
obtained with phylogenetic analyses (Geiser et al., 2000).
Vegetative compatibility group (VCG) was believed to
be a strong barrier to genetic exchange but recent studies
found that VCGs are able to outcross, leading to new VCGs
and thereby increased diversity (Leslie, 1993). Aspergillus
flavus is typically composed of isolates from hundreds of
different VCGs which reflect phenotypic differences (or
similarity) among individuals. Individuals (genotypes) of a
fungal species having the same heterokaryon or vegetative
incompatibility loci can fuse and undergo genetic exchange
through parasexuality (Glass et al., 2000). Fungal isolates
that form stable heterokaryons are considered to belong
31
to the same VCGs. In A. flavus populations, most of the
variations in morphology and mycotoxin production can be
attributed to differences among VCGs (Olarte et al., 2012).
However, molecular techniques have shown that there is a
high biodiversity, and that taxa and taxonomy are difficult
to discuss based only on their morphological features and
characteristics (Murakami, 1979; Al-Musallam, 1980).
Molecular Methods for the Characterization
of Aspergillus Species
Molecular characterization of the Aspergillus species
remains resilient models for studying basic questions
in eukaryotic biology and its diversity. Undoubtedly,
Aspergillus genomics will enlighten fundamental insights
into cell biology as well as have important implications for
agriculture, industry, and medicine. Development and iden-
tification of different molecular markers such as random
amplified polymorphic DNA (RAPD), restriction fragment
length polymorphism (RFLP), amplified fragment length
polymorphism (AFLP), and single nucleotide polymor-
phism (SNP) flourished rapidly. RFLP analysis was used
by Accensi et al. (1999) and Martinez-Culebras and Ramon
(2007) for the detection and characterization of Aspergillus
japonicus from Aspergillus aculeatus and to differentiate
five species, respectively. PCR-RFLP marker was used
for the detection of A. niger from Aspergillus tubingensis
(Parenicova et al., 2001). PCR is the most commonly used
molecular tool with specific primers, that is, AFLP mark-
ers (Schmidt et al., 2004), RAPD sequences (Fungaro et al.,
2004), calmodulin gene (Susca et al., 2007), ITS regions
(Patino et al., 2005), polyketide synthase (PKS) sequence
(Dao et al., 2005; Spadaro et al., 2011), frequently used
over the decades for the characterization of biodiversity
in the different Aspergillus species. Real-time PCR assays
were developed, able to detect A. carbonarius, the main
producer of OTA in grape (Gonzalez-Salgado et al., 2005;
Mule et al., 2006; Atoui et al., 2007; Selma et al., 2008).
Gonzalez-Salgado et al. (2009) developed a very highly
efficient quantitative PCR assay using SYBER Green I and
TaqMan for the detection of A. carbonarius from grapes.
There is also the most common feature to sequence the
PCR-amplified products with the Sanger method and com-
pare the sequence with the help of several bioinformat-
ics tools to detect the diversity status among the different
isolates.
Traditional Sequencing of Aspergillus Species
Sequencing-based molecular techniques provide better res-
olution at the intragenus and above level, while frequency
data from markers such as RAPD, AFLP, and microsatel-
lites provide the means to classify individuals into nominal
genotypic categories and are mostly suitable for intraspecies
32 SECTION | I Biology and Biodiversity
genotypic variation study (Robinson and Harris, 1999).
DNA sequencing is the determination of the order of the
nucleotide bases—A (adenine), G (guanine), C (cytosine),
and T (thymine) present in a target molecule of DNA.
Currently, the dye-terminator sequencing technique is the
standard method in automated sequencing analysis (Olsvik
et al., 1993). The dye-terminator sequencing method, along
with automated high-throughput DNA sequence analyzers,
is now being used for the vast majority of sequencing work.
Dye-terminator sequencing utilizes labeling of the chain
terminator ddNTPs, which allows sequencing in a single
reaction, rather than four reactions as in the previously used
labeled-primer method. In dye-terminator sequencing, the
four dideoxy-nucleotide chain terminators are labeled with
fluorescent dyes, each with a different wavelength of fluo-
rescence emission.
Gene Level
Aspergillus has been the subject of a large number of taxo-
nomic studies using DNA sequence data. Many of these
studies focused on specific groups (species, sections, sub-
genera) within Aspergillus and the number of phylogenetic
studies at the genus level and above are limited. Berbee et al.
(1995) studied the possible monophyly of Penicillium using
ITS and 18S rDNA sequences. In this study, Eupenicillium
javanicum (Penicillium javanicum), Monascus purpureus,
Neosartorya fischeri (Aspergillus fischeri), Eurotium
rubrum (Aspergillus ruber), and A. fumigatus form a
well-supported clade (98% bootstrap value, bs) indicating
the close relationship among these species. Furthermore,
A. ruber, A. fumigatus, and A. fischeri were placed together
on a branch with moderate statistical support (77% bs),
indicating that Aspergillus is monophyletic. Similar results
were found by Ogawa et al. (1997); in their phylogeny based
on 18S rDNA data, E. rubrum, N. fischeri, and A. fumigatus
also formed a well-supported clade (99% bs), distinct from
Penicillium and Monascus. Tamura et al. (2000) determined
the relationships within Aspergillus using 18S rDNA. Using
a larger sample size, data indicate that Aspergillus is mono-
phyletic, but the overall resolution was limited. This spe-
cies diversity was also revealed by sequence analyses of
partial calmodulin (660 bp) and β-tubulin (1360 bp) genes,
which confirmed a significant molecular divergence of
Aspergillus “uniseriate” group from other Aspergillus spe-
cies. The description of a new species named Aspergillus
uvarum isolated only from grape has been recently submit-
ted (Perrone et al. 2008). Sequencing of the ITS regions
and with RFLP analysis of rRNA by Magnani et al. (2005),
β-tubulin gene sequencing is more applicable and proved to
be more efficient for species identification. Peterson (2008)
studied the phylogenetic relationships within Aspergillus.
A phylogeny based on 5.8S rDNA, 28S rDNA, and the RPB2
sequences resolved Aspergillus into three main clades, but
the relationship among these clades was not statistically
supported. These clades roughly corresponded with the sub-
genera of Aspergillus, with one clade including the subgen-
era Circumdati and Fumigati, one representing subgenus
Nidulantes, and another containing a member of subgenus
Aspergillus. Houbraken and Samson (2011) and Houbraken
et al. (2014) characterized diversity in Aspergillus by using
a four-gene phylogeny (RPB1, RPB2, Tsr1, and Cct8) and
were based on similar data sets.
Sequence analyses of ITS, cytochrome oxidase subu-
nit 1 (cox1), β-tubulin, and calmodulin genes have been
widely used for the detection of diversity in Aspergillus
species (Samson et al., 2004; Varga et al., 2007; Perrone
et al., 2011). A new species named Aspergillus ibericus,
from atypical A. carbonarius strains isolated in Spain and
Portugal that do not produce OTA, was identified by using
ITS and calmodulin sequences and AFLP analyses (Serra
et al., 2006). Geiser et al. (2007) and Samson et al. (2007)
used integrating multilocus sequence analysis data with
morphological and physiological characters to delineate
new Aspergillus species.
Whole-Genome Sequencing
The published Aspergillus genome sequences (A. nidulans,
A. fumigatus, A. oryzae) and further sequence data from
A. clavatus, N. fischeri, A. flavus, A. niger, A. parasiticus, and
A. terreus are the first from a group of related filamentous
fungi (Jones, 2007). A number of sequenced Aspergillus
genomes have genomic data which are accessible via a num-
ber of public web resources (AspGD: http://www.aspgd.
org; CADRE: http://www.cadre-genomes.org.uk; Ensembl
Genomes ftp site: ftp://ftp.ensemblgenomes.org/pub/fungi;
FungiDB web resource: http://fungidb.org/fungidb). The
A. nidulans genome was sequenced and annotated in 2005
(Galagan et al., 2005; Machida et al., 2005; Nierman et al.,
2005) and the genome annotation has been updated from
time to time. Despite this wealth of data and manual cura-
tion efforts (Wortman et al., 2009), genome annotations are
largely based upon computational gene model predictions
which may be inaccurate, a particular problem for complex
genes containing multiple introns. The main advantages
of this technique are its robustness, automation, and high
accuracy (>98%). On the other hand, the limitations of this
technique include dye effects due to differences in the incor-
poration of the dye-labeled chain terminators into the DNA
fragment. Such incorporation of dye can result in unequal
peak heights and shapes in the electronic DNA sequence
trace chromatogram after capillary electrophoresis. Another
drawback is its inability to handle long sequences; however,
it can reliably sequence up to approximately 900-nucleo-
tide-long DNA fragments in a single reaction. The advent of
new-generation sequencers with solid-state chemistry has
significantly overcome these problems.
 Understanding the Diversity of Aspergillus by Next-Generation Sequencing Chapter | 2
Next-Generation Sequencing
The development of next-generation sequencing tech-
nologies (NGSTs) has probably had more impact on
our understanding of fungal molecular biology than any
other eukaryotic species group. By 2012, the genomes
of approximately 200 fungal species had been sequenced
(www.fungalgenomes.org) using different sequencing plat-
forms. More recently, RNA-seq, or high-throughput cDNA
sequencing technology, has been increasingly adopted as a
method of obtaining large amounts of transcriptomic data
with high sensitivity, high quality, and at relatively low
cost (Nagalakshmi et al., 2008; Mortazavi et al., 2008). As
new genome sequences became available, the application
of comparative analysis allowed the identification of gene
families with roles in fungal virulence (Butler et al., 2009)
and the identification of potential new lead compounds for
drug discovery (Abadio et al., 2011; Hodkinson and Grice,
2015).
The filamentous fungus A. nidulans is a model organism
for many aspects of molecular cell biology, genetics, and
diversity analysis. Additionally, the Aspergilli themselves
include fungi of biomedical, agricultural, and industrial sig-
nificance (Nierman et al., 2005; Machida et al., 2005; Pel
et al., 2007; Fedorova et al., 2008; Andersen et al., 2011).
