Biology of Microfungi 2016 PDF

Fungal Biology
De-Wei Li Editor
Biology of
Microfungi
Fungal Biology
Series Editors:
Vijai Kumar Gupta, PhD
Molecular Glycobiotechnology Group, Department of Biochemistry,
School of Natural Sciences, National University of Ireland Galway,
Galway, Ireland
Maria G. Tuohy, PhD

Molecular Glycobiotechnology Group, Department of Biochemistry,
School of Natural Sciences, National University of Ireland Galway,
Galway, Ireland
More information about this series at http://www.springer.com/series/11224

De-Wei Li
Editor
Biology of Microfungi
Editor
De-Wei Li
The Connecticut Agricultural Experiment
Station Valley Laboratory
Windsor, CT, USA
Co-Innovation Center for Sustainable

Forestry in Southern China
Nanjing Forestry University
Nanjing, Jiangsu, China
ISSN 2198-7777 ISSN 2198-7785 (electronic)

Fungal Biology
ISBN 978-3-319-29135-2 ISBN 978-3-319-29137-6 (eBook)
DOI 10.1007/978-3-319-29137-6
Library of Congress Control Number: 2016933679
Springer Cham Heidelberg New York Dordrecht London

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Preface
To me, mycology is not only a branch of biological science but also an acquired
interest and passion. It has led me to cross the Pacific Ocean from China to Canada
27 years ago and later to the USA to pursue my professional dream. It was a long
trip, full of challenges and sometimes doubts. When I was teaching plant pathology
at Olds College in Alberta, I was very grateful to Olds College for offering me my
first job in North America after my graduate studies and postdoctoral work. At the
same time, I missed mycological research and assumed that mycology might have
parted from me. During the pinnacle of public health concerns to indoor molds
along with a mounting number of indoor mold-related litigations in the USA in the
early 2000s, an industry job provided me an opportunity to return to mycology by
working on indoor molds, most of which are microfungi. I have been working on
microfungi and aeromycology ever since. This is one of the major reasons why the
book was titled “Biology of Microfungi.”
The focus of this book is principally on covering the latest development of
research on microfungi from both systematic and practical aspects. In a broad sense,
Mesofungi were also covered. It is not an overstatement that microfungi are in our
daily life, but we normally do not realize the presence of these microfungi. It is
almost impossible and impractical for us to live a microfungi-free environment,
whether we recognize it or not. Fungi are ubiquitous. Spores of microfungi are pres-
ent in the air. We breathe them in and out all the time. Microfungi are both our
friends and foes. Without microfungi, we would not be able to enjoy our bread,
mantou (steamed bun), other baked food, fermented food, preserved food, alcoholic
beverages (beer, wine, liquor, tequila, rice wine, etc.), and access to some modern
medication, such as penicillin and cyclosporin, etc., which are secondary metabo-
lites of microfungi. Microfungi have been directly used as medicinal herbs in
Chinese medicine to treat various diseases for over 1000 years. A number of micro-
fungi have been used as biocontrol agents to manage plant insects and diseases,
such as Trichoderma spp., Clonostachys rosea (Link) Schroers et al. (≡ Gliocladium
rosea Bainier) for plant disease control, Beauveria bassiana (Bals.-Criv.) Vuill., and
Metarhizium anisopliae (Metchnikoff) Sorokin for controlling insects. Without
microfungi as decomposers, our planet would be buried by numerous mountains of
v
vi Preface
plant litter and nutrient flow/recycle in our ecosystem would be interrupted or even
stopped. Without the microfungi of Glomeromycota to form arbuscular mycorrhi-
zae, the host plants’ adaptability to adverse environments would be significantly
reduced and some may not even be able to survive. For these aspects, microfungi are
our friends. On the negative aspects, some microfungi are pathogenic to humans,
animals, or plants. Some microfungi, such as Stachybotrys chartarum, Fusarium
spp., Aspergillus flavus, are able to produce mycotoxins, which are detrimental to
the health of humans and animals. Microfungi, especially airborne fungal spores,
can be allergens to some individuals. Thus, these microfungi are our foes. Some
microfungi can be both beneficial and detrimental to human beings. For example,
Tilletia hordei, Ustilago crameri, Ustilago maydis (DC.) Corda, and Ustilago nuda
(C.N. Jensen) Rostrup cause smuts on a number of cereal crops and grasses, but
they are used as medicinal remedies in Chinese medicine.
Have we fully explored the resources of microfungi? The answer is definitely NO.
This book is a collective effort of a team of mycologists who either contributed
chapter(s) to this book or reviewed manuscripts to make this book possible. I am so
fortunate that such a wonderful group of mycologists accepted my invitation and
committed themselves and their time to contribute to this book. At the time of com-
pleting this book, I am very grateful to have had this opportunity to edit it. I have
learnt so much from what the chapter authors have covered. However, any errors in
the book are mine.
The editor would like to express his gratitude to Dr. Bryce Kendrick for his men-
torship and friendship. Without his courage and support, I would not have been able
to take on this editorial task. The editor is very appreciative to the authors for their
excellent contributions. Without their collaboration, it would be impossible to see
this book in print. I am very grateful to the former and current directors of The
Connecticut Agricultural Experiment (CAES), Dr. John Anderson, the late Dr.
Louis A. Magnarelli, and Dr. Theodore G. Andreadis and Chief Scientist of Valley
Laboratory, Dr. James LaMondia, for their support. I am very privileged to work at
CAES as a mycologist and enjoy my mycological research. I am very much indebted
to my wife, Jin Zhang (ᕐ⪮) for her unreserved love and persistent support.
Windsor, CT De-Wei Li
Nanjing, Jiangsu, China ᶄᗭՕ
Contents
1 Introduction: Advances and Predicament ............................................ 1

De-Wei Li
2 Recent Changes in Fungal Nomenclature and Their Impact
on Naming of Microfungi ....................................................................... 7
Walter Gams
3 Future Perspectives and Challenges of Fungal Systematics
in the Age of Big Data ............................................................................. 25
Zheng Wang, R. Henrik Nilsson, Timothy Y. James, Yucheng Dai,
and Jeffrey P. Townsend
4 Molecular Techniques in Mycological Studies and Sequence
Data Curating: Quality Control and Challenges ................................. 47
R. Henrik Nilsson, Kessy Abarenkov, and Urmas Kõljalg
5 Challenges and Future Perspectives in the Systematics
of Kickxellomycotina, Mortierellomycotina, Mucoromycotina,
and Zoopagomycotina.............................................................................. 65
Gerald L. Benny, Matthew E. Smith, Paul M. Kirk, Eric D. Tretter,
and Merlin M. White
6 Entomophthoromycota: A New Overview of Some
of the Oldest Terrestrial Fungi............................................................... 127
Richard A. Humber
7 Latest Developments in the Research of Rust Fungi
and Their Allies (Pucciniomycotina) ..................................................... 147
Merje Toome-Heller
8 Conidiogenesis: Its Evolutionary Aspects in the Context
of a Philosophy of Opportunity (Lectics) .............................................. 169
Richard C. Summerbell and James A. Scott
vii
viii Contents
9 Fungal Diversity of Central and South America.................................. 197

Rafael F. Castañeda-Ruiz, Gabriela Heredia, Luis F.P. Gusmão,
and De-Wei Li
10 Mesofungi................................................................................................. 219
Bryce Kendrick
11 Evolution of Fungi and Update on Ethnomycology ............................. 237
De-Wei Li, R.F. Castañeda-Ruiz, and James LaMondia
12 Phylogenetic Diversity of Fungi in the Sea including
the Opisthosporidia .................................................................................. 267
Ka-Lai Pang and E.B. Gareth Jones
13 Biology and Ecology of Freshwater Fungi ............................................ 285
Clement K.M. Tsui, Christiane Baschien, and Teik-Khiang Goh
14 Dispersal Strategies of Microfungi ........................................................ 315
Donát Magyar, Máté Vass, and De-Wei Li
15 Microfungi in Indoor Environments: What Is Known
and What Is Not ...................................................................................... 373
Chin Yang, Sepideh Pakpour, John Klironomos, and De-Wei Li
16 Biology of the Whiskey Fungus ............................................................. 413
James A. Scott and Richard C. Summerbell
17 Allergenic Microfungi and Human Health: A Review
on Exposure, Sensitization, and Sequencing Allergenic Proteins ....... 429
Mercedes Amado and Charles Barnes
18 What’s Old is New: Recognition of New Fungal Pathogens
in the Era of Phylogenetics and Changing Taxonomy
and Implications for Medical Mycology ............................................... 451
Nathan P. Wiederhold and Deanna A. Sutton
19 Mycotoxins in Food and Feed: A Challenge
for the Twenty-First Century ................................................................. 469
J. David Miller
20 Inhalation Exposure and Toxic Effects of Mycotoxins ........................ 495
Harriet M. Ammann
21 Fungi in Fermentation and Biotransformation Systems ..................... 525
Carla C.C.R. de Carvalho
22 Microfungi in Biofuel and Bioenergy Research ................................... 543
Richa Raghuwanshi, Shalini Singh, Mohd. Aamir, Amrita Saxena,
Vijai Kumar Gupta, and R.S. Upadhyay
Contents ix
23 Interactions of Microfungi and Plant-Parasitic Nematodes ............... 573

James LaMondia and Patricia Timper
24 Pathogenic Microfungi Associated with Spartina in Salt Marshes ..... 615
Wade H. Elmer
Index ................................................................................................................. 631

Contributors
Mohd. Aamir, Ph.D. Department of Botany, Banaras Hindu University, Varanasi,

India
Kessy Abarenkov, Ph.D. Natural History Museum, University of Tartu, Tartu, Estonia
Mercedes Amado, M.D. Pediatric Allergy & Immunology, Allergy Asthma and
Immunology, Children’s Mercy Hospitals & Clinics, Kansas City, MO, USA
University of Missouri-Kansas City School of Medicine, Kansas City, MO, USA
Harriet M. Ammann, Ph.D. Department of Environmental & Occupational
Health Sciences, School of Public Health and Community Medicine, University of
Washington, Seattle, WA, USA
Charles Barnes, Ph.D. University of Missouri-Kansas City School of Medicine,
Kansas City, MO, USA
Division of Allergy/Immunology Laboratory, Children’s Mercy Hospitals and
Clinics, Kansas City, MO, USA
Christiane Baschien, Ph.D. Department of Microbial Ecology and Diversity,
Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures,
Braunschweig, Germany
Gerald L. Benny, Ph.D. Department of Plant Pathology, University of Florida,
Gainesville, FL, USA
Rafael F. Castañeda-Ruiz, Ph.D. Instituto de Investigaciones Fundamentales en
Agricultura, Tropical ‘Alejandro de Humboldt’ (INIFAT), Académico Titular de la
Academia de Ciencias de Cuba, Santiago de Las Vegas, C. Habana, Cuba
Yucheng Dai, Ph.D. Institute of Microbiology, Beijing Forestry University,
Beijing, China
Carla C.C.R. de Carvalho, Ph.D. iBB-Institute for Bioengineering and Biosciences,
Department of Bioengineering, Instituto Superior Técnico, Universidade de Lisboa,
Av. Rovisco Pais, Lisbon, Portugal
xi
xii Contributors
Wade H. Elmer, Ph.D. Department of Plant Pathology and Ecology, The

Connecticut Agricultural Experiment Station, New Haven, CT, USA
Walter Gams, Ph.D. Formerly The CBS-KNAW Fungal Biodiversity, The Netherlands
Molenweg, CK Baarn, The Netherlands
Teik-Khiang Goh, Ph.D. School of Biological Sciences, Faculty of Integrative
Sciences & Technology, Quest International University Perak, Perak Darul Ridzuan,
Malaysia
Vijai Kumar Gupta, Ph.D. Molecular Glyco-biotechnology Group, Discipline of
Biochemistry, School of Natural Sciences, National University of Ireland Galway,
Galway, Ireland
Luis F.P. Gusmão, Ph.D. Laboratorio de Micologia, Departamento de Ciências
Biológicas, Universidade Estadual, Feira de Santana, Brazil
Gabriela Heredia, Ph.D. Red de Biodiversidad y Sistemática, Instituto de
Ecología, Xalapa, Veracruz, Mexico
Richard A. Humber, Ph.D. USDA-ARS Emerging Pests and Pathogens Research
Unit, Robert W. Holley Center for Agriculture and Health, Ithaca, NY, USA
Timothy Y. James, Ph.D. Department of Ecology and Evolutionary Biology,
University of Michigan, Ann Arbor, MI, USA
E.B. Gareth Jones, Ph.D. Botany and Microbiology Department, College of
Science, King Saud University, Riyadh, Saudi Arabia
Bryce Kendrick, Ph.D., D.Sc., F.R.S.C. 8727 Lochside Drive, Sidney-by-the-Sea,
BC, Canada
Department of Biology, University of Waterloo, Waterloo, ON, Canada
Paul M. Kirk, Ph.D. Mycology Section, Kew Gardens, Royal Botanic Gardens,
Kew, Richmond, Surrey, UK
John Klironomos, Ph.D., F.R.S.C. Department of Biology, University of British
Columbia, Okanagan campus, Kelowna, BC, Canada
I.K. Barber School of Arts and Sciences, The University of British Columbia,
Okanagan Campus, Kelowna, BC, Canada
Urmas Kõljalg, Ph.D. Natural History Museum, University of Tartu, Tartu, Estonia
James LaMondia, Ph.D. The Connecticut Agricultural Experiment Station Valley
Laboratory, Windsor, CT, USA
De-Wei Li, Ph.D. The Connecticut Agricultural Experiment Station Valley
Laboratory, Windsor, CT, USA
Co-Innovation Center for Sustainable Forestry in Southern China, Nanjing Forestry
University, Nanjing, Jiangsu, China
Contributors xiii
Donát Magyar, Ph.D. Department of Air Hygiene and Aerobiology, National

Public Health Center, Budapest, Hungary
J. David Miller, Ph.D. Department of Chemistry, Carleton University, Ottawa,
ON, Canada
R. Henrik Nilsson, Ph.D. Department of Biological and Environmental Sciences,
University of Gothenburg, Göteborg, Sweden
Sepideh Pakpour Department of Biology, University of British Columbia,
Okanagan campus, Kelowna, BC, Canada
Okanagan Campus, Kelowna, BC, Canada
Ka-Lai Pang, Ph.D. Institute of Marine Biology and Center of Excellence for the
Oceans, National Taiwan Ocean University, Keelung, Taiwan (R.O.C.)
Richa Raghuwanshi, Ph.D. Department of Botany, Mahila Mahavidyalaya,
Banaras Hindu University, Varanasi, India
Amrita Saxena, Ph.D. Department of Botany, Banaras Hindu University, Varanasi,
India
James A. Scott, Ph.D. Division of Occupational and Environmental Health, Dalla
Lana School of Public Health, University of Toronto, Toronto, ON, Canada
Sporometrics Inc., Toronto, ON, Canada
Shalini Singh, Ph.D. Department of Botany, Banaras Hindu University, Varanasi,
India
Matthew E. Smith, Ph.D. Department of Plant Pathology, University of Florida,
Gainesville, FL, USA
Richard C. Summerbell, Ph.D. Sporometrics Inc., Toronto, ON, Canada
Dalla Lana School of Public Health, Gage Occupational and Environmental Health
Unit, University of Toronto, Toronto, ON, Canada
Deanna A. Sutton, Ph.D. Fungus Testing Laboratory, Department of Pathology,
University of Texas Health Science Center at San Antonio, San Antonio, TX, USA
Patricia Timper, Ph.D. USDA ARS, Tifton, GA, USA
Merje Toome-Heller, Ph.D. Plant Health and Environment Laboratory, Ministry
for Primary Industries, Auckland, New Zealand
Jeffrey P. Townsend, Ph.D. Department of Biostatistics, Yale School of Public
Health, Yale University, New Haven, CT, USA
Department of Ecology and Evolutionary Biology, Yale University, New Haven,
CT, USA
Program in Computational Biology and Bioinformatics, Yale University, New
Haven, CT, USA
xiv Contributors
Eric D. Tretter, M.S. Department of Biological Sciences, Boise State University,

Boise, ID, USA
Clement K.M. Tsui, Ph.D. Department of Pathology and Laboratory Medicine,
University of British Columbia, Vancouver, BC, Canada
R.S. Upadhyay, Ph.D. Department of Botany, Banaras Hindu University, Varanasi,
India
Máté Vass Department of Ecology and Genetics, Limnology, Uppsala University,
Uppsala, Sweden
Zheng Wang, Ph.D. Department of Biostatistics, Yale School of Public Health, Yale
University, New Haven, CT, USA
Merlin M. White, Ph.D. Department of Biological Sciences, Boise State University,
Boise, ID, USA
Nathan P. Wiederhold, Ph.D. Fungus Testing Laboratory, Department of
Pathology, University of Texas Health Science Center at San Antonio, San Antonio,
TX, USA
Chin Yang, Ph.D. Prestige EnviroMicrobiology, Inc., Voorhees, NJ, USA
Chapter 1
Introduction: Advances and Predicament
De-Wei Li
What Are Microfungi?
Microfungi are fungi that develop small (microscopic) fruiting bodies (Kirk et al.
2008). These fungi are also referred to as Micromycetes or microscopic fungi. They
need to be observed and morphologically studied under a microscope. Fungi and
mycology were at one point considered a branch of botany. In 1969, Robert
Whittaker segregated fungi from Kingdom Plantae and erected a new fifth king-
dom: Kingdom Fungi, or Eumycota, to the biological classification when phyloge-
netic studies indicated that fungi (true fungi) are closer to animal than to plants
(Whittaker 1969). This is the reason why Dr. Bryce Kendrick named his popular
mycology textbook as The Fifth Kingdom (Kendrick 1985). Some fungi tradition-
ally studied by mycologists belong to two other Kingdoms (Protozoa and Chromista).
However, this book will mainly cover the microfungi in the Kingdom Fungi.
At present, fungi are still covered in textbooks of botany or plant sciences. The
nomenclature of fungi remains governed under the latest code: The International
Code of Nomenclature for algae, fungi, and plants (Melbourne Code) adopted by
the Eighteenth International Botanical Congress Melbourne, Australia, July 2011
(formerly The International Code of Botanical Nomenclature) (McNeill et al. 2012).
The change to Article 59 to terminate the two name system for sexual and asexual
states of fungi in the Melbourne Code and that “one fungus equals to one name” was
adopted. The significance and subsequent implications of the change are discussed
in the book.
D.-W. Li, Ph.D. (*)

The Connecticut Agricultural Experiment Station Valley Laboratory,
153 Cook Hill Road, Windsor, CT 06095, USA
University, Nanjing, Jiangsu 210037, China
e-mail: dewei.li@ct.gov
© Springer International Publishing Switzerland 2016 1