Thus it is important to extend our understanding of A. nidu-
lans to facilitate analysis of key processes which underpin
fungal pathogenicity and biotechnological applications.
Next-generation platforms do not rely on Sanger
chemistry (Sanger et al., 1977) as did the first-generation
machines used for the last 30 years (Schuster, 2008). The
first of this kind of second-generation of sequencing tech-
nique appeared in 2005 with the landmark publication of the
sequencing-by-synthesis technology developed by 454 Life
Sciences (Margulies et al., 2005) based on pyrosequencing
(Ronaghi et al., 2006; Nyren, 2007). Commercial second-
generation sequencing methods are generally distinguished
by the role of PCR in library preparation for the sequenc-
ing procedures. There are four main platforms; all being
amplification-based: (1) Roche 454 GS FLX, (2) Illumina
Genome Analyzer IIx, (3) ABI SOLiD 3 Plus System, and
(4) Polonator G.007 (Lerner and Fleischer, 2010).
Pyrosequencing is faster, approximately 20–30 times
less expensive than Sanger sequencing, and does not require
cloning. The 454 pyrosequencing technique has recently
been used to characterize fungal diversity (Lim et al. 2010).
Furthermore, Lim et al. (2010) assessed pyrosequencing of
forest soils to reveal unexpectedly high fungal diversity in
the soil fungal communities. A single-molecule sequencing
method (also known as third generation) is independent of
PCR (Blow, 2008). A single-molecule sequencing mode
of sequencing protocol was recently developed by Helicos
Genetic Analysis System using the technology developed
by Braslavsky et al. (2003). Oxford Nanopore Technology
33
(www.nanoporetech.com) has been developing a label-free,
electrical, single-molecule genuinely revolutionary DNA
sequencing method. This technique is aimed at obviating
the need for amplification or labeling by instead detecting
a direct electrical signal (Clarke et al., 2009). However, this
technique is still in the development stage. The recently
developed Helicos third-generation high-throughput and
low-cost direct single-molecule RNA sequencing method
without requiring prior conversion of RNA to cDNA opened
the door for a comprehensive and bias-free understanding
of transcriptomes (Ozsolak and Milos, 2011). A different
sequencing plate form used in the next-generation sequenc-
ing (NGS) is discussed in following subheading and com-
parative analysis of platform mentioned in Table 2.1.
Roche 454 FLX Pyrosequencer
The main principle of the Roche 454 FLX pyrosequencer
is detection of pyrophosphate (PPi) that is released during
DNA synthesis. The generation of visible light intensity
is proportional to the number of nucleotides incorporated.
The FLX instrument applies 100 flows for each nucleotide.
The resulting reads of the GS FLX Titanium XL+ yield up
to 700 MB of data. The read length provided by the latest
454 platform can reach 1000 base pairs (bp). This technique
has higher output compared to traditional cloning sequenc-
ing approaches, pyrosequencing allows the detection of
rare bacterial and archaea genera. Recently, this method
has been extensively used to characterize composition and
diversity of soil microbial communities (Eilers et al., 2012)
and different fungal communities.
Illumina Genome Analyzer
This platform is based on the parallel, fluorescence-based
readout of millions of immobilized libraries that are
sequenced using reversible terminator chemistry. Recently,
Illumina offered four sequencers: HiSeq 2000, HiSeq 2500,
MiSeq platform Genome, and Analyzer IIx. The HiSeq 2500
sequencer is the most powerful sequencer among them,
delivering up to 600 GB of data with a maximum of 6 billion
reads per run and read length of approximately 2 × 100 bp.
Illumina sequencers have shorter reads compared to the
Roche 454 FLX pyrosequencer, but a much higher through-
put, which makes them very appropriate for gene expres-
sion studies of complex environments such as soils. This
technique has allowed the characterization of a number of
microbial communities (Fierer et al., 2012) and established
a low-cost access to DNA from organisms with low relative
abundances (Bartram et al., 2011). The main disadvantage
of this platform is that the length of reads is shorter, which
makes the taxonomic assignment less accurate, although
the output is much higher than pyrosequencing. Still, a few
studies have used this method to measure diversity in micro-
bial communities. Recently, Madsen et al. (2015) reported
TABLE 2.1 Summary of the Five Major Next-Generation Sequencing Platform Families
Platform Clonal Amplification Chemistry Highest Average Run Time Error Output Cost Aspergillus NGS Platform
Family Read Length Rate Per Run Reference Reference
454 Emulsion PCR Pyrosequencing 700 bp (paired- 23 h Low 0.7 GB Low Aspergillus oryzae Loman et al.
(seq-by-synthesis) end sequencing De Gannes et al. (2012)
available) (2013) Mardis (2008)
Illumina Bridge amplification Reversible dye 300 bp (overlapping 3–11 days Low 120–600 GB Low Aspergillus alliaceus Loman et al.
terminator (seq- paired-end Haynes et al. (2012) (2012)
by-synthesis) sequencing Aspergillus sp. MF297-2,
available) Li et al. (2012)
SOLiD Emulsion PCR Oligonucleotide 75 bp (paired- Up to 8 days High 150 GB Moderate A. oryzae RIB40, Shokralla et al.
8-mer chained end sequencing Umemura et al. (2013) (2012)Magi
ligation (seq-by- available) et al. (2010)
ligation)
Ion Torrent/ Emulsion PCR Proton detection 400 bp (bidirectional 3h Medium From High Aspergillus carbonarius Loman et al.
PGM Semiconductor (seq-by-synthesis) sequencing 20–400 MB (36 Mbp) Cabanes (2012); Egan
sequencing technology available) et al. (2015) et al. (2012)
SMRT N/A (single molecule) Phospholinked 8500 bp 2h High 230 GB Low A. fumigatus Schadt et al.
fluorescent Losada et al. (2013) (2010)
nucleotides (seq-
by-synthesis)
 Understanding the Diversity of Aspergillus by Next-Generation Sequencing Chapter | 2
fungal diversity by NGS of different samples and found the
highest numbers of reads for the problematic dust and the
workers belonging to A. fumigatus and the genera Rhizopus,
Mucor, and Lichtheimia.
Applied Biosystem Sequencing by Oligonucleotide
Ligation and Detection (SOLiD) Sequencer
Similarly to the 454 platform, the Sequencing by
Oligonucleotide Ligation and Detection (SOLiD) sys-
tem uses emulsion PCR to produce clone libraries. In this
sequencing method initially, complementary sequence
hybridizes with a fluorescently labeled probe then DNA
ligase is added to join the dye-labeled probe to the primer.
Ligated probes are identify by fluorescence imaging.
Billions of short sequence reads (2 × 60 bp) are generated
by this platform at once (120 GB of data). In a recently
study, SOLiD was used to sequence the genome of the
Pectobacterium sp. strain SCC3193, which is known for
causing soft rot and blackleg disease in different plants.
This platform is used to overcome homopolymer and
assembly errors that are generated by 454 sequencing. The
main demerit of this platform is the difficult assemblage of
short reads, which also applies for the Illumina platform.
However, its two-base sequencing technology provides
the highest accuracy of all platforms. It is widely used for
transcriptomics and epigenomics. Recently, Umemura et al.
(2013) performed de novo assembly of the A. oryzae RIB40
genome using only SOLiD read data of 50 bp generated
from mate-paired libraries with 2.8- or 1.9-kb insert sizes.
The assembled scaffolds showed an N50 value of 1.6 MB, a
22-fold increase compared with those obtained using only
SOLiD short read in other published reports.
Ion Personal Genome Machine (PGM)
The chemistry of this platform is that it uses semiconductor
sequencing technology, each time a proton is released after
incorporation of a nucleotide into the DNA, and the sub-
sequent change in pH is measured by a pH-sensitive field
effect transistor. The advantages of this platform are that
no labeled nucleotides are used and synthesis is detected
directly, and it offers shorter run times when compared
to systems based on fluorescence detection. Currently, it
delivers 400 bp reads in 4 h. Recently, the Personal Genome
Machine (PGM) platform was proposed to assess bacterial
and archaeal community dynamics (Whiteley et al., 2012).
Further, with the help of Ion Torrent sequencing technology,
a useful technology in whole-genome sequencing of bacte-
rial genomes (5 Mbp) was used for the sequencing of the
whole fungal genome of A. carbonarius (36 Mbp), a nono-
chratoxin A-producing strain, using the reference genome
Acv3 of a toxigenic A. carbonarius strain. The study also
reported the detection of the OTA production in the A. car-
bonarius strains, single nucleotide and deletion–insertion
35
polymorphisms, copy number variation, and PKS and
NRPS encoding gene analyses of the Aspergillus (Losada
et al., 2013). NGSTs have been used for the improvement of
the genome annotation procedures by RNAseq under a wide
range of conditions and to characterize diversity in A. fumig-
atus at the genomic level by sequencing almost 50 different
isolates. There is a findings from the NIAID-funded project
and reported on different gene content and gene expression
variations, SNP analysis, and gene model improvements in
A. fumigatus. Cabanes et al. (2015) sequenced the whole
fungal genome (36 bp) of A. carbonarius, a nonochratoxin
A-producing strain and found that the atoxigenic strain has
a high accumulation of nonsense and missense mutations in
several genes.
Heliscope Single-Molecule Sequencer
The main innovation of this platform is the direct sequenc-
ing of DNA/RNA fragments, therefore no amplification
is needed. It involves fragmenting the template DNA and
hybridizing on disposable glass flow cells. Each of the 25
channels on one standard flow cell can be addressed indi-
vidually for the addition of samples. Once the flow cells
have been prepared, they are inserted in the HeliScope
Sequencing system along with all the reagents necessary for
sequencing by synthesis and imaging. This system can gen-
erate billions of reads per run that range from 25 to 35 bp,
and the data output is over 1 GB per day. Heliscope might
contribute to genome biology through direct sequencing of
nucleic acids. For example, Kapranov et al. (2012) obtained
sequence information for counting the abundance of short
RNA (sRNAs) and discovery of new sRNAs through the
HeliScope sequencer in cultured cells. This technique
counts with a high error rate that goes between 3% and 4%.
Compared to other methods it has a higher cost than other
platforms and is still not popular in the NGS market.