D.-W. Li (ed.), Biology of Microfungi, Fungal Biology,
DOI 10.1007/978-3-319-29137-6_1
2 D.-W. Li
The history of mycology has been covered in detail by several authors (Lütjeharms
1936; Ramsbottom 1941; Braun 1965; Ainsworth 1976; Rippon 1982; Bridge
2002), but early evolutionary history remains less studied due to the lack of fungal
fossils. However, new molecular technology has made molecular clock dating pos-
sible and provides a much better tool to study the evolution of fungi. No doubt,
DNA sequencing, next-generation sequencing (high-throughput sequencing tech-
nologies), and genomic studies of fungi have led to major developments in the sys-
tematics, diversity, biochemistry, and ecology of fungi in the last 20 years. Fungal
fossils are scarce in comparison with organisms in the Kingdoms of plants and
animals for studies of fungal evolution. New technology allows us to overcome the
difficulties in insufficient fossil records to understand fungal evolution. At the same
time, more and more new data from research on fossils and archeological evidence
allow us to better understand the relationship between fungi and human beings,
especially the role fungi played in pre- and early historic periods of human beings.
Studies in the last two decades have led to discoveries of several major fungal
groups previously unknown to science such as Cryptomycota (Jones et al. 2011) or
substantial changes to certain taxonomic groups, such as former Zygomycota (zygo-
mycetous fungi) from which new phyla Glomeromycota (Schüßler et al. 2001) and
Entomophthoromycota and subphyla Kickxellomycotina, Mortierellomycotina,
Mucoromycotina, and Zoopagomycotina were segregated (Benny et al. 2014).
Several new phyla have been erected. Several chapters will cover the latest develop-
ment of fungal systematics and the subsequent changes to the major taxonomic
groups related to microfungi. Microsporidia (ca. 1500 species described) is a phy-
lum newly transferred to Kingdom Fungi from Kingdom Protista based on phyloge-
netic studies. However, this new development has an unwanted implication. It
would render ca. 1000 species invalid, since these species were named following
zoological nomenclature based on the view of Microsporidia belonging to Protista,
because fungi are subject to the rules of botanical nomenclature (Keeling 2009).
Such destabilization of these names will ultimately lead to huge chaos to these taxa
of Microsporidia. To avoid an unnecessary large number of name changes and a
contentious chaotic situation for Microsporidia, the phylum Microsporidia is
excluded from governance by the Melbourne Code and continue to be covered by
the International Code of Zoological Nomenclature, despite their phylogenetic posi-
tion (McNeill et al. 2012). This is a necessary solution, but an imperfect one. It has
brought back the old question should all nomenclature codes for life [International
Code of Nomenclature for algae, fungi, and plants (ICN), International Code of
Zoological Nomenclature (ICZN), International Code of Nomenclature of Bacteria
(ICNB), International Committee on Taxonomy of Viruses (ICTV)] be unified?
Such unification will break down some barriers or boundaries and expedite cross-
field collaborations. No doubt this is a huge undertaking.
A recent fungal biogeographical study by Tedersoo et al. (2014) on patterns of
biodiversity of soil fungi worldwide using a mixture of six forward primers analo-
gous to ITS3 and a degenerate reverse primer analogous to ITS4 demonstrated an
exponential decline in fungal species richness and the plant-to-fungus richness ratio
with distance from the equator, and at the same time the diversity of a number of
1 Introduction: Advances and Predicament 3
fungal groups depended more on the abundance of host plants than host diversity or
geography. This study indicated an enormous gap between described species and
the actual numbers of distinct fungi in the soils at the global scale.
Discussions on this study indicated that the fungal diversity of certain fungal
groups may not be accurately estimated due to limitations of the molecular method
used. Tedersoo et al. (2014) acknowledged that Tulasnellaceae and Microsporidia
would not be amplified with the primer set they used, and ca. 30 % Tulasnella have
approximately two mismatches and Archaeorhizomycetes. Thus, Schadt and Rosling
(2015) pointed out that there is a likely bias caused by mismatched polymerase
chain reaction (PCR) primer for Archaeorhizomycetes in the soils. It was previously
shown that the ITS4 reverse primer has at least two mismatches to all known spe-
cies in the Archaeorhizomycetes (Rosling et al. 2011). These mismatches resulted in
at least a tenfold underrepresentation of Archeaorhizomycetes. Thus, Schadt and
Rosling (2015) opined that Archaeorhizomycetes are not low-diversity, low-
abundance soil fungi as reported by Tedersoo et al. (2014), but the opposite.
Fungal diversity can be overestimated also. Cloning or massive parallel sequenc-
ing may significantly overestimate fungal diversity if there are sufficient differences
among these sequences within a fungal individual or taxon due to the fact that these
sequences were derived from single alleles (Lindner and Banik 2011; Lindner and
Banik 2011; Lücking et al. 2014; Větrovský et al. 2015). Větrovský et al. (2015)
indicated that operational taxonomic unit (OTU) counts and relative abundance of
the Basidiomycota were highly overestimated by ITS. The variable length of the
ITS region represents another important problem as there is a strong PCR bias
against species with longer amplicons (Ihrmark et al. 2012). Thus, the methods to
study fungal diversity should be improved to better reflect fungal diversity. At pres-
ent this is the area where more studies should be conducted on validation or calibra-
tion of certain methods.
It is our intention that all authors can use this book as a platform to discuss not
only the development in their areas, but also to express their opinions on weak-
nesses or gaps in microfungi research and identify the future directions and areas
which remain overlooked.
There are several different estimates of the number of fungi (Blackwell 2011).
A widely accepted estimate for fungal diversity is that there are 1.5 million species
of fungi on our planet (Hawksworth 1991). At present, approximately 100,000 spe-
cies (<7 % of total fungal taxa) have been described. Among the undescribed fungal
taxa, majority are microfungi. There is no doubt that mycologists have a long way
to go and a large number of fungi are waiting for us to find and describe. Tropics
and subtropics are very rich in mycota with a higher fungal diversity. Microfungal
Diversity of Central and South America covered in Chap. 9 will show you not only
the beauty of microfungi and their diversity and habitats, but also the methodology
to collect and study them. Among these fungi, many were found from the areas as
new taxa in the last three decades. Unfortunately, the tropic and subtropical areas in
Central and South America, Asia, and Africa remain less studied at present. Not
only do we have to describe a huge number of fungi which remain unknown to sci-
ence, but also to rescue the endangered fungal species from extinction due to global
4 D.-W. Li
warming and loss of habitats from human activities such as population growth,
overexploitation, and overdevelopment. Deforestation in the Amazon River area
and South Asia is depleting the habitats of not only fungi, but all life in the areas and
is reducing biodiversity (Foley et al. 2007; Davidson et al. 2012; Zhai et al. 2014).
Amazonian forests have a significant impact on regional and global climates and
deforestation in the area can be a driver of climate change (Malhi et al. 2008).
Reduced habitats will result in decline of fungal diversity and cause some fungal
taxa to disappear prior to even having a chance to describe them.
There are about two dozen fungi listed as rare, threatened, and endangered fungi
in the webpage of Mushroom Observer. All these species on the list are macrofungi
(Mushroom Observer 2015). The International Union for Conservation of Nature
(IUCN) red list of endangered species listed four lichens: Anzia centrifuga, Cladonia
perforata (Florida perforate reindeer lichen), Erioderma pedicellatum (boreal felt
lichen), Gymnoderma insulare, and Pleurotus nebrodensis (white Ferula mushroom)
as endangered fungal species (IUCN 2014). At present, 77 species are suggested for
a Global Red List Assessment (GFRLI 2015). Among those species, they are all
either macrofungi or lichens while not a single microfungus is present. The main
embarrassment is that we do not have enough data on microfungi to assess and pro-
pose to the Global Red List. One simple assessment method is that for each plant
species we lose, at least six fungal species would be lost. Among the lost species,
about 89 % would be microfungi. This conservative estimate is based on the number
of described fungi and the ratio of macrofungi and microfungi described (Kirk et al.
2008). This is an area which is severely overlooked. New initiatives for research on
endangered microfungi should be promoted. The key issue is how to balance nature
conservation and economic development.
The biggest challenge to mycology is the continuous decline of the mycological
profession. It is rather depressing to see that many mycologist positions have disap-
peared in the academic and teaching institutions in the past two decades in North
America. The membership of Mycological Society of America (MSA) has dropped
29 % from 1342 to 953 members between 2002 and 2015 (Cantrell 2014, 2015). It
is shocking to watch MSA lose so many members in a decade. Crisis may not be an
overstatement. Since so many advances have been made in mycology in the past
decade, why have so many mycologists left the fields of mycology and why is there
not enough new members to fill in? No doubt it is an alarming and embarrassing
trend to all mycologists and society. Without fungi, the biosphere and biodiversity
would be incomplete. What are the key reasons or leading factors behind this incli-
nation? Lack of research funding and shrinking job market for mycologists may be
considered the major contributing factors by a majority of mycologists, but what
else? Can we reverse it? How? Microfungi, like macrofungi, are a fascinating group
of organisms to study from morphology, ecology, and phylogenetics. It is our obli-
gation to inspire more young talented individuals’ interests in mycology, and these
promising future mycologists can be trained to explore and study not only the
biological significance, but also the beauty of microfungi from both morphological
and molecular perspectives.
1 Introduction: Advances and Predicament 5
This book comprises of 24 chapters. Several chapters update the latest development
of fungal systematics, especially the phyla related to microfungi, and fungal diver-
sity. The perspectives and challenges we are facing in the molecular age, such as
data curating and quality control, are also discussed. At the same time, this book
focuses on the ecological and functional aspects of microfungi. This part covers a
broad spectrum of areas from indoor to freshwater fungi, nematode fungi, and aller-
genic fungi to mycotoxins. However, there are several areas that were less covered
in the past, such as marine fungi, whisky fungus, and spartina fungi. If you are
wondering what is whisky fungus, please read Chap. 16 to find out how it was dis-
covered and described and its relationships with people and whisky making. It is a
fascinating story about an alcoholphilic fungus. Can a microfungus become alco-
holic? This chapter will answer this question.
It is our intention that information on recent advances in mycology presented in
this book will be useful to identify the needs in mycological research and to determine
the direction or niches for future research. If the book can strike some sparks for novel
ideas and in-depth discussion, its objective has been reached.
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Chapter 2
Recent Changes in Fungal Nomenclature
and Their Impact on Naming of Microfungi
Walter Gams
The Previous Situation
As documented in the International Code of Botanical Nomenclature (the ICBN,

Article 59, latest edition by McNeill et al. 2006), fungal nomenclature had an
unusual situation for several decades: the often very different forms of sporulation
(morphs) of a fungus could have different valid and legitimate names in different
genera, which were either teleomorph or anamorph typified. The name attached to
the teleomorph had precedence over older anamorph-based names. The basic idea
behind this rule was that only with the knowledge of the teleomorph could a fungus
be inserted in a natural taxonomic system while that of anamorphs was deemed to
remain more or less artificial (e.g., Gams 1995). The most complete compilation of
anamorph–teleomorph connections is presented for hyphomycete anamorph genera
by Seifert et al. (2011). Another comparable compilation was provided by
Wijayawardene et al. (2012).
This situation of dual nomenclature was in conflict with the time-honored prin-
ciple IV of the Code: one organism–one name. It was also abnormal because a qual-
ity was demanded for the type: presence of a (sexual) teleomorph in the diagnosis
and specimen to make it acceptable as a teleomorph name; otherwise it was anamor-
phic. In the era of molecular work, this abnormality appeared inappropriate,
although the dual system had many advantages for the (morphological) identifica-
tion of a fungus. A change toward unification was postulated mainly by molecular
phylogeneticists, while other taxonomists were afraid of a flood of name changes
inevitably following such a change.
W. Gams, Ph.D. (*)

Formerly The CBS-KNAW Fungal Biodiversity, 3584 CT Utrecht, The Netherlands
Molenweg 15, 3743 CK Baarn, The Netherlands
e-mail: walter.gams@online.nl

DOI 10.1007/978-3-319-29137-6_2
8 W. Gams
Preparations for a Unification
In Studies in Mycology 45 (Seifert et al. 2000), several authors explored how

molecular studies affected the classification of Ascomycetes and showed how far the
discrepancy between anamorph and teleomorph taxonomy still persisted. This
incongruence of anamorph-defined and teleomorph-defined genera was in fact one
of the major obstacles toward a unification. Grégoire Hennebert, in a philosophical
text published temporarily on the CBS website, outlined several scenarios for a pos-
sible change, according to which the nomenclatural changes might be reduced to a
bearable minimum. Keith Seifert and Paul Cannon convened a symposium at the
Oslo IMC (Seifert et al. 2003), during which two teams, defending either unification
or the conservative side, debated heavily. The audience then was requested to vote,
resulting in a majority of 121:84 for retaining the dual system.
Since 2000 the molecular-based classification of fungi has made dramatic prog-
ress. The Genealogical Concordance Phylogenetic Species Recognition (GCPSR)
concept introduced by Taylor et al. (2000) has become broadly recognized and
allows an almost final phylogenetic reconstruction, only inferior to an entire
genome-based classification. In contrast to plant species, some 95 % of fungal spe-
cies are estimated to be undescribed (Hawksworth 2004b). Less than 20 % of the
known fungal species are represented with sequences in GenBank (Hawksworth
2004b). Only ca. 15.5 % of the known (<3 % of the estimated) species have known
ITS sequences which are considered as universal barcodes (Schoch et al. 2012),
although they lack resolution in some species-rich groups such as Colletotrichum,
Fusarium, or Trichoderma. The largely varying quality of available DNA data is not
sufficiently considered in molecular-based classification. Up to 20 % of fungal ITS
sequences in GenBank are labeled with wrong species names (Nilsson et al. 2006).
The different technical quality of sequencing (reading errors, ambiguous positions,
simple reads vs. double reads, chimeric sequences) also leads to ambiguity of data
interpretation. Sequences available in GenBank are often used without checking
whether they have undergone a review process or are unpublished. Sequences
derived from vouchers identified by other (primary) means than comparisons with
DNA data are obviously superior to sequences only identified (secondarily) by
comparison with other already available sequences. Schoch et al. (2014) are making
laudable efforts to select and re-annotate a set of marker reference sequences that
represent each currently accepted order of fungi.
At the International Botanical Congress at Vienna in 2005, just one issue was
resolved toward unification: A proposal (Hawksworth 2004a) was passed that
allowed epitypification of a previously anamorphic taxon with teleomorphic mate-
rial in order to avoid unnecessary name changes. For this procedure Redhead
(2010b) introduced the term teleotypification. Gams et al. (2010a, b) discussed
some problems associated with the procedure and wanted to restrict the mechanism
to cases where no appropriate teleomorph genus was yet available.
At the Vienna Congress, a Special Committee on the Nomenclature of Fungi
with a Pleomorphic Life History, headed by Scott Redhead, was established and
given the mandate of finding solutions for the problems surrounding Article 59.
2 Recent Changes in Fungal Nomenclature and Their Impact… 9
The members of this committee were rather evenly distributed from both opposing
camps and did not reach a conclusion (Redhead 2010a), in contrast to the permanent
Nomenclature Committee for Fungi (NCF), which in a ballot reached a majority
favoring unification. Redhead (2010b) then prepared a set of alternative proposals
to be presented and voted on at the forthcoming International Botanical Congress
(IBC18) in Melbourne.
Pedro Crous, for a long time one of the strongest defenders of the dual system,
became converted to the unifying camp under the influence of David Hawksworth,
John Taylor, Keith Seifert, and others. Rossman and Seifert (2011) edited a further
volume 68 of Studies in Mycology with contributions honoring the retirement of
Gary J. Samuels. The contributors experimented with various modes of moving
toward a unified nomenclature, and some of them introduced genera for holomorphs
typified by anamorphic species. In April 2011, Crous convened a symposium in
Amsterdam “One fungus–one name.” At this occasion the Amsterdam Declaration
(Hawksworth et al. 2011) was signed by some 80, mainly practically oriented
mycologists as a strong plea for immediate unification. The opponents, who
regarded the time for unification as not yet ripe, were no less active and published
cogent opposed arguments (Gams et al. 2011) in a paper signed by some 70 mycologists,
mainly those involved in morphological taxonomy. All this was done to prepare for
the crucial decisions at the IBC in Melbourne in July 2011.
The Crucial Sessions at Melbourne
In the week preceding the main part of the Melbourne Congress, the nomenclature
delegates convened and discussed 338 proposals to modify the Code. Hawksworth’s
proposal (Hawksworth et al. 2009) and lively discussions intended to do justice to
mycology in the title of the Code resulted in the new name: International Code of
Nomenclature for algae, fungi, and plants (the ICN, McNeill et al. 2012), in which
the fungi and algae (in the broadest sense) appear for the first time in the title.
A further important decision concerned a compulsory registration of all nomencla-
tural novelties for their validity (Hawksworth et al. 2010). Any fungal specimen can
now serve as type material (including permanently preserved, inactivated fungal
cultures). But environmental samples (in spite of Hibbett et al. 2011) and DNA
material alone (in spite of Reynolds and Taylor 1992) remain unacceptable as types
of formal fungal names. Norvell (2011), McNeill and Turland (2011), Gams (2013),
and for phytopathogenic fungi Zhang et al. (2013) have surveyed the major results
of this congress.
To introduce the most contentious item, Article 59, Scott Redhead, chair of the
Special Committee dealing with Article 59, had prepared a set of proposals to be
presented in succession. He started with the most drastic one, which implied the
complete abolition of dual nomenclature and precedence of teleomorph-based
names over those for anamorphs. A short presentation of pros and cons was
permitted before the vote. Expecting that this drastic step would fail, some less
drastic procedures would then be presented subsequently. To the great surprise of all
10 W. Gams
participants, the first vote ended immediately in an overwhelming yes result.

The botanical majority of voters did not seem sufficiently aware of the intricacies of
the situation and simply voted for a simple and drastic solution. This caused great
consternation among the many mycologists who had opposed the move and regarded
this step as premature, while too few fungi were still sufficiently characterized in
the phylogenetic system. Others (Hawksworth 2011; Wingfield et al. 2012) did
afterwards not hide their triumph.
The new situation briefly implies the following: Names introduced indepen-
dently for different morphs of a pleomorphic fungus remain legitimate but a choice
must be made for one of them.
59.1. A name published prior to 1 January 2013 for a taxon of non-lichen-forming
Ascomycota and Basidiomycota, with the intent or implied intent of applying to or being
typified by one particular morph (e.g. anamorph or teleomorph), may be legitimate even if
it otherwise would be illegitimate under Art. 52 on account of the protologue including a
type (as defined in Art. 52.2) referable to a different morph. If the name is otherwise legiti-
mate, it competes for priority (Arts. 11.3 and 11.4; see also Art. 57.2).
On or after 1 Jan 2013, the introduction of a name for a morph different from that
previously named for the same species is illegitimate, at least if the later-named
morph includes in its protologue the type (or name) of the earlier-named morph.
This ruling does therefore not preclude the possibility that two morphs of one
fungus inadvertently receive two different legitimate names also after 2012 (Braun
2012). When two names for different morphs pertaining to the same new fungus are
simultaneously introduced, neither is validly published (Art. 36.2). Quite generally
the principle of priority prevails, no matter whether the original type of a taxon was
teleomorphic or anamorphic. The present Code still contains the clause:
57.2. In pleomorphic fungi (including lichenicolous fungi, but excluding lichen-forming
fungi and those fungi traditionally associated with them taxonomically, e.g.
Mycocaliciaceae), in cases where, prior to 1 January 2013, both teleomorph-typified and
anamorph-typified names were widely used for a taxon, an anamorph-typified name that
has priority is not to displace the teleomorph name(s) unless and until a proposal to reject
the former under Art. 56.1 or 56.3 or to deal with the latter under Art. 14.1 or 14.13 has been
submitted and rejected.
This is a remnant of the previous time-honored rule of precedence for

teleomorph-based names; it now finds little appreciation and may soon be abolished
(Hawksworth 2014).
Whatever the implementation is, the new ruling has started to bring about drastic
nomenclatural changes. To avoid complete chaos, two new additions were made to
Articles 14 and 56:
14.13. In the interest of nomenclatural stability, for organisms treated as fungi (including
lichenicolous fungi, but excluding lichen-forming fungi and those fungi traditionally asso-
ciated with them taxonomically, e.g. Mycocaliciaceae), lists of names may be submitted to
the General Committee, which will refer them to the Nomenclature Committee for Fungi
(see Div. III) for examination by subcommittees established by that Committee in
consultation with the General Committee and appropriate international bodies. Accepted
names on these lists, which become Appendices of the Code once reviewed and approved
by the Nomenclature Committee for Fungi and the General Committee, are to be listed with
their types together with those competing synonyms (including sanctioned names) against
which they are treated as conserved (see also Art. 56.3).
56.3. In the interest of nomenclatural stability, for organisms treated as fungi (including
lichenicolous fungi, but excluding lichen-forming fungi and those fungi traditionally asso-
ciated with them taxonomically, e.g. Mycocaliciaceae), lists of names to be rejected may be
submitted to the General Committee, which will refer them to the Nomenclature Committee
for Fungi (see Div. III) for examination by subcommittees established by that Committee in
consultation with the General Committee and appropriate international bodies. Names on
these lists, which become Appendices of the Code once reviewed and approved by the
Nomenclature Committee for Fungi and the General Committee, are to be treated as
rejected under Art. 56.1 and may become eligible for use only by conservation under Art. 14
(see also Art. 14.13).
These paragraphs imply that the previous, entirely rule-dominated nomenclature

is now changed to a committee- and list-based classification and nomenclature
(Hawksworth 2012b). Suggestions to minimize the effects were presented by Gams
et al. (2012) and Braun (2012).
The Committee-dominated Era
For several major groups of pleomorphic fungi, ad hoc working groups of experts
began to establish themselves or were convened at the 2012 and 2013 CBS spring
symposia or commissioned by the ICTF. Hawksworth (2012a) proposed a time
schedule for this work in order to promote the activity of various committees in
view of the International Mycological Congress in Bangkok in 2014 and the
International Botanical Congress in China in 2017, when the outcome of this work
should be vetted.
The International Commission on the Taxonomy of Fungi (ICTF) is strongly
involved in coordinating these efforts (Seifert and Miller 2012, 2013), and most of
the discussion papers listed below are also located on the ICTF site www.fungaltax-
onomy.org/lists, where they will be most easily found. The lists resulting from these
efforts will subsequently remain freely accessible through the Internet.
The committees work with the terms accepted names (which are protected) and
suppressed names. There is an important difference from the other system of con-
served vs. rejected names, which are irreversible and universally binding. The new
lists will remain open for additions and possibly changes. This system is also differ-
ent from the previously proposed and then defeated system of “Names in current
use” (Hawksworth and Greuter 1998), where only current use would determine a
rather arbitrary selection of names to be retained and protected.
Several groups of mycologists have already done their work. The guiding
principles for the choice of genera are well-supported monophyletic clades which
comprise morphologically and ecologically rather homogeneous taxa. Normally the
priority of names, no matter whether teleomorph- or anamorph-based, decides the
choice. But deviations are proposed in some cases in the spirit of nomenclatural
parsimony (Seifert and Miller 2012; Rossman 2014). The frequency of usage of a
name may also be taken into account, but a more important criterion is the number
of necessary name changes when a particular name is given preference.
12 W. Gams
A problem is whether in cases where the oldest epithet for a species was given in
the suppressed genus, this epithet must be recombined, replacing a well-known
binomial in the accepted genus. Thus Trichoderma reesei would have to be replaced
by a newly combined T. jecorinum and T. citrinoviride by a new T. schweinitzii.
This was not done by Jaklitsch and Voglmayr (2014), and Gams et al. (2012) also
advised against it. Subsequently Samuels (2014) prepared formal proposals for
conservation of these younger names and a few similar cases and all recognized
Trichoderma names were listed by Bissett et al. (2015). For preferential genera of
the Leotiomycetes Johnston et al. (2014), quite generally made the new combina-
tions for the oldest available epithet.
Discussion Papers
Discussion papers (published or unpublished) with lists of preferred generic names

are now available for the following groups of ascomycetes (Pezizomycotina)
dealing mainly with genera, while the phylogenetic delimitation of species would
deserve priority (Braun 2012).
Orbiliomycetes
Debates are ongoing concerning the Orbiliales (Baral et al. 2016). A generic
segregation of the nematode trapping, so far mainly anamorph-based species from
the large genus Orbilia, would be possible in the narrowest of the generic concepts
discussed.
Pezizomycetes
Tedersoo et al. (2013) and for the largest family, the Pyronemataceae, Hansen et al.
(2013) provide phylogenetic insights, and in spite of the occurrence of anamorphs,
teleomorph classification clearly dominates.
Dothideomycetes
An account of preferential names for pleomorphic genera of the class was compiled
by Rossman et al. (2015b).
Dothideales
Thambugala et al. (2014) delimit the Dothideaceae (including the Dothioraceae)

against the newly coined Aureobasidiaceae, a family now comprising seven genera.
Pleosporales
In the review by Hyde et al. (2013) and complete table by Wijayawardene et al.
(2014), some genera are still controversial, e.g., the speciose and still heterogeneous
Pleospora vs. the well-delimited Stemphylium. The genus Alternaria will have to
comprise species of so far separate genera like Ulocladium, Embellisia, Nimbya,
etc. (Woudenberg et al. 2013), when only phylogeny counts.
Capnodiales
Crous et al. (2009a, b), Crous (2010). The family name Cladosporiaceae is resur-
rected and Cladosporium obviously deserves preference over the associated teleo-
morph genus Davidiella. Species of Mycosphaerella s. str. are placed in Ramularia,
whereas a bulk of species still remains in the teleomorph genus.
Botryosphaeriales
Phillips et al. (2013) place the Phyllostictaceae in this order and give preference to
Phyllosticta over Guignardia. In the Botryosphaeriaceae Slippers et al. (2013) recog-
nize 17 genera on a phylogenetic basis, among which Diplodia, Neodeightonia,
Lasiodiplodia, Sphaeropsis, Macrophomina, Neoscytalidium, and Neofusicoccum are
all sufficiently distinct and keyed out based on anamorph features. Teleomorph features
are insufficient to distinguish phylogenetically significant genera morphologically.
Eurotiomycetes
Chaetothyriomycetidae, Chaetothyriales
Réblová and Untereiner (2013) introduce the new anamorph-based family

Cyphellophoraceae for the expanded genus Cyphellophora, which now comprises
species with septate and nonseptate conidia. Two new genera are introduced for
some former Cyphellophora species. Gueidan et al. (2014) include four orders,
Celotheliales ad int., Chaetothyriales, Pyrenulales, and Verrucariales, and ten
14 W. Gams
families (Adelococcaceae, Celotheliaceae, Chaetothyriaceae, Cyphellophoraceae,