RNA Seq (NGST) Obtaining and making sense out of ITS
sequences generated by NGS implies several steps, both
at the lab bench and at the computer desk. DNA has to
be extracted from the cells and the ITS region amplified
by PCR (a process that generates many copies of a target
region) for each sample separately. This step adds specific
barcodes to the different samples. This is important later to
distinguish which species came from each sample. The PCR
products from each sample are then cleaned, combined, and
read by an NGS machine. The result is a big computer file
with hundreds of thousands to millions of sequences. The
sequences are then separated by sample (using the barcode
mentioned above) and compared to a sequence database
with the goal of identifying the detected fungi.
NGSTs have been used to sequence the genomes of
some of the species, such as Aspergillus kawachii (Futagami
et al., 2011) and Aspergillus sojae (Sato et al., 2011). NGSTs
36 SECTION | I Biology and Biodiversity
have also been used to sequence the genomes of additional
isolates from already-sequenced species (Gibbons et al.,
2012). NGST applications, such as RNA-Seq (Rokas et al.,
2012), have been employed to characterize the structure
and variation of the Aspergillus transcriptome (Wang et al.,
2010a,b; Yu et al., 2011; Delmas et al., 2012). Gibbons et al.
(2012) sequenced the genomes of seven A. oryzae and seven
A. flavus isolates as well as three of the transcriptomes of
each species. Gibbons et al. (2012) identified the candidate
gene of the Aspergillus by RNA-Seq and identified different
genes. Primarily, sequencing and mapping of up to 10 mil-
lion sequence reads against the A. fumigatus transcriptome
identified 3728 differentially regulated genes in the two
conditions (biofilm [BF] growth and liquid plankton [PL]
growth). Many of these genes, like transcription factors,
stress response, the ribosome, and the translation machin-
ery, likely reflecting the different growth demands in the two
conditions, were identified. The experiment also identified
hundreds of candidate genes for the observed differences
in morphology and pathobiology. Furthermore, upregu-
lated genes showed significant spatial structure across the
A. fumigatus genome. The results also add valuable insight
into the genetics of biofilm formation in A. fumigatus and
other filamentous fungi.
Yu et al. (2011) used RNA-seq to determine the tran-
scriptional response of 55 secondary metabolite clusters
in A. flavus to changes in temperature. Expression of 11
clusters, including the aflatoxin cluster, is induced at 30°C
relative to 37°C. It was further reported that most genes
in the aflatoxin cluster are expressed at very low levels at
the higher temperature. Transcriptome studies in the same
species also reveal an abundance of small transcripts;
although some of these transcripts have significant similar-
ity to sequences from other species, most of them appear
to be uniquely present in A. fumigatus (Rokas et al., 2012).
Similarly, sequencing of noncoding transcripts shorter than
500 base pairs in A. fumigates identified a few dozen non-
coding RNAs, several of which appear to also be develop-
mentally regulated (Jochl et al., 2008). In the last few years,
several other advantages have been aided by RNA-Seq
and high-throughput proteomics approaches (Doyle, 2011;
Rokas et al., 2012), we predict that the identification and
functional characterization of these small transcripts and
proteins will become an indispensable part of the study of
Aspergillus development and pathogenicity (Rokas et al.,
2012). Muller et al. (2012) studied the comparison of the
transcriptome and proteome of an A. fumigatus wildtype
strain with an mpkA-null mutant strain revealed that 70.4%
of the genome was found to be expressed and that MpkA
plays a significant role in the regulation of many genes
involved in cell wall remodeling, oxidative stress, and iron
starvation response, and secondary metabolite biosynthesis.
Moreover, absence of the mpkA gene also strongly affects
the expression of genes involved in primary metabolism.
The data were further processed to evaluate the potential
of the mRNA-Seq technique. We comprehensively matched
up our data to published transcriptome studies and were
able to show improved data comparability of mRNA-Seq
experiments independently of the technique used. Analysis
of transcriptome and proteome data revealed only a weak
correlation between mRNA and protein abundance. Jain
et al. (2011) also studied the comparison of transcript levels
in both the wildtype strain and the ΔmpkA mutant strain by
repeating the experiment. The result suggests that the RNA
deep-sequencing technique is more sensitive in finding dif-
ferentially expressed genes than the microarray technique.
This might be explained by the greater dynamic range cov-
ered by the RNA technique (Wang et al., 2009; Feng et al.,
2010). Sibthorp et al. (2013) carried out sequencing of both
the whole transcriptome and transcript 5′ ends from the
filamentous fungus A. nidulans using five different growth
conditions.
RNA-Seq has been applied to several Aspergillus spe-
cies to show transcriptional responses to: biofilm growth in
A. fumigatus (Gibbons et al., 2012); growth on lignocellu-
loses and germination of conidia in A. niger (Delmas et al.,
2012; Novodvorska et al., 2013; van Leeuwen et al., 2013);
and temperature changes and 5-azacytidine in A. flavus (Yu
et al., 2011; Lin et al., 2013). These and other studies (Wang
et al., 2010a,b; Rokas et al., 2012) have also explored
Aspergillus transcriptomes more generally and improved
genome annotation.
Pacific Biosciences SMRT DNA Sequencer
Pacific Biosciences launched in 2010 a single-molecule
real-time sequencing platform. This platform uses a struc-
ture called Zero Mode Waveguide or ZMW. This structure
allows the observation of a single nucleotide of DNA being
incorporated by the DNA polymerase fixed at the ZMW.
Each nucleotide has been marked with different fluorescent
dyes. A detector reads the fluorescent signal of the nucleo-
tide incorporated. SMRT has been used mainly in genome
sequencing, resequencing, and methylation detection
(Myllykangas et al., 2012). English et al. (2012) presented
new software (PBJelly) for upgrading genome assemblies
based on long-read sequence form Pacific Bioscience RS.
This software will improve annotation problems in gap-
associated regions. Further improvements have been done
for genome assembly. Chin et al. (2013) discussed the use
of short reads to correct errors in the long SMRT reads. This
assembly requires at least two different libraries and a vari-
ety of sequencing runs.
CONCLUSION
Aspergillus diversity is measured by a variety of techniques,
including traditional plate culture, phospholipid and fatty
acid analysis, denaturing gradient gel electrophoresis, gel
 Understanding the Diversity of Aspergillus by Next-Generation Sequencing Chapter | 2
electrophoresis of microbial proteins, phylo arrays, DNA
fingerprinting techniques, restriction analysis, markers
based on PCR and Sanger sequencing. High-throughput
sequencing technologies now play a significant role in
microbial ecology studies and next-generation techniques,
such as 454 pyrosequencing, facilitate the detection of
Aspergillus and their diversity. By directly sequencing
single molecules of DNA or RNA, Helicos True Single-
Molecule Sequencing (tSMS) technology significantly
increased the speed of sequencing, while also decreasing
the cost. Using the Helicos DNA Barcoding protocol, sci-
entists at Helicos were able to multiply the system’s sample
throughput fivefold (from 50 to 250 samples per run), with-
out compromising accuracy or representational bias. DNA
sequencing data from next-generation platforms typically
present shorter read lengths, higher coverage, and differ-
ent error profiles compared with Sanger sequencing data.
Most of the coming research in Aspergillus fungal diversity
is going the NGS way and the reality is that gathering data
is no longer the limiting factor for understanding different
genes and their functions.
REFERENCES
Abadio, A.K., Kioshima, E.S., Teixeira, M.M., Martins, N.F., Maigret, B.,
Felipe, M.S., 2011. Comparative genomics allowed the identification of
drug targets against human fungal pathogens. BMC Genomics. 12, 7.
Accensi, F., Cano, J., Figuera, L., Abarca, M.L., Cabanes, F.J., 1999. New
PCR method to differentiate species in the Aspergillus niger aggre-
gate. FEMS Microbiol. Lett. 180, 191–196.
Al-Musallam, A., 1980. Revision of the black Aspergillus species. PhD
thesis. State University Utrecht, The Netherlands.
Andersen, M.R., Salazar, M.P., Schaap, P.J., van de Vondervoort, P.J.,
Culley, D., Thykaer, J., et al., 2011. Comparative genomics of cit-
ric-acid-producing Aspergillus niger ATCC 1015 versus enzyme-pro-
ducing CBS 513.88. Genome Res. 21, 885–897.
Aspergillus Genome Database (AspGD) at <http://www.aspgd.org/>.
Atoui, A., Mathieu, F., Lebrihi, A., 2007. Targeting a polyketide synthase
gene for Aspergillus carbonarius quantification and ochratoxin A
assessment in grapes using real-time PCR. Int. J. Food. Microbiol.
115, 313–318.
Baker, S.C., 2006. Aspergillus niger genomics: past, present and into the
future. Med. Mycol. 44, 517–521.
Baker, S.E., Bennett, J.W., 2008. An overview of the genus Aspergillus. In:
Goldman, G.H., Osmani, S.A. (Eds.), The Aspergilli: genomics, medi-
cal applications, biotechnology, and research methods CRC Press,
Boca Raton, FL, pp. 3–13.
Balajee, S.A., Houbraken, J., Verweij, P.E., Hong, S.B., Yaghuchi, T.,
Varga, J., et al., 2007. Aspergillus species identification in the clinical
setting. Stud. Mycol. 59, 39–46.
Bartram, A.K., Lynch, M.D.J., Stearns, J.C., Moreno-Hagelsieb, G.,
Neufeld, J.D., 2011. Generation of multimillion-sequence 16S
rRNA gene libraries from complex microbial communities by assem-
bling paired-end Illumina reads. Appl. Environ. Microbiol. 77,
3846–3852.
Berbee, M.L., Yoshimura, A., Sugiyama, J., Taylor, J.W., 1995. Is
Penicillium monophyletic? An evaluation of phylogeny in the family
37
Trichocomaceae from 18S, 5.8S and ITS ribosomal DNA sequence
data. Mycologia 87, 210–222.
Blow, N., 2008. DNA sequencing: generation next-next. Nat. Methods. 5,
267–274.
Braslavsky, I., Hebert, B., Kartalov, E., Quake, S.R., 2003. Sequence infor-
mation can be obtained from single DNA molecules. Proc. Natl. Acad.
Sci. USA 100, 3960–3964.
Buee, M., Reich, M., Murat, C., Morin, E., Nilsson, R.H., Uroz, S., et al.,
2009. 454 pyrosequencing analyses of forest soils reveal an unexpect-
edly high fungal diversity. New Phytol. 184, 449–456.
Butler, G., Rasmussen, M.D., Lin, M.F., Santos, M.A., Sakthikumar, S.,
Munro, C.A., et al., 2009. Evolution of pathogenicity and sexual
reproduction in eight Candida genomes. Nature 459, 657–662.
Cabanes, F.J., Sanseverino, W., Castella, G., Bragulat, M.R., Cigliano,
R.A., Sanchez, A., 2015. Rapid genome resequencing of an atoxigenic
strain of Aspergillus carbonarius. Sci. Rep. 5, 9086.
CADRE. <http://www.cadre-genomes.org.uk/>.
Chang, P.K., Ehrlich, K.C., 2010. What does genetic diversity of
Aspergillus flavus tell us about Aspergillus oryzae? Int. J. of Food
Microbiol. 138, 189–199.
Chin, C.S., Alexander, D.H., Marks, P., Klammer, A.A., Drake, J., Heiner,
C., et al., 2013. Nonhybrid, finished microbial genome assemblies
from long-read SMRT sequencing data. Nat. Methods. 10, 563–569.
Chulze, S.N., Magnoli, C.E., Dalcero, A.M., 2006. Occurrence of ochra-
toxin a in wine and ochratoxigenic mycoflora in grape and dried vine
fruits in South America. Int. J. Food. Microbiol. 111, S5–S9.
Clarke, J., Wu, H.C., Jayasinghe, L., Patel, A., Reid, S., Bayley, H., 2009.
Continuous base identification for single-molecule nanopore DNA
sequencing. Nat. Nanotechnol. 4, 265–270.
Cotty, P.J., 1997. Aflatoxin-producing potential of communities of
Aspergillus section Flavi from cotton producing areas in the United
States. Mycol. Res. 101, 698–704.
Cotty, P.J., Bayman, D.S., Egel, D.S., Elias, K.S., 1994. Agriculture,
aflatoxins and Aspergillus. In: Powell, K.A., Renwick, A., Peberdy,
J.F. (Eds.), The Genus Aspergillus: From Taxonomy and Genetics to
Industrial Applications Plenum Press, New York, NY, pp. 1–27.
Dalcero, A., Magnoli, C., Hallak, C., Chiacchiera, S.M., Palacio, G., Rosa,
C.A.R., 2002. Detection of ochratoxin A in animal feeds and capacity
to produce this mycotoxin by Aspergillus section Nigri in Argentina.
Food Addit. Contam. 19, 1065–1072.
Dao, H.P., Mathieu, F., Lebrihi, A., 2005. Two primer pairs to detect OTA
producers by PCR method. Int. J. Food Microbiol. 104, 61–67.
De Gannes, V., Eudoxie, G., Hickey, W.J., 2013. Insights into fungal com-
munities in composts revealed by454- pyrosequencing: implications
for human health and safety. Front. Microbiol. 164, 4.
Delmas, S., Pullan, S.T., Gaddipati, S., Kokolski, M., Malla, S., Blythe,
M.J., et al., 2012. Uncovering the genome-wide transcriptional
responses of the filamentous fungus Aspergillus niger to lignocellu-
lose using RNA sequencing. PLoS Genet. 8, e1002875.
Denning, D.W., 1998. Invasive aspergillosis. Clin. Infect. Dis. 26, 781–803.
Domsch, K.H., Gams, W., Anderson, T.H., 2007. Compendium of Soil
Fungi, second ed. IHW-Verlag, Eching, The Netherlands, pp. 860.
Doyle, S., 2011. Fungal proteomics: from identification to function. FEMS
Microbiol. Lett. 321, 1–9.
Egan, A.N., Schlueter, J., Spooner, D.M., 2012. Applications of next-
generation sequencing in plant biology. Am. J. Bot. 99, 175–185.
Egel, D.S., Cotty, P.J., Elias, K.S., 1994. Relationships among isolates of
Aspergillus sect. Flavi that vary in aflatoxin production. Phytopatology
84, 906–912.
38 SECTION | I Biology and Biodiversity
Eilers, K.G., Debenport, S., Anderson, S., Fierer, N., 2012. Digging deeper
to find unique microbial communities: The strong effect of depth on
the structure of bacterial and archaeal communities in soil. Soil. Biol.
Biochem. 50, 58–65.
English, A.C., Richards, S., Han, Y., Wang, M., Vee, V., Qu, J., et al., 2012.
Mind the gap: Upgrading genomes with pacific biosciences RS long-
read sequencing technology. PLoS. ONE. 7, e47768.
Ensembl Genomes ftp site. <ftp://ftp.ensemblgenomes.org/pub/fungi/>.
Fedorova, N.D., Khaldi, N., Joardar, V.S., Maiti, R., Amedeo, P., Anderson,
M.J., et al., 2008. Genomic islands in the pathogenic filamentous fun-
gus Aspergillus fumigatus. PLoS Genet. 4, e1000046.
Feng, L., Liu, H., Liu, Y., Lu, Z., Guo, G., Guo, S., et al., 2010. Power of
deep sequencing and agilent microarray for gene expression profiling
study. Mol Biotechnol 45, 101–110.
Fierer, N., Leff, J.W., Adams, B.J., Nielsen, U.N., Bates, S.T., Lauber,
C.L., et al., 2012. Cross-biome metagenomic analyses of soil micro-
bial communities and their functional attributes. Proc. Natl. Acad. Sci.
USA. 109, 21390–21395.
Frisvad, J.C., Larsen, T.O., de Vries, R., Meijer, M., Houbraken, J.,
Cabanes, F.J., et al., 2007. Secondary metabolite profiling, growth pro-
files and other tools for species recognition and important Aspergillus
mycotoxins. Stud. Mycol. 59, 31–37.
Fungaro, M.H.P., Vissotto, P.C., Sartori, D., Vilas-Boas, L.A., Furlaneto,
M.C., Taniwaki, M.H., 2004. A molecular method for detection of
Aspergillus carbonarius in coffee beans. Current Microbiol. 49,
123–127.
FungiDB web resource. <http://fungidb.org/fungidb/>.
Futagami, T., Mori, K., Yamashita, A., Wada, S., Kajiwara, Y., Takashita, H.,
et al., 2011. Genome sequence of the white koji mold Aspergillus
kawachii IFO 4308, used for brewing the Japanese distilled spirit sho-
chu. Eukaryot. Cell. 10, 1586–1587.
Galagan, J.E., Calvo, S.E., Cuomo, C., Ma, L.J., Wortman, J.R., Batzoglou, S.,
et al., 2005. Sequencing of Aspergillus nidulans and comparative
analysis with A. fumigatus and Aspergillus oryzae. Nature. 438,
1105–1115.
Geiser, D.M., Arnold, M.L., Timberlake, W.E., 1996. Wild chromosomal
variants in Aspergillus nidulans. Curr. Genet. 29, 293–300.
Geiser, D.M., Dorner, J.W., Horn, B.W., Taylor, J.W., 2000. The phyloge-
netics of mycotoxin and sclerotium production in Aspergillus flavus
and Aspergillus oryzae. Fungal Genet. Biol. 31, 169–179.
Geiser, D.M., Klich, M.A., Frisvad, J.C., Peterson, S.W., Varga, J., Samson,
R.A., 2007. The current status of species recognition and identification
in Aspergillus. Stud. Mycol. 59, 1–10.
Gibbons, J.G., Beauvais, A., Beau, R., McGary, K.L., Latge, J.P., Rokas, A.,
2012. Global transcriptome changes underlying colony growth in the
opportunistic human pathogen Aspergillus fumigatus. Eukaryot. Cell.
11, 68–78.
Glass, N.L., Jacobson, D.J., Shiu, P.K.T., 2000. The genetics of hyphal
fusion and vegetative incompatibility in filamentous ascomycete
fungi. Annu. Rev. Genet. 34, 165–186.
Gonzalez-Salgado, A., Patino, B., Vazquez, C., Gonzalez-Jaen, M.T.,
2005. Discrimination of Aspergillus niger and other Aspergillus spe-
cies belonging to section Nigri by PCR assays. FEMS Microbiol. Lett.
245, 353–361.
Gonzalez-Salgado, A., Patino, B., Gil-Serna, J., Vazquez, C., Gonzalez-
Jaen, M.T., 2009. Specific detection of Aspergillus carbonarius by
SYBR Green and TaqMan quantitative PCR assays based on the mul-
ticopy ITS2 region of the rRNA gene. FEMS Microbiol. Lett. 295,
57–66.
Haynes, S.W., Gao, X., Tang, Y., Walsh, C.T., 2012. Assembly of asperli-
cin peptidyl alkaloids from anthranilate and tryptophan: a two-enzyme
pathway generates heptacyclic scaffold complexity in asperlicin E. J.
Am. Chem. Soc. 134, 17444–17447.
Hodkinson, B.P., Grice, E.A., 2015. Next-generation sequencing: a review
of technologies and tools for wound microbiome research. Adv.
wound Care 4 (1), 50–58.
Houbraken, J., Samson, R.A., 2011. Phylogeny of Penicillium and the
segregation of Trichocomaceae into three families. Stud. Mycol. 70,
1–51.
Houbraken, J., Vries, R.P., Samson, R.A., 2014. Modern taxonomy of bio-
technologically important Aspergillus and Penicillium species. Adv.
Appl. Microbiol. 86, 199–249.
Jain, R., Valiante, V., Remme, N., Docimo, T., Heinekamp, T., Hertweck,
C., et al., 2011. The MAP kinase MpkA controls cell wall integrity,
oxidative stress response, gliotoxin production and iron adaptation in
Aspergillus fumigatus. Mol. Microbiol. 82, 39–53.
Jochl, C., Rederstorff, M., Hertel, J., Stadler, P.F., Hofacker, J.L., Schrettl,
M., et al., 2008. Small ncRNA transcriptome analysis from Aspergillus
fumigatus suggests a novel mechanism for regulation of protein syn-
thesis. Nucleic Acids. Res. 36, 2677–2689.
Jones, M.G., 2007. The first filamentous fungal genome sequences:
Aspergillus leads the way for essential everyday resources or dusty
museum specimens? Microbiology 153, 1–6.
Jumpponen, A., Jones, K.L., 2009. Massively parallel 454 sequencing indi-
cates hyperdiverse fungal communities in temperate Quercus macro-
carpa phyllosphere. New Phytol. 184, 438–448.
Kapranov, P., Ozsolak, F., Milos, P.M., 2012. Profiling of short RNAs
using helicos single-molecule sequencing Next-Generation micro-
RNA Expression Profiling Technology. Springer-Verlag, Berlin,
Germany.219–232
Keller, N.P., Turner, G., Bennett, J.W., 2005. Fungal secondary metabo-
lism-from biochemistry to genomics. Nat. Rev. Microbiol. 3, 937–947.
Klich, M.A., 2002. Biogeography of Aspergillus species in soil and litter.
Mycologia 94, 21–27.
Latge, J.P., 1999. Aspergillus fumigatus and aspergillosis. Clin. Microbiol.
Rev. 12, 310–350.
Lerner, H.R.L., Fleischer, R.C., 2010. Prospects for the use of next-genera-
tion sequencing methods in Ornithology. The Auk. 127, 4–15.
Leslie, J.F., 1993. Fungal vegetative compatibility. Annu. Rev. Phytopathol.
31, 127–150.
Li, S., Anand, K., Tran, H., Yu, F., Finefield, J.M., Sunderhaus, J.D., et al.,
2012. Comparative analysis of the biosynthetic systems for fungal
bicyclo[2.2.2]diazaoctane indole alkaloids: the (+)/(−)-notoamide,
paraherquamide and malbrancheamide pathways. Medchemcomm. 3
(8), 987–996.
Lim, Y.W., Kim, B.K., Kim, C., Jung, H.S., Kim, B.S., Lee, J.H., et al.,
2010. Assessment of soil fungal communities using pyrosequencing.
J. Microbiol. 48, 284–289.
Lin, J.Q., Zhao, X.X., Zhi, Q.Q., Zhao, M., He, Z.M., 2013. Transcriptomic
profiling of Aspergillus flavus in response to 5-azacytidine. Fungal.
Genet. Biol. 56, 78–86.
Loman, N.J., Constantinidou, C., Chan, J.Z., Halachev, M., Sergeant, M.,
Penn, C.W., et al., 2012. High-throughput bacterial genome sequenc-
ing: an embarrassment of choice, a world of opportunity. Nat. Rev.
Microbiol. 10, 599–606.
Losada L., Fedorova N., Joardar V., Pakala S., Pakala S., Zafar N.,
et al. (2013). Community Resources for Aspergillus fumigatus: an
NIAID funded genome sequencing project. The Tenth International
 Understanding the Diversity of Aspergillus by Next-Generation Sequencing Chapter | 2
Aspergillus Meeting Asilomar Conference Grounds, Pacific Grove,
CA, March 12–17. pp. 138.
Machida, M., Asai, K., Sano, M., Tanaka, T., Kumagai, T., Terai, G., et al.,
2005. Genome sequencing and analysis of Aspergillus oryzae. Nature
438, 1157–1161.
Madsen, A.M., Zervas, A., Tendal, K., Nielsen, J.L., 2015. Microbial
diversity in bioaerosol samples causing ODTS compared to refer-
ence bioaerosol samples as measured using Illumina sequencing and
MALDI-TOF. Environ. Res. 140, 255–267.