Epibryaceae fam. nov., Herpotrichiellaceae, Pyrenulaceae, Requienellaceae,
Trichomeriaceae, and Verrucariaceae) to the subclass. To resolve the very diffi-
cult complex of Capronia–Exophiala–Rhinocladiella–Phialophora in the
Herpotrichiellaceae, no solution is yet offered.
Eurotiales
The Aspergillaceae are now distinguished from the Trichocomaceae (Samson et al.
2011). Penicillium (including the teleomorph genus Eupenicillium and anamorphs
previously classified in Eladia, Torulomyces, and Thysanophora) is clearly sepa-
rated from Talaromyces, which now also incorporates anamorphic taxa (formerly
Penicillium subgen. Biverticillium) (Samson et al. 2011). Debates are going on
between a majority of members of the Penicillium–Aspergillus working group who
prefer recognizing one large genus Aspergillus (so far linked to over ten teleomorph
genera) and other mycologists who prefer several, mostly teleomorph-linked and
ecologically very distinct genera, among which Aspergillus s. str. would have to
retain a conserved type that represents the former section Circumdati and not the
original genus in the sense of Eurotium (Pitt and Taylor 2014). Another debatable
case is the choice between Byssochlamys and Paecilomyces.
Leotiomycetes
In a voluminous survey Johnston et al. (2014) propose several cases of preferential tele-
omorph names that are younger than the associated anamorph names, such as Ascocoryne
over Coryne, Dematioscypha over Haplographium, Dermea over Sphaeronaema,
Diplocarpon over Entomosporium, Gremmeniella over Brunchorstia, Monilinia over
Monilia, Neofabraea over Phlyctema, and Pyrenopeziza over Cylindrosporium, but they
retain the older Hyphodiscus over Catenulifera, Pezicula over Cryptosporiopsis,
Phacidium over Ceuthospora, Phialocephala over Phaeomollisia, Pilidium over (Disco-
)Hainesia, Rhytisma over Melasmia, and Vibrissea over Anavirga.
Erysiphales
Braun (2013) proposes conservation of the teleomorph-based name Blumeria over

the older anamorph-based Oidium and several more teleomorph-based epithets over
correlated older anamorph names. Thus the names of powdery mildew genera are
now consistently teleomorph-based, although anamorph features also contribute to
genus delimitation.
Phacidiales
Crous et al. (2014) raise the family Phacidiaceae to ordinal level, segregated from
the formerly paraphyletic Helotiales and synonymize the younger anamorph genus
Ceuthospora with Phacidium.
Sordariomycetes
Xylariomycetidae, Xylariales
In the Xylariales the teleomorph-based taxonomy is quite clearly the guiding rule of
generic classification negating that of anamorphs (Stadler et al. 2013). The authors
are retaining teleomorph-generic names throughout the order (exception Virgaria
preferred over Ascovirgaria), while certain anamorph features also correlate with
generic delimitation. Debatable cases include Arthrinium vs. Apiospora,
Monographella vs. Microdochium, Seiridium vs. Eutypa, and a few others.
Hypocreomycetidae
Hypocreales
Rossman et al. (2013) list several genera of Hypocreales as candidates for protec-
tion while avoiding many hot irons in this group. Nectria clearly deserves prefer-
ence over Tubercularia. The so far vaguely defined genus Cylindrocarpon (although
a nomen conservandum) is sacrificed in favor of several associated teleomorph-
based genera. Gliocladium s.str. is replaced by Sphaerostilbella, but the morpho-
logically similar Clonostachys will outlive the associated older teleomorph name
Bionectria. The very important genus Fusarium cannot be sacrificed for its teleo-
morph Gibberella (Geiser et al. 2013), but how many clades will remain in this
genus is still uncertain, while some of them were already excluded and transferred
to other genera by Gräfenhan et al. (2011). The controversy over the taxonomic
identity of the speciose F. solani clade and the appropriate name for such a clade, if
it were to be recognized as distinct from Fusarium, remain to be solved. Lombard
et al. (2015) propose the name Neocosmospora for this clade and follow a narrow
generic concept.
In Hypocrea, according to the former rule of teleomorph precedence, several
species have been described recently, some of which lacked an anamorph altogether
or had deviating anamorphs, but all of them are now transferred to the broadly pre-
ferred anamorph-based genus Trichoderma (Jaklitsch and Voglmayr 2014) as listed
for the whole genus by Bissett et al. (2015). The introduction of a teleomorph genus
for Volutella by Luo and Zhuang (2012) obviously becomes redundant after the
work by Gräfenhan et al. (2011), but its publication in 2012 does not render the
16 W. Gams
name Volutellonectria illegitimate. For the difficult complex of Acremonium–

Emericellopsis–Stilbella–Gliomastix (Summerbell et al. 2011), no workable solu-
tion is yet in sight.
The Clavicipitaceae s. l. are treated in several papers: In the Clavicipitaceae s.
str., the teleomorph-based genus Epichloë is older than Neotyphodium for associated
anamorphs, and these taxa can easily be merged (Leuchtmann et al. 2014). Kepler
et al. (2013) broaden the concept of Polycephalomyces and Kepler et al. (2014) cre-
ate a large genus Metarhizium including former Nomuraea and, although strongly
deviating, some paecilomyces-like species. Dealing with the Ophiocordycipitaceae,
Quandt et al. (2014) give preference to the younger teleomorph name Ophiocordyceps
over several older anamorph-based generic names. Tolypocladium is now conceived
in a wider frame that required numerous new combinations, including some from a
teleomorph genus.
The genera to be distinguished in the Cordycipitaceae are the most controversial.
Cladistically minded mycologists wish to recognize no more than 11 rather widely
defined monophyletic genera, while the morphology-trained and ecologically
oriented mycologists demand a much finer distinction of genera.
Microascales
For the medically relevant genera around Pseudallescheria, Lackner et al. (2014)
recognize the generic names Parascedosporium, Lomentospora, Petriella,
Petriellopsis, and Scedosporium (displacing Pseudallescheria, but still debated). For
the mainly phytopathogenic taxa around Ceratocystis, de Beer et al. (2014) distin-
guish several genera, Ceratocystis sensu stricto, Chalaropsis, Endoconidiophora,
Thielaviopsis, and Ambrosiella, and the new genera, Davidsoniella and Huntiella,
most of which have chalara-like anamorphs.
Glomerellales
The older genus name Colletotrichum is to be protected against the teleomorph

genus Glomerella (Cannon et al. 2012).
Sordariomycetidae
Diaporthales
In the Magnaporthaceae Luo et al. (2014) distinguish three major lines: (1)
Ophioceras, (2) Pyricularia (suppressing Magnaporthe, but still debated), (3)
Gaeumannomyces (Harpophora), Magnaporthiopsis, and distinct anamorph-based
genera Nakataea and Pseudophialophora. A survey of preferential names in
pleomorphic genera of the order was compiled by Rossman et al. (2015a).
Ophiostomatales
De Beer et al. (2013, see Conclusions below) give a nomenclature of all presently
recognized ophiostomatoid taxa.
Some of these lists were briefly presented at the 10th International Mycological
Congress in Bangkok during three nomenclature sessions (Redhead et al. 2014),
which were too short for a detailed discussion. The lists still have to be scrutinized
by the Nomenclature Committee for Fungi before being published in their final
form in the Internet and presented to and sanctioned by the next Botanical Congress
in China (Hawksworth 2012b). Thus the present years can only be regarded as a
transitional period (Zhang et al. 2013), but it is likely that the examples listed here
will be fixed as described.
Conclusions
The unification of fungal nomenclature has been pushed through in order to provide
for the Fungi a natural system just like for plants and animals. Sooner or later this
move had to come, ideally at a time when a majority of fungal species is known to
science. The most urgent task of mycology—discovery and careful description of
new species—is now placed into second place by raising the issue of unified nomen-
clature to the top. At this moment phylogenetic knowledge is not sufficiently devel-
oped throughout the fungal system to provide clear-cut solutions for problematic
cases. The present hectic activity at least enforces a useful stocktaking of what is so
far known.
Mycologists are making enormous efforts to minimize the chaos ensuing from the
somewhat prematurely introduced unification by generating meaningful lists of
names. Lists of protected names are being produced and will become established.
They are not the last word in fungal taxonomy, and mycologists cannot be forced to
adopt a particular taxonomy when they do not agree with it. It is presently impossible
to effectively squeeze all known species into recognized, available, and strictly
monophyletic genera. Braun (2012) rightly emphasizes the permanent legitimacy of
“suppressed” generic names as long as for many species evidence for their affinity
with a list-accepted genus is missing. In addition I wish to emphasize that recogni-
tion of paraphyletic genera as being a natural phenomenon will do much more justice
to a classification based on morphological and ecological criteria.
Unification was expected to facilitate the study of fungal systematics by students.
However, this is not the case, as the knowledge of both sexual and asexual morphs
of a fungus (and associated names) remains indispensable, even when only one
generic name is recognized and the alternative one retains the role of a morphologi-
cal descriptor and often also is the basis for the names of higher taxonomic ranks.
As suggested by Seifert et al. (2000), some of these presently suppressed names will
continue to be used as descriptive adjectives or nouns. We will just have phialoph-
ora-like and acremonium-like, but not phaeoacremonium-like or simplicillium-like
18 W. Gams
because the latter two are recognized genera. Thus a certain duality of names for
one genus is bound to persist after this move.
A complete move to unified nomenclature will require the recombination of all
included species into a single recognized genus for a particular group. This has so
far only been achieved consistently in Trichoderma (Jaklitsch and Voglmayr 2014;
Bissett et al. 2015) and in Tolypocladium (Quandt et al. 2014). It has also been done
for Penicillium and Talaromyces (Samson et al. 2014), but for Aspergillus remains
controversial (Pitt and Taylor 2014). An interesting solution is chosen by de Beer
et al. (2013) for ophiostomatoid fungi, where the authors list the species of mono-
phyletic clades but leave them with their original binomial; e.g., in Ophiostoma they
retain species of Sporothrix, Raffaelea, and even “Leptographium,” although that
genus phylogenetically belongs to a different clade. This procedure has much to
recommend it, especially when not all fungi in question have yet been revised phy-
logenetically. This is preferable to imposing countless new generic combinations
for controversial cases which at present can hardly be satisfactorily resolved.
A debatable proposal concerns morph pairs with identical epithets, for which
Hawksworth et al. (2013) suggest a mechanism by which newly discovered alter-
nate morphs are generally to be declared as new combinations based on the type of
the older name. This would replace the hitherto compulsory introduction of a new
species in the appropriate genus for the newly discovered morph, which created
heterotypic names for contiguous morphs. However, this heterotypic situation has
the advantage that the connection can be viewed critically in each case and need not
always be recognized (Braun 2012); a global adoption of the proposed mechanisms
has little chance of stabilizing names.
Taxonomic decisions can never be made by rules of nomenclature. If a name is
placed on a list for suppression, this does not mean that it cannot be used when
required. This is the important difference of the other system of officially conserv-
ing vs. rejecting names that is universally binding and irreversible (also Seifert and
Miller 2012). These subtle differences are, however, not easily understood by
authors, editors, and reviewers, not to say students, and may cause unnecessary
conflicts about how to proceed. Presently, lists of names to be protected are pre-
pared, and parallel official conservation proposals are published often by members
of the same team (e.g., Samuels 2014), which further confuses these two different
ways of formalizing names.
Publication of new names is now facilitated by eliminating the hurdles of Latin
diagnoses and print publication so that the number of validly but qualitatively
poorly published names is increasing. Some knowledge of Latin does remain indis-
pensable for understanding the old literature and correctly coining new names for
new taxa.
Taxonomic knowledge of numerous fungal groups is still quite inadequate and
often does not yet allow decisions about the delimitation of natural taxa. The ten-
dency by many mycologists to ignore morphological characters when introducing
fungal taxa is not helpful when striving for a natural classification. The morpho-
logical knowledge gathered by older workers and that to be gathered for recent
material remains an indispensable basis for establishing a natural system for fungi
that guarantees stability and allows predictions of properties of related taxa. A careful
morphological analysis and permanent preservation of the material studied are indis-
pensable prerequisites to assure that a sequence obtained really applies to the fungus
in question. Do not forget that genotype and phenotype are two sides of the same
coin. Much more material needs to be collected and thoroughly studied to enhance
mycological knowledge. Are the taxonomists of the future prepared to meet this
challenge in all of its dimensions? The fungal world remains alive in its native envi-
ronment, awaiting discovery. Conditions must urgently be created to enable mycolo-
gists to get out of the boardrooms and back into the field.
Acknowledgments For critical reading of the manuscript, I am indebted to Keith A. Seifert,