Magi, A., Benelli, M., Gozzini, A., Girolami, F., Torricelli, F., Brandi,
M.L., 2010. Bioinformatics for next generation sequencing data.
Genes 1, 294–307.
Magnani, M., Fernandes, T., Prete, C.S.E.C., Homechim, M., Ono, E.Y.S.,
Vilas-Boas, L.A., et al., 2005. Molecular identification of Aspergillus
spp. isolated from coffee beans. Scientia Agric 62, 45–49.
Mardis, E.R., 2008. The impact of next-generation sequencing technology
on genetics. Trends Genet. 24, 133–141.
Margulies, M., Egholm, M., Altman, W.E., Attiya, S., Bader, J.S., Bemben,
L.A., et al., 2005. Genome sequencing in microfabricated high-density
picolitre reactors. Nature 437, 376–380.
Martinez-Culebras, P.V., Ramon, D., 2007. An ITS-RFLP method to iden-
tify black Aspergillus isolates responsible for OTA contamination in
grapes and wine. Int. J. Food Microbiol. 113, 147–153.
Midorikawa, G.E.O., Pinheiro, M.R.R., Vidigal, B.S., Arruda, M.C., Costa,
F.F., Pappas Jr., G.J., et al., 2008. Characterization of Aspergillus flavus
strains from Brazilian Brazil nuts and cashew by RAPD and ribosomal
DNA analysis. Lett. Appl. Microbiol. 147, 12–18.
Mortazavi, A., Williams, B.A., McCue, K., Schaeffer, L., Wold, B., 2008.
Mapping and quantifying mammalian transcriptomes by RNA-Seq.
Nat. Methods 5, 621–628.
Mule, G., Susca, A., Logrieco, A., Stea, G., Visconti, A., 2006. Development
of a quantitative real-time PCR assay for the detection of Aspergillus
carbonarius in grapes. Int. J. Food Microbiol. 111, S28–S34.
Muller, S., Baldin, C., Groth, M., Guthke, R., Kniemeyer, O., Brakhage,
A.A., et al., 2012. Comparison of transcriptome technologies in the
pathogenic fungus Aspergillus fumigatus reveals novel insights into
the genome and MpkA dependent gene expression. BMC Genomics
13, 519.
Murakami, H., 1979. Some experimental methods and cultural characteris-
tics of the black Aspergilli. Taxonomic studies on Japanese Industrial
strains of the Aspergillus. J. Soc. Brew. 74, 323–327.
Myllykangas, S., Buenrostro, J., Ji, H.P., 2012. Overview of sequenc-
ing technology platforms Bioinformatics for High Throughput
Sequencing. Springer-Verlag, Berlin, Germany.11–25
Nagalakshmi, U., Wang, Z., Waern, K., Shou, C., Raha, D., Gerstein, M.,
et al., 2008. The transcriptional landscape of the yeast genome defined
by RNA sequencing. Science 320, 1344–1349.
Nierman, W.C., Pain, A., Anderson, M.J., Wortman, J.R., Kim, H.S.,
Arroyo, J., et al., 2005. Genomic sequence of the pathogenic and
allergenic filamentous fungus Aspergillus fumigatus. Nature 438,
1151–1156.
Novodvorska, M., Hayer, K., Pullan, S.T., Wilson, R., Blythe, M.J., Stam,
H., et al., 2013. Trancriptional landscape of Aspergillus niger at
breaking of conidial dormancy revealed by RNA-sequencing. BMC
Genomics. 14, 246.
Nyren, P., 2007. The history of pyrosequencing. Methods Mol. Biol. 373,
1–14.
Ogawa, H., Yoshimura, A., Sugiyama, J., 1997. Polyphyletic origins of
species of the anamorphic genus Geosmithia and the relationships
39
of the cleistothecial genera: evidence from 18S, 5S and 28S rDNA
sequence analyses. Mycologia 89, 756–771.
Okuda, T., Klich, M.A., Seifert, K.A., Ando, K., 2000. Media and incu-
bation effect on morphological characteristics of Penicillium and
Aspergillus. In: Samson, R.A., Pitt, J.I. (Eds.), Integration of Modern
Taxonomic Methods for Penicillium and Aspergillus Classification
Harwood Academic publishers, Amsterdam, pp. 83–99.
Olarte, R.A., Horn, B.W., Dorner, J.W., Monacell, J.T., Singh, R., Stone,
E.A., et al., 2012. Effect of sexual recombination on population diver-
sity in aflatoxin production by Aspergillus flavus and evidence for
cryptic heterokaryosis. Mol. Ecol. 21, 1453–1476.
Olsvik, O., Wahlberg, J., Petterson, B., Uhlén, M., Popovic, T., Wachsmuth,
I.K., et al., 1993. Use of automated sequencing of polymerase chain
reaction-generated amplicons to identify three types of cholera toxin
subunit B in Vibrio cholerae O1 strains. J. Clin. Microbiol. 31, 22–25.
Opik, M., Metsis, M., Daniell, T.J., Zobel, M., Moora, M., 2009. Large-
scale parallel 454 sequencing reveals host ecological group specificity
of arbuscular mycorrhizal fungi in a boreonemoral forest. New Phytol.
184, 424–437.
Ozsolak, F., Milos, P.M., 2011. RNA sequencing: advances, challenges
and opportunities. Nat. Rev. Genet. 12, 87–98.
Parenicova, L., Skouboe, P., Frisvad, J., Samson, R.A., Rossen, L., ten
Hoor-Suykerbuyk, M., et al., 2001. Combined molecular and biochem-
ical approach identifies Aspergillus japonicus and Aspergillus aculea-
tus as two different species. Appl. Environ. Microbiol. 67, 521–527.
Patino, B., Gonzalez-Salgado, A., Gonzalez-Jaen, M.T., Vazquez, C., 2005.
PCR detection assays for the ochratoxin producing Aspergillus car-
bonarius and Aspergillus ochraceus species. Int. J. Food. Microbiol.
104, 207–214.
Payne, G.A., Nierman, W.C., Wortman, J.R., Pritchard, B.L., Brown, D.,
Dean, R.A., et al., 2006. Whole genome comparison of Aspergillus
flavus and A. oryzae. Med Mycology 44 (Suppl. 1), 9–11.
Payne, G.A., Yu, J., Nierman, W.C., Machida, M., Bhatnagar, D.,
Cleveland, T.E., et al., 2007. A first glance into the genome sequence
of Aspergillus flavus. In: Osmani, S., Goldman, G. (Eds.), The
Aspergilli: Genomics, Medical Aspects, Biotechnology and Research
Methods CRC Press, Boca Ratan, FL, pp. 15–23.
Pel, H.J., de Winde, J.H., Archer, D.B., Dyer, P.S., Hofmann, G., Schaap,
P.J., et al., 2007. Genome sequencing and analysis of the versatile cell
factory Aspergillus niger CBS 513.88. Nat. Biotechnol. 25, 221–231.
Perrone, G., Susca, A., Cozzi, G., Ehrlich, K., Varga, J., Frisvad, J.C., et al.,
2007. Biodiversity of Aspergillus species in some important agricul-
tural products. Stud. Mycol. 59, 53–66.
Perrone, G., Varga, J., Susca, A., Frisvad, J.C., Stea, G., Kocsube, S., et al.,
2008. Aspergillus uvarum sp. nov., an uniseriate black Aspergillus spe-
cies isolated from grapes in Europe. Int. J. Syst. Evol. Microbiol. 58,
1032–1039.
Perrone, G., Stea, G., Epifani, F., Varga, J., Frisvad, J.C., Samson, R.A.,
2011. Aspergillus niger contains the cryptic phylogenetic species
A. awamori. Fungal Biol 115, 1138–1150.
Peterson, S.W., 2008. Phylogenetic analyses of Aspergillus species using
DNA sequences from four loci. Mycologia 100, 205–226.
Pitt, J.I., 1994. The current role of Aspergillus and Penicillium in human
and animal health. J. Med. Vet. Mycol. 32, 17–32.
Raper, K.B., Fennell, D.I., 1965. The Genus Aspergillus. Williams &
Wilkins Company, Baltimore.
Robinson, J.P., Harris, S.A., 1999. In: Gillet, E.M. (Ed.), Which DNA Marker
for Which Purpose. Institut für Forstgenetik und Forstpflanzenzüchtung,
Universität Göttingen, Göttingen, Germany, pp. 1–27.
40 SECTION | I Biology and Biodiversity
Rokas, A., Gibbons, J.G., Zhou, X., Beauvais, A., Latge, J.P., 2012. The
diverse applications of RNA-seq for functional genomic studies in
Aspergillus fumigates. Ann. N. Y. Acad. Sci. 1273, 25–34.
Ronaghi, M., Karamohamed, S., Pettersson, B., Uhlen, M., Nyren, P.,
2006. Real-time DNA sequencing using detection of pyrophosphate
release. Anal. Biochem. 242, 84–89.
Samson, R.A., Houbraken, J.A.M.P., Kuijpers, A.F.A., Frank, J.M.,
Frisvad, J.C., 2004. New ochratoxin A or sclerotium producing spe-
cies in Aspergillus section Nigri. Stud. Mycol. 50, 45–61.
Samson, R.A., Noonim, P., Meijer, M., Houbraken, J., Frisvad, J.C., Varga,
J., 2007. Diagnostic tools to identify black Aspergilli. Stud. Mycol.
59, 129–146.
Sanger, F., Nicklen, S., Coulson, A.R., 1977. DNA sequencing with
chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74,
5463–5467.
Sato, A., Oshima, K., Noguchi, H., Ogawa, M., Takahashi, T., Oguma, T.,
et al., 2011. Draft genome sequencing and comparative analysis of
Aspergillus sojae NBRC4239. DNA Res. 18, 165–176.
Schadt, E.E., Turner, S., Kasarskis, A., 2010. A window into third-genera-
tion sequencing. Hum. Mol. Genet. 19, R227–R240.
Schmidt, H., Taniwaki, M.H., Vogel, R.F., Niessen, L., 2004. Utilization
of AFLP markers for PCR-based identification of Aspergillus carbon-
arius and indication of its presence in green coffee samples. J. Appl.
Microbiol. 97, 899–909.
Schuster, S.C., 2008. Next-generation sequencing transforms today’s biol-
ogy. Nat. Methods. 5, 16–18.
Selma, M.V., Martínez-Culebras, P.V., Aznar, R., 2008. Realtime PCR
based procedures for detection and quantification of Aspergillus car-
bonarius in wine grapes. Int. J. Food Microbiol. 122, 126–134.
Serra, R., Cabanes, J., Perrone, G., Kozakiewicz, Z., Castella, G., Venancio,
A., et al., 2006. Aspergillus ibericus: a new species of the Section
Nigri isolated from grapes. Mycologia 98, 295–306.
Shokralla, S., Spall, J.L., Gibson, J.F., Hajibabaei, M., 2012. Next-
generation sequencing technologies for environmental DNA research.
Mol. Ecol. 21, 1794–1805.
Sibthorp, C., Wu, H., Cowley, G., Wong, P.W.H., Palaima, P., Morozov,
I.Y., et al., 2013. Transcriptome analysis of the filamentous fungus
Aspergillus nidulans directed to the global identification of promoters.
BMC Genomics 14, 847.
Spadaro, D., Patharajan, S., Kartikeyan, M., Lore, A., Garibaldi, A.,
Gullino, M.L., 2011. Specific PCR primers for the detection of iso-
lates of Aspergillus carbonarius producing ochratoxin A on grapevine.
Ann. Microbiol. 61, 267–272.
Susca, A., Stea, G., Mule, G., Perrone, G., 2007. PCR identification of
Aspergillus niger and Aspergillus tubingensis based on calmodulin
gene. Food Addit. Contam. 24, 1154–1160.
Tamura, M., Kawahara, K., Sugiyama, J., 2000. Molecular phylogeny
of Aspergillus and associated teleomorphs in the Trichocomaceae
(Eurotiales). In: Samson, R.A., Pitt, J.I. (Eds.), Integration of Modern
Taxonomic Methods for Penicillium and Aspergillus Classification
Harwood Academic publishers, Amsterdam, pp. 357–372.
The Aspergillus and Candida Genome Databases, AspGD. <http://www.
aspgd.org>.
Trail, F., Mahanti, N., Linz, J., 1995. Molecular biology of aflatoxin bio-
synthesis. Microbiology. 141, 755–765.
Umemura, M., Koyama, Y., Takeda, I., Hagiwara, H., Ikegami, T., Koike,
H., et al., 2013. Fine de novo sequencing of a fungal genome using
only SOLiD short read data: verification on Aspergillus oryzae RIB40.
PLoS ONE. 8 (5), e63673.
van Leeuwen, M.R., Krijgsheld, P., Bleichrodt, R., Menke, H., Stam, H.,
Stark, J., et al., 2013. Germination of conidia of Aspergillus niger is
accompanied by major changes in RNA profiles. Stud. Mycol. 74,
59–70.
Varga, J., Kocsube, S., Toth, B., Frisvad, J.C., Perrone, G., Susca, A., et al.,
2007. Aspergillus brasiliensis sp. nov., a biseriate black Aspergillus
species with world-wide distribution. Int. J. Syst. Evol. Microbiol. 57,
1925–1932.
Wang, B., Guo, G., Wang, C., Lin, Y., Wang, X., Zhao, M., et al., 2010a.
Survey of the transcriptome of Aspergillus oryzae via massively paral-
lel mRNA sequencing. Nucl. Acids Res. 38, 5075–5087.
Wang, K., Singh, D., Zeng, Z., Coleman, S.J., Huang, Y., Savich, G.L.,
et al., 2010b. MapSplice: accurate mapping of RNA-seq reads for
splice junction discovery. Nucl. Acids Res. 38 (18), e178.
Wang, Z., Gerstein, M., Snyder, M., 2009. RNA-seq: a revolutionary tool
for transcriptomics. Nat. Rev. Genet. 10, 57–63.
Whiteley, A.S., Jenkins, S., Waite, I., Kresoje, N., Payne, H., Mullan, B.,
et al., 2012. Microbial 16S rRNA ion tag and community metagen-
ome sequencing using the Ion Torrent (PGM) platform. J. Microbiol.
Methods. 91, 80–88.
Wortman, J.R., Gilsenan, J.M., Joardar, V., Deegan, J., Clutterbuck, J.,
Andersen, M.R., et al., 2009. The 2008 update of the Aspergillus nidulans
genome annotation: a community effort. Fungal Genet. Biol. 46, 2–13.
Yu, J., Fedorova, N.D., Montalbano, B.G., Bhatnagar, D., Cleveland, T.E.,
Bennett, J.W., et al., 2011. Tight control of mycotoxin biosynthesis
gene expression in Aspergillus flavus by temperature as revealed by
RNA-Seq. FEMS Microbiol. Lett. 322, 145–149.
Chapter 3
Molecular Evolution of Aspergillus
A.C. Flores-Gallegos, F. Veana-Hernandez, M. Michel-Michel, F. Lara-Victoriano and R. Rodríguez-Herrera
Universidad Autónoma de Coahuila, Saltillo, Coahuila, México
ASPERGILLUS GENERALITIES
Aspergillus is a saprophytic fungus, but it can also be a
benign parasite of plants and animals, human included.
There are few species capable of being facultative sapro-
phytic. Species of Aspergillus are mainly present in the
asexual phase (anamorph), but it is estimated that a third
of them have a sexual phase. The perfect phase is known
for some species, corresponding to Eurotium, Sartorya, or
Emericella genera. Most of the Aspergillus species have
their reproduction through a parasexual cycle, except for
Aspergillus fumigatus which has sexual and asexual pro-
cesses for spore production (Nahlik et al., 2005). The cycle
consists of hyphae fusion and heterokaryon formation with
haploid nucleus that may fuse to form a diploid. A het-
erozygote is originated and its chromosomes are recom-
bined during mitosis. The parasexual cycle is important for
Aspergillus evolution (Kim et al., 2002). On the other hand,
the sexual cycle is initiated due to differentiation of veg-
etative mycelia aggregations and formation of Hülle cells.
Cleistothecium is formed from those aggregations, which is
the origin of the asci that contain the ascospores.
Identification of these fungi has been based on morpho-
logical characteristics, however, molecular identification
has been used in recent years. Aspergillus belongs to the
Hyphomycetes class and is characterized by the formation
of conidiophores with large stipe, gross walls, and swol-
len apex or vesicles. They can be spherical and have phi-
alides or metula. Conidia are produced by the phialides,
and can be located directly over the vesicle (uniseriate) or
over the metula (biseriate). Aspergillus conidia are formed
in specialized cell chains, and are hyaline, oval, or ellipti-
cal. Conidia ornamentation could be like alternate spines
or in grooves, which is called equinulation. There is also a
pod-type ornamentation, which is smooth, but ornamenta-
tion may be absent.
Studies of ascoma (fruiting body) have not been accom-
panied of genetic analysis in most ascomycetos. Molecular
mechanisms that control fruiting body formation have been
studied in only a few organisms, Aspergillus nidulans for
New and Future Developments in Microbial Biotechnology and Bioengineering. DOI: http://dx.doi.org/10.1016/B978-0-444-63505-1.00003-8
© 2016 Elsevier B.V. All rights reserved.
example (Busch and Braus, 2007). During ascoma develop-
ment, simple vegetative hyphae are differentiated into 15 cell
types that form the complex structure of the fruiting body
(Bistis et al., 2003). This process is influenced by external
conditions such as light, temperature, nutrient supplementa-
tion, and internal factors as specific signaling molecules.
Aspergillus is widely distributed in nature and can use
more than 100 carbon compounds (sugars, alcohols, oligo-
and polysaccharides, organic acids, amino acids, and nucle-
otides) (Kevei et al., 1996). Its growth temperature range
varies from 6°C to 47°C, with an optimal of 35–37°C. Spore
germination occurs at 35°C and 0.77 water activity (Ayest,
1966). Mature conidia can survive adverse conditions of
temperature, humidity, and air dispersion (Dijksterhuis and
Samsón, 2002). It has been found that Aspergillus can tol-
erate high acid concentration (>200 g/L citrate) and have
great adaptation versatility to complex substrates, which is
of interest for industry.
Some Aspergillus species are known as fermentation
agents able to produce valuable metabolic products, includ-
ing organic acids (citric acid), enzymes (amylase), and
secondary metabolites such as gryseofulvin, which is an
antagonist for dermatophytes. Biosynthesis of these products
is associated with development and cellular differentiation
of this fungal species. Better biochemistry and molecular
Aspergillus comprehension has led to improvement of pro-
duction processes and developments of new products.
ASPERGILLUS ROLE IN NATURE
Aspergillus is a filamentous saprophytic genus of fungi
whose essential role in nature is aerobic decomposition of
organic material. Aspergillus is a truly ubiquitous fungus.
In addition to being found in soil and plant remains, it is
found throughout the year at homes, schools, workplaces,
libraries, and in such improbable places as desert (Schuh
and Hogaboam, 2005). Aspergillus species are widely dis-
tributed in nature and can cause different diseases based
on their degree of immune competence. Pathogenic fungus
41
42 SECTION | I Biology and Biodiversity
grows easily and quickly in routine culture media used for
bacteria and fungi (Garcés Jarque et al., 2003).
Aspergillus as Pathogen
While the vast majority of Aspergillus species do not repre-
sent a threat to humans, at least 19 species are known oppor-
tunistic pathogens. The most important of the pathogenic
strains is A. fumigatus (Latgé, 2001). This is the most com-
mon species recovered from cases of invasive aspergillosis.
The next most commonly recovered species are Aspergillus
flavus, Aspergillus niger, and Aspergillus terreus (Hope
et al., 2005; Walsh et al., 2008).
In environments outside of a host, Aspergillus produces
conidia (asexual spores) that can be easily dispersed in the
air. Inhalation of conidia in aerosol form is usually the initial
route of entry for Aspergillus infection (Weigt et al., 2013).
Aspergillus species have emerged as important causes of
morbidity and mortality in immune-compromised patients.
Invasive aspergillosis currently constitutes the most com-
mon pneumonic cause of infectious mortality in patients
undergoing hematopoietic stem cell transplantation (HSCT)
and is an important cause of opportunistic respiratory dis-
eases and disseminated infection in other immune-compro-
mised patients. Moreover, Aspergillus species also produce
a wide range of chronic, saprophytic, and allergic condi-
tions. Although other forms of aspergillosis, such as allergic
bronchopulmonary aspergillosis (ABPA), allergic sinusitis,
and saprophytic infection, are also causes of morbidity, they
are rarely fatal (Walsh et al., 2008; Yoshida et al., 2015).
Aspergillus fumigatus is a saprophytic fungus, with
prevalence worldwide. It is one of the most common species
of fungi in the air. Their floating airborne spores (conidia)
are responsible for its ability to use various carbon sources,
thermotolerance, and allow it to survive in areas with very
different climates and environments (Ramirez-Ortiz et al.,
2011). The infection can spread hematoma generously,
TABLE 3.1 Some Microbial Enzymes from Different Aspergillus Species
Source Enzyme
A. niger Proteases
A. niger MTCC5889 Tannase
A. niger HN-1 Cellulase
A. oryzae IPT-301 Pectinase
A. awamori GHRTS Fructosyltransferase
A. japonicus β-Fructofuranosidase
A. oryzae KB β-Fructofuranosidase
A. oryzae CFR 202 Fructosyltransferase
resulting in outbreaks of fungal growth in other organs, par-
ticularly in the brain (Moore, 2013). Epidemiological stud-
ies illustrate a montage risk for developing diseases related
to fungi worldwide.
Aspergillus Usefulness for Humankind
Aspergillus fungus has different uses in industry, such as in
production of metabolites and enzymes (Sangeetha et al.,
2005).
Enzyme and Metabolite Production
Aspergillus metabolites and enzymes have been widely used
during food, pharmaceutical, biofuel, pesticide, and deter-
gent processes (Tamano, 2014). Different Aspergillus spe-
cies have been exploited in industrial processes. Aspergillus
niger has a long tradition of safe use in the production of
enzymes and organic acids (Soares de Castro et al., 2015).
The Aspergillus produces different types of enzymes
such as: pectinases (Meneghel et al., 2014), fructofuranosi-
dases (Kurakake et al., 2008), fructosyltransferases (Sathish
and Prakasham, 2013), tannases (Viswanath et al., 2015),
and proteases (Soares de Castro et al., 2015). One of the
strategies considered to be effective for enhancing the pro-
duction of primary metabolites is the expression of genes
involved in metabolite synthesis (Tamano, 2014). Several
fungal strains, especially those from Aspergillus genus, are
known to produce a variety of enzymes. In Table 3.1 it can
be observed that the better temperature for the production of
metabolites is between 28–30ºC.
ASPERGILLUS DIVERSITY
Aspergillus genera includes about 250 species (Klic, 2009),
50 of which were described in the last 10 years (Table 3.2).
Since 1965, “The genus Aspergillus” from Raper and Fennell
Temperature References
30°C Soares de Castro et al. (2015)
28–30°C Viswanath et al. (2015)
30°C Ahlawat et al. (2014)
28°C Meneghel et al. (2014)
30°C Sathish and Prakasham
(2013)
26°C, 30°C, 34°C Mussatto et al. (2013)
30°C Kurakake et al. (2008)
30°C Sangeetha et al. (2005)
Another random document with
no related content on Scribd:
The Project Gutenberg eBook of H. van
Brakel, Ing. B.O.W
This ebook is for the use of anyone anywhere in the United
States and most other parts of the world at no cost and with
almost no restrictions whatsoever. You may copy it, give it away
or re-use it under the terms of the Project Gutenberg License
included with this ebook or online at www.gutenberg.org. If you
are not located in the United States, you will have to check the
laws of the country where you are located before using this
eBook.