Martina Réblová, Roland Kirschner, Marc Stadler, Uwe Braun, and for a final correction to Nick
Turland.
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Chapter 3
Future Perspectives and Challenges of Fungal
Systematics in the Age of Big Data
Zheng Wang, R. Henrik Nilsson, Timothy Y. James, Yucheng Dai,

and Jeffrey P. Townsend
Fungal Diversity and Systematics
The beginning of wisdom is to call things by their proper name (Chinese proverb: ਽↓䀰亪)
Z. Wang, Ph.D. (*)

Department of Biostatistics, Yale School of Public Health, Yale University,
135 College Street, New Haven, CT 06510, USA
e-mail: wangzhengff@gmail.com; wang.zheng@yale.edu
R.H. Nilsson, Ph.D.
Department of Biological and Environmental Sciences, University of Gothenburg,
Box 461, Göteborg 405 30, Sweden
e-mail: henrik.nilsson@bioenv.gu.se
T.Y. James, Ph.D.
Department of Ecology and Evolutionary Biology, University of Michigan,
1147 Kraus Natural Science Building, 830 North University, Ann Arbor,
MI 48109-1048, USA
e-mail: tyjames@umich.edu
Y. Dai, Ph.D.
Institute of Microbiology, Beijing Forestry University, Beijing 100083, China
e-mail: yuchengd@yahoo.com
J.P. Townsend, Ph.D. (*)
Department of Biostatistics, Yale School of Public Health, Yale University,
135 College Street, New Haven, CT 06510, USA
Department of Ecology and Evolutionary Biology, Yale University,
165 Prospect Street, New Haven, CT 06520, USA
Program in Computational Biology and Bioinformatics, Yale University,
165 Prospect Street, New Haven, CT 06520, USA
e-mail: jeffrey.townsend@yale.edu

DOI 10.1007/978-3-319-29137-6_3
26 Z. Wang et al.
The mission and agenda of fungal systematists are to discover, describe, and
inventory the global species diversity of one of the most diverse groups on earth.
The circumscription of the fungi has evolved over time. Fungi are most closely
related to animals and share a more recent common ancestor with them than with all
other major groups of eukaryotes. The majority of the fungal kingdom is composed
of heterotrophic, non-photosynthetic eukaryotes with cell walls containing chitin
and β-glucans and, when present, a single flagellum (Stajich et al. 2009). Fungi can
occur both as single-celled and multicelled organisms and can reach sizes typically
associated with plants and animals. For example, the largest single fungal fruiting
body on record was found to be nearly 500 kg in weight (Dai and Cui 2011), and the
oldest and largest mycelium described covers 15 ha of area and is over 1500 years
old (Smith et al. 1992). Life cycles of many fungi include a vegetative growth phase
that spreads throughout its environment by extension of hyphae and/or release of a
large number of asexual spores from simple structures and by a more complex,
transient sexual phase producing smaller numbers of resistant sexual spores from
well-developed fruiting bodies. Fungal diversity is estimated to comprise 1.5–7.1
million species. An increasing number of new taxa continue to be reported world-
wide (Blackwell 2011; Bass and Richards 2011), and fungi have been isolated from
almost all kinds of ecosystems on Earth (Stajich et al. 2009). This fungal diversity
is described by systematics, which is the science not only of naming fungi but also
of positioning the species among other existing names to represent their evolution-
ary relationships. To properly describe the substantial diversity of the Kingdom
Fungi, mycologists have been updating its classification and systematics, based on
accumulated knowledge of fungal biology interpreted within new concepts and
approaches that are emerging from evolutionary biology.
In contrast to large aboveground organisms that can be easily spotted and
counted, fungi are major components of underground diversity. Their study is often
made difficult by their microscopic structures and shortage of discriminatory mor-
phological characters. Traditional biological information used for classifying fungi
into major groups includes morphology, ultrastructure, physiology, tissue biochem-
istry, and ecological traits. Early synthesis of this information yielded major fungal
groups that have remained comparatively stable over a very long time period in the
twentieth century. Some morphological and ecological traits, such as the structure
of the cell wall and hyphal septa, sexual reproduction and meiotic spores, nutri-
tional modes, as well as geographic distribution, have proven to be relatively con-
served and informative, especially for high-level classification. However, phenotypic
plasticity of traits and fast-evolving traits have caused considerable uncertainty
regarding lower-level phylogenies based on morphology and ecology (Lutzoni
et al. 2004). Starting in the 1970s, but gaining momentum in the late 1990s, the use
of DNA sequence data to infer phylogenetic relationships among fungal lineages
brought about a revolution in terms of taxonomic resolution and scientific reproduc-
ibility (de Bertoldi et al. 1973; Bruns et al. 1991; Bridge et al. 2005; Blackwell et al.
2006). Initial molecular studies, typically based on a single gene region, were
followed by a wave of multilocus phylogenetic studies including all major fungal
groups. The new phylogenies facilitated several major taxonomic revisions,
3 Future Perspectives and Challenges of Fungal Systematics in the Age of Big Data 27
including new lineages at the phylum and class level (James et al. 2006a; Hibbett
et al. 2007; Kirk et al. 2008; Schoch et al. 2009a, b; Rosling et al. 2011; James and
Berbee 2012; Matheny et al. 2007). More changes and many new taxa were added
to lower-level fungal groups. In addition, much novel diversity was revealed in
sequence data collected from environmental samples and identified as operational
taxonomic units (OTUs) (Blaxter et al. 2005). The quantity of novel OTUs in most
environmental samples hints at a massive, inconspicuous, undescribed, and thriving
fungal diversity (Hibbett et al. 2011). Classifying and naming this huge fungal
diversity is a necessary step toward understanding the functions of these fungi in the
ecosystems. Thus, finding ways to take full advantage of the power afforded by
next-generation sequencing approaches to integrate environmental DNA sequences
has become one of the major challenges for fungal systematics. Simultaneously, a
complementary aspect of the future of the fungal systematics is the integration of
systematics, the evolution of complex traits, and functional genomics to understand
the comparative biology of fungi and to create a holistic view of the fungus and how
it evolves.
Currently, efficient communication regarding fungal species rests upon on the
use of scientific names constructed based on a system of hierarchical ranks. Within
this system, one of the major purposes of fungal taxonomy and systematics is to
create and position nomenclatural units unique for each fungal species. While a
stable name as a symbol for communication is always appreciated by researchers—
especially for the widely used industrial, medical, plant pathogenic, and model
species—systematics must also continue to refine and revise the application of
names to reflect continual gains in knowledge about the evolutionary histories of all
taxa. We make no attempt here to cite all papers on development of fungal taxonomy
and systematics nor to summarize recent systematic progress within and among the
major fungal phyla. Instead, we have chosen to highlight recent research that enables
us to illustrate specific points about perspectives and challenges of fungal systematics
in the age of big data.
Integrative Taxonomy and Current Fungal Systematics
Traditionally, morphological and sometimes ecological traits have been used to

classify fungi into hierarchical ranks and groups. However, evolutionary relation-
ships derived from these traits, whose ontology is often inferred from a phyloge-
netic hypothesis, can be problematic, especially for lower-level phylogeny, where
diverse fungal groups can have plesiomorphic or convergent morphologies. One
problem in reconstructing fungal evolutionary history is a lack of paleontological
information due to the scarcity of well-preserved fungal fossils (Bidartondo et al.
2011). This scarcity makes it extremely difficult to evaluate the evolutionary history
of morphological traits for fungal systematics, especially for morphologically sim-
ple groups. The meager fossil record also makes it difficult to precisely calibrate
molecular phylogenies. Nevertheless, information on molecular evolutionary
28 Z. Wang et al.
events, such as mutation and gain or loss of nucleotide characters, has been well
preserved in gene sequences. Molecular phylogenies using single genetic markers
or multilocus data have led to dramatic advances in the systematics of a range of
taxonomic levels within the fungal kingdom over the past three decades. However,
systematic hypotheses based on molecular phylogenetic data alone can be ques-
tioned, especially when in conflict with morphological evidence. Ideally, evidence
from different lines, such as morphology, ecology, and molecular data, can be
evaluated jointly to robustly define taxa at all ranks. This approach has been
called integrative taxonomy and has been advocated by Will et al. (2005) and Pante
et al. (2015).
A major driver of new advances in the molecular phylogeny of fungi was the
Assembling the Fungal Tree of Life (AFTOL) project, funded by the National
Science Foundation (NSF) of the United States and organized by mycologists at
several leading laboratories. This project sprung out of an NSF-funded research
coordination network known as Deep Hypha and culminated in significant gains in
the study of evolution and molecular phylogenetics of fungi (Lutzoni et al. 2004;
Blackwell et al. 2006; James et al. 2006a; Hibbett et al. 2007; Schoch et al. 2009a).
Among the very first multilocus phylogenies targeting the majority of major fungal
lineages, Lutzoni et al. (2004) highlighted two major challenges in fungal systemat-
ics in the molecular age. One is achieving a balanced sampling of taxa and genetic
markers. The other is identifying and interpreting inconsistency between the evolu-
tion of morphology and molecular phylogeny. When standard PCR using degener-
ate primers and Sanger sequencing were the major tools for recovering DNA
sequences from fungal tissue, loci such as nuclear and mitochondrial rRNAs and
several widely used protein-coding genes, including subunits of elongation factors
and RNA polymerases, were selected by the AFTOL project. A six-gene phylogeny
using these markers, including data from 52 sequenced fungal genomes, was gener-
ated to assess early evolution of fungi, and ecological characters were mapped onto
the tree.
Groups recognized in the six-gene phylogeny were generally consistent with
traditional views of fungal systematics prior to the molecular systematic age, but
only for the fungi in Dikarya (James et al. 2006a). Non-monophyly of two of the six
recognized phyla led to the abandonment of one (Zygomycota) and the description
of two new phyla Blastocladiomycota, by James et al. (2006b), and
Neocallimastigomycota, by Hibbett et al. (2007). Similar sequence datasets, which
were often incomplete with missing sequences, were generated for a more inclusive
taxon sampling within each major fungal group at class level, and the resulting
phylogenetic classifications were collected in the special Deep Hypha issue of
Mycologia in 2006 (Blackwell et al. 2006). A comprehensive phylogenetic classifi-
cation of the fungi kingdom was later proposed by Hibbett et al. (2007), featuring
16 new taxa above the level of order. This classification was adopted by the latest
version of the Dictionary of the Fungi (Kirk et al. 2008). A more taxonomically
complete six-gene dataset for 420 ascomycetes was subsequently assembled and
analyzed. Key morphological and ecological characters were evaluated for useful-
ness in ascomycete systematics, and a new class was differentiated for two
earthtongue genera: Geoglossum and Trichoglossum (Schoch et al. 2009a, b). This
dataset made it possible to quantify phylogenetic informativeness (Townsend et al.
2008; Townsend and Lopez-Giraldez 2010; Lopez-Giraldez and Townsend 2011)
for several widely used genetic markers (Schoch et al. 2009a, b). With the release
of more fungal genome sequences and the ever-growing availability of data from
additional genetic markers, several multilocus phylogenies inferred using partially
or solely from genomic data (phylogenomics) have been published (Ebersberger
et al. 2012; Binder et al. 2013; Ortiz-Santana et al. 2013; Dutilh et al. 2007). Updated
classifications for major fungal groups were collected in Mycota VII—Systematics
and Evolution (McLaughlin et al. 2014).
The vast majority of molecular systematic studies of fungi have been based on
annotated (voucher) specimens of primarily sexual (teleomorphic) but also asexual
(anamorphic) collections. The accuracy of voucher specimens is particularly impor-
tant now, because in many modern studies, only molecular data are shared and
examined: fungal herbaria thus play important roles in keeping records for well-
annotated specimens (Bidartondo 2008; Schoch et al. 2014). Best-practice guide-
lines on how to appropriately use molecular data in mycology are readily available
(Lindahl et al. 2013; Hyde et al. 2013; Nilsson et al. 2012). Nevertheless, these
guidelines are not sufficiently frequently adhered to fungal molecular phylogeneti-
cists. Well-preserved and annotated collections are now mandatorily required by
journals for newly published morphological and molecular data (Seifert and
Rossman 2010). However, there has been no guarantee of accurate identification of
fungal collections, especially for microfungi, partially due to the problematic out-
comes of applying species concepts in fungi.
Morphological, biological, or phylogenetic species concepts all have limitations
when they are applied to fungal species (Taylor et al. 2000, 2006). In particular,
different mycologists often have different quantitative or qualitative interpretations
of data used to define species boundaries. For example, using several genetic mark-
ers, multiple species were identified within the single morphological and biological
species commonly known as the “turkey tail” fungus Trametes versicolor (Carlson
et al. 2014), and two species were recognized for North American Heterobasidion
annosum, which has been considered one of the most important forest pathogens in
the world (Otrosina and Garbelotto 2010). Another extraordinary and exciting
example would be that of the morel fungi, for which tens, if not hundreds, of new
species have been recognized within several original common names (Du et al.
2012; Richard et al. 2014). An increasing number of low-level classifications are
based on integrative approaches using both morphological and molecular data.
These approaches have been applied to solve identification issues of several com-
mercially important fungi (Cao et al. 2012; Wu et al. 2014; Zhang et al. 2005).
In many cases, the reference molecular data are directly downloaded from vari-
ous databases, assuming accurate identification without checking the resource spec-
imens. Cryptic species complexes are particularly likely for many species of
microfungi, in which case, dense samples from accurately annotated specimens will
be especially critical for proper species taxonomy. However, phylogenetic recogni-
tion of fungal species has proved to be reliable, reproducible, and increasingly
30 Z. Wang et al.
widely applicable, facilitating convenient naming of species or strains, especially