Title: H. van Brakel, Ing. B.O.W

Oorspronkelijke roman

Author: P. A. Daum

Release date: September 7, 2023 [eBook #71591]

Language: Dutch

Original publication: Leiden: A.W. Sijthoff, 1875

Credits: Jeroen Hellingman and the Online Distributed

Proofreading Team at https://www.pgdp.net/ for Project
Gutenberg (This book was produced from scanned
images of public domain material from the Google
Books project.)

*** START OF THE PROJECT GUTENBERG EBOOK H. VAN

BRAKEL, ING. B.O.W ***
H. VAN BRAKEL,
Ing. B. O. W.
 H. VAN BRAKEL,
Ing. B. O. W.

OORSPRONKELIJKE ROMAN

DOOR
MAURITS.

LEIDEN.—A. W. SIJTHOFF.
[1]
H. VAN BRAKEL,
Ingenieur B. O. W.
De lampen brandden in de achtergalerij, boven de nog gedekte tafel.
Vlug namen de bedienden de gerechten weg; ze hadden ditmaal
haast; ’t was immers de laatste arbeid des daags!

Het hoofd van Lucie zonk voorover op haar borst; haar oogen waren
dichtgevallen; zij kon zoo’n onoverwinnelijken slaap krijgen, ’s
avonds na het eten! Dan was het zoo rustig, zoo stil: de kinderen
sliepen; de huiselijke bedrijvigheid was ten einde.

Van Brakel had zijn lorgnet opgezet en las de courant. ’t Beviel hem
niet. Er stond weer iets in van den „strijkstok,” waaraan bij den
Waterstaat zooveel hangen bleef. Het doelde niet op hem,—volstrekt
niet; maar dan toch op z i j n ondergeschikten. Hm! ’t was beter, dat
die kerels, dacht hij, wat amusanter couranten maakten, dan zich
altijd te bemoeien met eens andermans zaken.

Toen hij ’t blad neerlei en zijn stoel achteruitschoof, schrikte Lucie

wakker.

„Ga je uit?” vroeg ze, zich de oogen wrijvende.

„Ja, nog ’n uurtje naar de soos.” [2]

„Ik ga naar bed.”

„Welzeker. Je bent moe.”

Hij zette zijn dienstpet op, nam een wandelstok uit een hoek en floot
zijn hond, die hem blaffend naar buiten volgde.
Toen Lucie haar buffet en haar dispenskast had gesloten, draaide zij
de lamp neer en ging naar haar kamer.

Het was er benauwd, en het rook er onfrisch.

Op een grooten divan stonden twee k o d j o n g s , en daaronder

sliepen als rozen de tweelingen, waarmede zij den gelukkigen
waterstaatsingenieur op St.-Nicolaas aangenaam had verrast.

Drie jaren waren zij getrouwd, en ze had nu vier kinderen.

Hun aantal „beren” echter was legio.

Vooreerst kon Van Brakel als vrijgezel de eindjes reeds niet

samenknoopen, en wat Lucie betreft,—op de koffie-onderneming
haars vaders had men steeds het huishouden „gedaan” uit het
werkkapitaal; dat „s t i m m t e ”, altijd volgens H e r r Drütlich.

Zij was een brave, ijverige huisvrouw; van dat de zon aan den hemel
kwam tot ’s avonds klokke acht, was ze in de weer; haar tweelingen
zoogde ze zelve, en ze zou, als een krachtige telg van
Germaanschen stam, in staat zijn geweest zeslingen te voeden
en.… over te houden. Zij had verstand van keuken en goedang-
zaken, als de beste uit Europa geïmporteerde huisvrouw. Zij zorgde
goed voor haar man en haar kleintjes. Als die maar „dik” waren, dan
leefde ze, en haar stelsel van vetmesten gelukte volkomen, ook wat
haarzelve betrof; de gansche familie zat terdege in het vleesch, en
Van Brakel, schoon hem de tweelingen aanvankelijk [3]zwaar op ’t
hart hadden gelegen, was geëindigd met er trotsch op te zijn.

Wanneer bezoekers deze welgeslaagde proeven van multiplicatie-

vermogen bewonderden, en de „engelen van kinderen” ’t vel van de
wangetjes kusten, dan kon Van Brakel er bij staan met een gezicht,
stralend van zelfvoldoening; een gezicht, waarop als het ware een
aanvraag stond te lezen om een gezegeld en geregistreerd
certificaat.

Sommige menschen, die wel wisten waar in dergelijke

omstandigheden de beroemde Abraham zijn geurigen mosterd
haalde, glimlachten spottend; maar de domme menigte kende den
ingenieur groote verdiensten toe.

Doch welk een goed vrouwtje Lucie ook was,—twee eigenschappen

miste zij: ze was niet erg zindelijk op haar huis, en zuinig was ze
evenmin; stof zag ze niet gauw, maar ze schreef verbazend snel een
bonnetje.

Dit eerste, en de eigenaardige geuren van de zuigelingen en van de

baboe, die vóór den divan op den grond lag te slapen, waren
oorzaak van de onfrissche lucht, die in het slaapvertrek heerschte.
Zij merkte het niet; ze was aan die soort van zaken gewoon; thuis,
toen haar moeder stierf, bleef ze met haar broertjes en zusjes
achter, en nu die groot waren, zat ze in een wip in haar eigen
kindertjes.

Zulke fijne reukorganen kwamen op haars vaders onderneming ook

niet te pas, en als Van Brakel thuis kwam, en een sterken
gemengden geur van tabak en brandy buiten en binnen de
k l a m b o e verspreidde, dan had ze daar zoo geen last van. Papa
Drütlich begroette steeds het opgaan der zon met zijn
F r ü h s c h o p p e n , rookte er groote pijpen zware [4]tabak bij, en
salueerde Morpheus met b r a n d y k r i n g en havana’s. Zoo’n
„heerenluchtje” hinderde haar niet; ze was het van kindsbeen af
gewoon.

En Van Brakel was een goed man. Hij hield veel van Lucie; net
zooveel als toen ze nog geëngageerd waren. Voor geen geld zou hij
haar ontrouw zijn geworden; zij wist, dat ze voor hem d e vrouw
was, en hij kwam daar altijd rond voor uit. Doch huiselijk van aard
was hij niet, en hij werd dat met elke maand minder. De sociëteit had
iets wonderlijk aantrekkelijks voor hem.

Sedert lang mopperde hij niet meer tegen den dienst. Men hoorde
hem niet meer afgeven op ongediplomeerde hoofdingenieurs en op
projecten, die toch nooit werden uitgevoerd. Hij ontplooide een
grooten ijver in het begrinten van wegen, het verven van
gouvernements-gebouwen, het witten en teeren van postloodsen. ’s
Morgens vroeg kon men hem reeds zien uitrijden in zijn bendy, hoe
verder, hoe liever. Het mocht dan waar wezen, dat hij geregeld elke
maand te kort kwam, en hij zijn „beren” even voordeelig zag groeien
en dik worden als zijn kroost,—het eenige wat nog strekken kon om
er niet al te diep onder te raken was een fatsoenlijk bedrag aan
declaraties elke maand.

Fluitend en pratend tegen zijn hond, die al blaffende om hem heen

sprong, liep hij voort in den helderen maneschijn; zijn blonde haren
krulden om zijn pet en de schaduw van zijn gezette figuur
dandineerde op het witte zand van den weg.

Het was een gewone avond in de sociëteit, want er werd geen

muziek gemaakt. [5]

De groote lokalen waren leeg. Uit de biljartzaal kwam het eentonig

getik der tegen elkaar loopende ballen. De kastelein, die de
verlichting uit zijn verdiensten moest betalen, was zoo vrij geweest
de lampen op „halve kracht” te stellen.

Er viel toch niets te verdienen op zoo’n avond!

Van Brakel ging door de lange voor- en binnengalerijen op het geluid

der biljartballen af. Daar ten minste was nog wat leven.
Hij trad binnen en knikte even met het hoofd een „goeden avond” in
het rond.

„Zoo,” zei de assistent-resident van politie, een vroolijk celibatair.

„Wat kom jij hier doen?”

„Ik kom eens zien of jullie je niet misdraagt,” lachte Van Brakel.

„Nou,” zei een ander ingenieur, die met den redacteur van een
dagblad aan het biljarten was, „we zijn altijd blij als we je rechtop
naar huis zien loopen.”

Men schertste, en critiseerde het spel, waarvan de spelers

uitmuntend geoefend waren, en men dronk er de eeuwige brandy-
soda bij. Het was een „vast clubje.” Van Brakel was het eenige
getrouwde lid. Zijn collega, de journalist en de assistent-resident
hielden trouw den gehuwden staat een „kleine vrouw” voor, waarop
Hymen telkens verschrikt en beschaamd de vlucht nam.

„Willen we?” vroeg, toen de partij uit was, een van de club, terwijl hij
met duim en vinger een beweging maakte, als wilde hij iets laten
tellen.

Zij glimlachten allen en keken elkaar aan; zij glimlachten, [6]zooals

verstandige, goed ontwikkelde en beschaafde menschen doen,
wanneer ze willen overgaan tot iets, wat ze weten dat verkeerd is,
dat strijdt tegen hun beschaving, ontwikkeling en verstand; zij
glimlachten als menschen, die heel goed weten, welke in het leven
de verboden vruchten zijn, maar er zich niettemin in koelen bloede
aan te goed gaan doen.