for microfungi. The huge undisclosed fungal diversity and the difficulty of reconcil-
ing species concepts in fungi can make the application of the International Code of
Nomenclature (McNeill and Turland 2012) very challenging—to the extent that it
can ironically slow down, rather than speed up, mycological progress. Recently, for
instance, instead of following the code to use the teleomorph genus name for mono-
phyletic groups, mycologists advocated recognizing the genus Fusarium as the sole
name for groups that have been studied under that name but are not monophyletic
(Geiser et al. 2013). Such challenges will become more significant as more invisible
diversity is discovered within diverse environmental samples. These challenges
should aid the community in pushing for the development of standards for sequence-
based classification (Hibbett and Taylor 2013). A recent review of the impacts of
the nomenclatural code on the scientific names that have been adopted is available
for plant pathogenic fungi (Zhang et al. 2013).
Systematics and Classification for Invisible Diversity
Fungi are widely distributed in all terrestrial and aquatic ecosystems. About 100,000
fungal species have been discovered and documented. They play critical roles in
inorganic and organic nutrition, nutrient cycling, and especially in the decay of
carbon compounds that were fixed and integrated into complex compounds by
plants. Furthermore, fungi are frequently intimate partners in coevolving biotic and
trophic relationships with other organisms, notably through mycorrhizal associa-
tions with plants; almost all land plants form symbiotic associations with mycor-
rhizal fungi (Stajich et al. 2009; van der Heijden et al. 2015). However, only a small
portion of the total fungal diversity has been documented based on specimens/
strains deposited in herbaria, culture collections, or in personal collections all over
the world. Indeed, a modest ~1000 new species are described per year (Hibbett et al.
2011), which would require 5000 years of cataloging at this rate, should the 5.1 mil-
lion estimate of species diversity hold.
The challenges to description of this undescribed fungal diversity are threefold.
First, there are few mycological researchers and little research to study this unde-
scribed diversity. Second, many of these undescribed species whose morphology
can be characterized are actually cryptic species hidden within species previously
described on the basis of morphological characters; morphological characters might
not separate the genetic species, as discussed for Trametes versicolor and Morchella
spp. above. Third, the majority of the extant fungal diversity produces no distin-
guishing morphological structures that are visible or describable, e.g., these fungi
carry out their lives mostly or entirely as unculturable and morphologically indistin-
guishable yeasts or vegetative hyphae that cannot be described formally. If these
fungi are unculturable as well as morphologically and biochemically indistinguish-
able, only can molecular identification be used as a tool to classify this potentially
huge diversity. This kind of molecular-only identification leads to the absurd
situation where next-generation sequencing efforts of environmental substrates

reveal the existence of thousands upon thousands of new species of very high rele-
vance to phylogenetic and ecological characterization of the fungal kingdom—and
yet this huge diversity of species cannot be described. This inability to describe
these species effectively excludes them from further scientific scrutiny. Such
sequences are typically submitted to sequence databases labeled as “uncultured fun-
gus,” making unambiguous reference to those species across datasets and studies
problematic at best. This lack of linkage across studies in turn makes it difficult to
assemble data for these species; what countries, hosts, and substrates these indi-
vidual, unnamed species are known from cannot easily be compiled. In turn, this
lack of synthetic inferential power further complicates the eventual formal descrip-
tion of these species.
The UNITE database for molecular identification of fungi recently presented a
solution to this problem (Kõljalg et al. 2013). All fungal ITS sequences are clus-
tered to approximately the species level based on sequence similarity, and each such
OTU—called a species hypothesis—is assigned a unique, stable name of the acces-
sion number. Thus, regardless of whether the OTU has a formal Latin name or not,
unambiguous reference across publications—as well as data assembly for individ-
ual species—is possible and even automated. A recent study, based on 365 soil
samples collected from across the globe, identified 80,486 fungal OTUs and used
the UNITE species hypothesis system to analyze them. Although a very modest
4353 of the OTUs could be linked to highly similar reference sequences from her-
barium specimens or described culture collections, the underlying sequences of the
full results of the study are now integrated in UNITE for standardized reference
(Tedersoo et al. 2014; Wardle and Lindahl 2014). At the time of this writing,
GenBank has a collection of more than 600,000 fungal sequences from environ-
mental samples, chiefly the nuclear ribosomal internal transcribed spacer (ITS)
region. Among these, there are about 200,000 that have been identified as stemming
from an “uncultured fungus,” without an affiliation to any existing ranks.
It is hard to estimate how inclusion of this huge invisible diversity would affect
the fungal systematics that so far encompassed only just over 100,000 accepted
fungal species. Despite the challenges, it is clear that not including these extant but
unnamed species in molecular studies of fungi and fungal communities is detrimen-
tal to mycology. Nilsson et al. (2011) examined the topological effects of including
such environmental sequences in phylogenetic analyses that featured only sequences
from vouchered fruiting bodies and cultures. Their inclusion made a significant dif-
ference to the inferred topology and to the support of internodes. Similarly, the rela-
tively recent realization that aquatic ecosystems abound with uncharted fungal
diversity, particularly in the Chytridiomycota and Cryptomycota, could provide
taxonomic sampling that might provide resolution of this part of the fungal tree of
life, which has been plagued by low resolution and poor branch support (Wurzbacher
and Grossart 2012; Ishii et al. 2015). Recently a whole new class, Archaeorhizomycetes,
comprising hundreds of cryptically reproducing culturable filamentous fungi of
poorly understood ecology, has been discovered from soil samples (Rosling et al.
2011). Using multilocus analyses, they have been phylogenetically placed into the
32 Z. Wang et al.
species-poor group Taphrinomycotina of the Ascomycota. The recognition of the

Archaeorhizomycetes represents a major step forward in our understanding of soil
fungi, as these fungi seem to be common in soil samples throughout the world
(Porter et al. 2008; Rosling et al. 2013). At an even higher rank, the new fungal
phylum Cryptomycota, rich particularly in aquatic environments, is also known
almost exclusively from environmental DNAs (James and Berbee 2012; Jones et al.
2011). The systematics of the Archaeorhizomycetes and Cryptomycota will remain
hindered by the absence of complete genome sequences, which will be challenging
to obtain from these minute fungi. On the other hand, recent advances in obtaining
near-complete genome sequences from single cells hold promise for both placing
uncultured fungal lineages on the tree of life and for inferring their ecological roles
(Rinke et al. 2013).
For the majority of fungal lineages, ITS sequences provide a powerful and effi-
cient means of identification. Therefore, the ITS has been proposed and accepted as
a universal DNA barcode marker for fungi (Schoch et al. 2012). A DNA barcode,
however, is nothing more than a sequence that can be unambiguously linked to a
taxonomic label for a species. DNA barcodes do not promise a solution for nomen-
clatural classification of diversity. Such a solution might arise from digital codes
such as PhyloCode (de Queiroz and Gauthier 1994). However, this concept still
lacks a standardized real-life implementation (de Queiroz and Gauthier 1994; De
Oliveira Martins et al. 2014; Money 2013). While ITS is generally considered as
only informative for species recognition and low-lever phylogenetic analysis, clas-
sification of the environmental diversity typically relies on observations of high
sequence similarity to reference sequences from annotated specimens (Schoch et al.
2014). However, with the use of new tools to address some serious alignment issues
regarding the ITS region (Liu et al. 2009, 2012), ITS alignments have shown prom-
ise in use for intermediate-level phylogeny (Koetschan et al. 2010), providing com-
parable classification accuracy to some other frequently used gene markers, such as
the large subunit of rRNA sequence (Wang et al. 2011). Including proper reference
sequences would provide insights into evolutionary history and ecology for these
so-called invisible fungi (Wang et al. 2011; Porras-Alfaro et al. 2014; Del Olmo-
Ruiz and Arnold 2014). Automatic phylogenetic approaches, such as those imple-
mented in MOR (Hibbett et al. 2005) and WASABI (Kauff et al. 2007) would be
able to efficiently filter and classify environmental sequence data. Still, there might
be many environmental species that have no comparable characterized lineages,
such that they cannot be morphologically defined or easily systematically posi-
tioned. Moreover, the absence of barcodes of the ITS region associated with this
phylum is also an impediment, as many barcodes that cannot be assigned to a phy-
lum may belong to these poorly sampled basal lineages, which exist in databases
primarily as 18S rDNA sequences. To incorporate these taxa into fungal systemat-
ics requires developing methods for gathering informative sequence data that link
barcodes to darker regions of the fungal phylogeny and performing efficient phylo-
genetic analysis on large datasets.
Given the deep divergence of the major fungal lineages, plodding through taxa
using PCR with degenerate primers to fish for loci is a challenging, if not impossible,
approach toward recovering an effective diversity of protein-coding genes that will