„Nog één keer, en dan nooit weer,” zei de assistent-resident.

Ze lachten nu luid, en al schertsend en lachend gingen ze naar een

hoek van de binnengalerij, waar het uitverkoren plekje was voor hun
zonde.

De bedienden brachten hun glazen; de mandoor haalde het

draaibord; elk zette een „lapje” van tien gulden bij; men draaide.

Er werd weinig bij gesproken. Zij waren echte spelers; zij speelden
niet om het genoegen van het spel, maar alleen om te winnen. Een
half uur waren ze aan den gang, maar het hielp niet. De kans was
zeer grillig; ieder won op zijn beurt; er ging „niets om.”

„We konden best vijf en twintig zetten,” meende er een.

Men keek elkaar even aan en knikte goedkeurend.—Het scheen te

helpen, de kans richtte zich naar v e i n e en d é v e i n e ; er werd
gewonnen en verloren; de hartstocht werd opgewekt en met de
grootste aandacht werd de beweging van den draaienden wijzer
gevolgd.

Uit de leeskamer der sociëteit, die met een deur in de binnengalerij

uitkwam, schreed langzaam een heer en ging voorbij het tafeltje der
spelers; hij groette zeer beleefd. [7]

Van Brakel mompelde iets met saamgeknepen lippen, terwijl hij hem
woedend nakeek. „Het is nu de tweede maal, dat die ploert me dit
levert.”

„Misschien heeft hij er geen bedoeling bij,” zei zijn collega.

„Nu ja! I k zeg je, hij doet het met opzet. M i j kan ’t niet schelen.”

„Mij ook niet.”

„Waar zeur je dan over? Kom, zet op!” viel de assistent-resident in,
en gaf met zijn dikke vingers een krachtigen zet aan het draaitoestel.
Van Brakel eindigde dien avond met een paar honderd gulden
verlies, maar het scheen hem niet te hinderen. Ook sprak men daar
niet over. Iemand, die over zijn verlies zou hebben gesproken of
getoond zou hebben, dat hij daar niet tegen kon, ware, althans in
hun clubje, een onteerd man. Of liever het was ondenkbaar, want
dan kon hij tot dat clubje niet behooren.

Men ging gezamenlijk biljarten om geld: een rijksdaalder per

carambole; dat was een billijk tarief.

Van Brakel won er een kleinigheid mee, maar het werd den spelers
te warm.

Nog één keer dobbelden ze, wat hem zijn biljartwinst weer afhandig
maakte, en met het slaan van tweeën gingen de vrienden naar huis.

Het was nog altijd een heerlijke nacht. Van Brakel’s hond was
vroolijk en blafte als een razende tegen de maan; maar de baas
werd door onaangename gedachten geplaagd.

Den volgenden dag was het traktementsdag. Verwenschte dag! Dat

was nu, naar zijn gevoelen, de ellendigste der [8]geheele maand,
terwijl het eigenlijk de aangenaamste wezen moest.

Het was dan toch ook schandelijk van het Gouvernement om iemand
van zijn positie en zijn dienstjaren zóó slecht te betalen. Zelfs m e t
de declaratie-gelden kon men van zoo’n inkomen niet leven! Had hij
een paar honderd gulden meer in de maand, dan was er doorkomen
aan. Nu gaf het slechts een agglomeratie van beren, waaraan geen
einde kwam. Men kon er waarlijk het einde niet van zien.

Zóó wandelde hij naar huis in droevige stemming, het lot

verwenschend, dat hem als ingenieur B. O. W. in de klauwen had
doen vallen van het Indisch Gouvernement.
Hij had dan een geweldigen afkeer van rekeningen ten zijnen laste,
en, welk een goed en gemoedelijk man hij ook overigens was, zoo
kon hij buiten zichzelven geraken van woede, bij het zien van een
mandoer, die met een portefeuille of een trommel vol quitanties het
erf op kwam. Deze bruine broeders, wetende welke onwelkome
verschijningen ze waren bij den toean ingenieur, bogen als
knipmessen nog vóór ze iemand zagen; maar zóó vriendelijk konden
ze niet wezen of Van Brakel zei, met een paar groote oogen, tegen
zijn Lucie:

„Daar heb je weer zoo’n smeerlap!”

Zij moest er om lachen. Haar gemoedelijke aard verloochende zich

nooit. Het was immers niets! „Als men niet kan betalen”, zei ze altijd
heel leuk, „dan zegt men maar l a i n b o e l a n ”. De tokohouders
verdienden, vond zij, toch genoeg.

Maar Van Brakel kon er niet tegen, en daarom zorgde hij steeds met
den maandelijkschen Grooten Verzoendag op reis [9]te zijn om te
zien naar de dijkjes, de postloodsen en wat zich verder koesterde
onder de vleugelen zijner technische bekwaamheid.

Alles sliep toen hij thuis kwam; zelfs toen hij in bed stapte en zijn
gewicht de ijzeren staven van het ledikant deed knarsen, werd Lucie
niet wakker, maar bleef rustig voortslapen, haar dikke, blanke armen
boven het hoofd gekruist. Nog een oogenblik zat Van Brakel
overeind, bedenkend of hij haar wakker zou maken of niet.

Hij deed het niet, want „k a s i a n ,” dacht hij, „ze is zoo moe”.

Wèl was het reeds drie uren, vóór hij rustig insliep, maar dat belette
hem niet met het vallen van het ochtendschot weer op te staan. Zijn
ijzersterk gestel veroorloofde hem alles; wat een ander in de
gematigde luchtstreek doodziek zou hebben gemaakt, dat kon hij
zich in de tropen ongestraft veroorloven. Een uur later was hij reeds
op weg naar „het werk”, dat zes, acht palen van de hoofdplaats werd
uitgevoerd.

Maar in dat uur, welk een drukte en bedrijvigheid! Als Lucie sliep,
dan was ze moeilijk wakker te krijgen, doch eenmaal goed uitgerust
ontwaakt, scheen zij een voor den ganschen dag opgewonden
uurwerk, dat met een krachtige vaart afliep en ’s avonds stilstond.

Nog was de duisternis niet geheel geweken, toen reeds alles in rep
en roer was; de koffie werd gezet, de tafel voor het ontbijt gedekt, de
zuigelingen schreeuwden van den honger, de baboes liepen heen en
weer met vochtig en geïllustreerd beddegoed, de katten miauwden,
Lucie gaf met luide [10]stem vier, vijf bevelen te gelijk aan de
bedienden, Van Brakel zocht vloekend een gesp voor zijn schoone
pantalon,—het was of met den nieuwen dag Satan was losgebroken,
alsof het een huishouden was van Jan Steen; kleine Wilhelm, het
oudste zoontje, gilde als een bezetene, omdat hij niet wilde baden,
en de driejarige Lucie beet haar baboe in de wang.

Zoo was na vier jaren van ongestoord huwelijksgeluk het „kleine

Paradijs”, waarover Lucie haar oude vriendin Louise Van der Linden
met zooveel enthusiasme had geschreven, toen ze nog pas kort
waren getrouwd.

Toen eindelijk Van Brakel vertrokken was, en de kinderen gevoed en

gereinigd met de baboes aan ’t wandelen waren, begon het
eenigszins rustiger te worden. Dan weldra snorden de naaimachines
van Lucie en haar m e n d j a h i t , welk eentonig en toch zenuwachtig
geratel alleen werd afgebroken door de meer of min onaangename
besprekingen met weldra van alle zijden opdagende Chineezen en
rekeningloopers.
Het was een nare geschiedenis toch tegenwoordig. Vroeger kreeg zij
elke maand geld gezonden van haar vader, en dat was zoo heerlijk;
Van Brakel wist er wel zoo iets van, maar hij vroeg er niet naar, en zij
kon met dat geld soms zoo ongemerkt de leemten in de ménage
aanvullen. Doch daarvan was in den laatsten tijd geen sprake meer.
Het was, schreef H e r r Drütlich, nu „d i e v e r d a m m t e
K a f f e e l a u s ” zijn boomen vernielde, niet meer mogelijk om haar
etwas te zenden, en daar bleef het bij, terwijl toch de uitgaven elken
dag toenamen.

Na zijn tochtje, dat tot twaalf uren ’s middags duurde, reed Van
Brakel beslijkt en bestoven het erf op van de sociëteit. [11]De Club-
leden zaten er reeds. Zijn collega, iemand altijd even net en
bedaard, zoo in zijn uiterlijk als wat zijn manieren betreft, zag hem
opmerkzaam aan, toen hij uit zijn voertuig stapte.

„Vindt je niet dat Braak erg achteruitgaat?” vroeg hij den assistent-
resident.

„Och, het gaat nogal. ’n Beetje vet, anders niet.”

„Je hebt hem vroeger niet gekend. Te Delft was hij een van de netste
kerels. En nu heeft hij hier in Indië iets verschrikkelijk ordinairs
gekregen.”

„Kom, dat is zoo erg niet!—Dag Braak, ’n paitje?”

„Dat was nog zoo ’n kwaad idee niet,” meende Van Brakel.

Zij bleven zitten tot tegen twee uren. Sommigen hadden zich tot
weinig consumtie beperkt: het waren de ambtenaren, die nog naar
hun kantoren moesten; maar Van Brakel en de assistent-resident,
die t o c h m a a r naar huis gingen, hadden een „slordig bittertje”
gedronken.
Thuis vond hij Lucie met een van de tweelingen aan de borst, en
bezig een glaasje Spaanschen wijn te drinken, wat zoo goed was en
zoo versterkend; hij vond het erg gezellig en accompagneerde haar
met nog een weinig volksdrank, waarna zij aan tafel zwart Engelsch
bier dronk om zich te versterken en hij bruin Duitsch bier omdat hij
het lekker vond.

„Ik heb een brief gekregen van pa,” zei ze onder het eten.

„Zoo, hoe maakt hij het?”

„O heel goed.”

„En hoe gaat het met de K a f f e e l a u s ?”

„Akelig! Geen driehonderd pikols van ’t jaar.” [12]

„’t Is beroerd.”

„’t Is verschrikkelijk. Zoo’n mooi land!”

„En wat denkt hij nu te doen?”

„Ja, wat zal hij doen?”

Dat wist Van Brakel ook niet, en daarom zweeg hij maar liever.

Lang zouden ze geslapen hebben dien middag, zoo niet tegen

halfvijf een politie-oppasser was gekomen met een briefje van den
assistent-resident, die in vliegende haast meldde, dat hij per
telegram was overgeplaatst en in weinige dagen naar zijn nieuwe
standplaats moest vertrekken, omdat de overneming van den dienst
daar geen uitstel kon leiden.

„Kom, ik ga gauw baden,” zei Van Brakel tegen Lucie. „Je begrijpt,
dat wij hem in elk geval een heerendinertje moeten aanbieden.”