prove informative for deep phylogeny. Moreover, establishing linkages among
multiple independent genes that derive from the same OTU defined from environ-
mental DNA is nearly impossible at present. Thus, with the development of single-
cell genome sequencing, phylogenomic approaches might provide an alternative
and more powerful means to reconstruct a systematics of both the visible and the
invisible fungal diversity.
Fungal Genomes, Phylogenomics, and Phylotranscriptomics
The very first sequenced fungal genome was also the first sequenced eukaryotic
genome: that of the wine yeast Saccharomyces cerevisiae, an important genetic
model and an industrial workhorse. This comparatively small genome was pub-
lished in 1996 (Goffeau et al. 1996). Since then, following the technical progress in
genome sequencing, fungal genomes have been released at an ever-accelerating
rate. The number of available fungal genome sequences has increased by another
order of magnitude (Galagan et al. 2005). In GenBank (http://www.ncbi.nlm.nih.
gov) alone, there are currently fungal genomes representing 451 species. The
recently launched 1000 Fungal Genomes (1KFG) project (http://1000.fungalge-
nomes.org) plans to sequence representatives from more than 650 recognized fami-
lies of fungi (Kirk et al. 2008; Hibbett et al. 2013). The released genomes facilitate
assembly of closely related genomes against the reference genomes even in small
laboratories, and the sampled genomes of closely related organisms are designed to
enable comparative studies. Comparative genomics of closely related organisms
can provide a powerful approach to ascertain the genetic basis of diverse pheno-
types, such as fungi-host associations, secondary metabolic pathways, morphologi-
cal development, and fungal responses to environmental signals (Galagan et al.
2005; Hibbett et al. 2013; Sikhakolli et al. 2012; Andersen et al. 2011; Lehr et al.
2014; Nishant et al. 2010; Rodriguez-Romero et al. 2010; Heitman 2007). Many
comparative genomic studies focus on the biology and evolution of model fungi to
make inferences about basic biological processes in all eukaryotes. Studies that
analyze genomes in the context of their phylogenetic and evolutionary relationships
are accelerating research into the fundamental aspects of eukaryotic biology. As
stated in Delsuc et al. (2005) “…nothing in genomics makes sense except in the
light of evolution.”—large numbers of genomes alone do not provide much insight
into organismal biology, however. Many features of genomes need to be related to
organismal knowledge and understood in the context of their evolutionary history.
How can these fungal genomes empower fungal systematic research? The
genome itself comprises all informative genetic markers that could be sampled for
any individual. Access to this scale of genomic data for phylogenetic purposes
could potentially alleviate previous and present problems of phylogenetics that arise
from insufficient or biased sampling of genetic markers. With this massive increase
of potentially useful characters, the focus of phylogenetic inference must shift
34 Z. Wang et al.
toward development of new methodologies that can efficiently, accurately, and reli-
ably handle big data and toward approaches that facilitate a powerful sampling of
taxa (Philippe et al. 2011). Basic approaches and future challenges in phylogenom-
ics toward reconstruction of the larger tree of life were addressed 10 years ago
(Delsuc et al. 2005), and phylogenomic approaches and tree reconstruction methods
have been tested using different sets of fungal genomic data (Ebersberger et al.
2012; Dutilh et al. 2007; Medina et al. 2011). Development of phylogenomic
approaches for fungal phylogenetic inference has been addressed recently (Hibbett
et al. 2013; Taylor and Berbee 2014) and is beyond the scope of this review. Current
genome projects have sampled representative taxa in major lineages across fungal
kingdom, providing extensive datasets for resolving relationships between major
lineages of higher fungi. The current genomic projects might provide sufficient
taxon sampling to resolve some of the unsolved polytomies within Basidiomycota
and Ascomycota, as summarized in Hibbett et al. (2007). However, to resolve the
phylogeny of the earliest fungal lineages, it is already clear that densely sampled
genomes and the development of novel culture-independent methods will be criti-
cal. Recent phylogenomic analyses support the supergroup Opisthosporidia (Micro
sporidia + Cryptomycota + Aphelida) as the basal branch of all sequenced fungi
(Capella-Gutierrez et al. 2012; Haag et al. 2014; James et al. 2013; Karpov et al.
2014). This group is known to be highly diverse on the basis of environmental DNA
studies (Jones et al. 2011; Karpov et al. 2014) and also completely unculturable in
the absence of a host. Sufficient sampling of genomes is also important for under-
standing divergence and recent adaptation among very closely related species, espe-
cially to reveal cryptic species and enable genome-wide population studies (Ellison
et al. 2011; Park et al. 2011; Padamsee et al. 2012; Neafsey et al. 2010). Taking
advantage of next-generation sequencing techniques, genome-wide expressed
mRNA sequences can be easily generated without previous knowledge of genome
sequence or of specific gene regions. Phylotranscriptomics, the use sequences of
expressed messenger RNA sequences to infer phylogeny, has been shown to be a
promising approach to infer phylogenies in several non-fungal groups (Breinholt
and Kawahara 2013; Wickett et al. 2014). Similar applications in the fungal king-
dom are certainly looming on the horizon.
Despite increasing sequencing capacity, it remains the case that for the majority
of fungal species, genome-scale sequence is unlikely to be available soon. In most
of these cases, a multilocus phylogeny is now realistically affordable and is expected
to be informative enough for most systematic questions about these taxa. However,
previously used genetic markers for phylogenetic analysis were originally identified
by a trial and error process based on very limited data and often subsequently
sequenced in other taxa solely motivated by the desire for completion of particular
datasets. Thus, the phylogenetic usefulness of some genetic markers can be far from
optimal (Robert et al. 2011). Sequenced genomes make it possible to assess the
potential phylogenetic utility of many genetic markers as well as to enable more
successful primer design and PCR efficiency (Ye et al. 2012). Knowledge regarding
gene ontology and substitution rates is also critical for selecting proper markers for
resolution of divergences occurring on diverse time scales during disparate epochs.
Approaches for selecting robust sets of phylogenetic markers based on sequenced

genomes are starting to emerge and are urgently needed. For example, ranking
genes for their usefulness in phylogenetic inferences showed promise as a means of
solving phylogenies for some problematic fungal groups (Schoch et al. 2009a;
Binder et al. 2013; Robert et al. 2011; Hyde et al. 2014; Capella-Gutierrez et al.
2014).
Experimental Design and Analysis for Systematics Using

Genome Data
Phylogenetic inference can be improved either by use of better models or by obtain-

ing better data. For phylogenetic problems corresponding to short, deep internodes,
quality of data is often the limitation to successful resolution (Townsend et al. 2008;
Philippe et al. 2011; Su et al. 2014). Early fungal phylogenetic research expanded
the repertoire of genetic markers beyond the common rRNA markers by testing and
developing gene markers that had been found useful in other organisms. The first
AFTOL project selected six markers to sample from major fungal groups after
attempting to widely amplify more than 10 markers (Lutzoni et al. 2004; James
et al. 2006a; Hibbett et al. 2007; Matheny et al. 2007; Liu and Hall 2004). Testing a
small number of genetic markers on a small number of taxa using degenerate PCR
amplification is laborious but feasible; however, its use for evaluating a genome-
scale pool of genes for diverse taxonomic sampling would be infeasible. Identifying
the most informative candidate loci across the genome in advance can provide a
prioritized list for identification by degenerate PCR of novel promising markers or
for use in deciding on reference gene sets for genome-scale targeted capture meth-
odologies (e.g., Li et al. 2013). By adopting relaxed assumptions regarding the
model of molecular evolution and deriving theory based on asymptotic interest in
resolving short deep internodes of four taxon trees, a method for profiling phyloge-
netic informativeness over time of diverse gene markers was developed (Townsend
2007) and applied to the task of identifying better markers during the second
AFTOL project (Schoch et al. 2009a; Townsend et al. 2008).
This theory was generalized to resolve nodes based on rates of evolution of indi-
vidual characters or sets of characters onto the molecular evolutionary or chrono-
logical time scale of interest, weighing the accumulation of signal with internode
length versus the loss of signal on subtending branches of the phylogenetic tree
(Taylor and Berbee 2014; Su et al. 2014; Townsend et al. 2012; Feau et al. 2011;
Miadlikowska et al. 2014; Walker et al. 2012). Binder et al. (2013) performed a
thorough analysis of candidate loci to identify optimal experimental design for reso-
lution of phylogenetic hypotheses. In this comprehensive study, among 356 single-
copy genes, 25 markers ranked at the top for phylogenetic informativeness and
probability to resolve key epochs were selected to resolve the problematic phylog-
eny of wood-decay fungi. As demonstrated in that study, gene markers selected
36 Z. Wang et al.
from sequenced genomes should be evaluated for their site rate distributions, phy-
logenetic informativeness, and predicted signal and noise. Markers then can be
quantified for predicted utility compared to the worst possible performance or ran-
dom sampling of taxa and genes. For a given phylogenetic hypothesis, the process
should rank additional taxa whose genome sequences would provide the most
power for resolving these nodes and then predict which taxon-gene elements of a
presumed data matrix would provide the most power for resolving these nodes. The
result minimizes the effort for resolving the given nodes (and simultaneously mini-
mizes the probability of error) by assessing phylogenetic performance for top taxon-
gene combination until a robust phylogeny is reached.
The advent of big data in phylogenomics has brought renewed attention not only
to issues of phylogenetic signal but also to issues of phylogenetic noise and bias
(Townsend and Lopez-Giraldez 2010; Lopez-Giraldez and Townsend 2011; Lopez-
Giraldez et al. 2013). In data-limiting analyses, it was always possible to quiet con-
cerns about the relative efficacy of some data over other data with a plaintive call
for more data. In the genomic era, with the availability of big data, due to known
issues such as inconsistency of substitution rates, horizontal gene transfer, and
unclear gene ontology, it has become clear that big data results bulwarked by the
traditional hallmarks of strong support are sometimes in conflict with each other
(Salichos and Rokas 2013). The resolution of this conflict requires rigorous thought
about the sources of noise and consequently the relative power of data to address
phylogenetic hypotheses. At the same time, the growing resource of publicly avail-
able sequenced genes and genomes should in principle provide some guidance as to
how to optimally design a phylogenetic sequencing study. For example, genes can
be chosen from sequenced genomes of known phylogeny and then ranked for their
performance in accurately inferring phylogenetic relationships—this approach is an
extension of the practice of traditional marker selection facilitated by automatic
computer programs (Capella-Gutierrez et al. 2014). Performance of these analyses
is facilitated by the web application PhyDesign (Fig. 3.1) (Lopez-Giraldez and
Townsend 2011). PhyDesign evaluates gene performance based on sequence align-
ment and a chronogram to predict signal and noise and the best-possible perfor-
mance, where the metric of interest is the amount of support provided for the given
nodes. Providing a means for prioritizing gene sequencing and taxon sampling and
for sorting the “wheat from the chaff” in large phylogenomic studies, this applica-
tion of the theory for phylogenetic study design would robustly improve the scope
of data collection and analysis, the overall cost-effectiveness, and the probability of
correct inference of a phylogenetic study. In addition, phylogenetic inferences are
increasingly required to be robust to differential gene divergence under the multi-
species coalescent, necessitating informed choices not only on what genetic mark-
ers to employ but also on what analysis approaches to take (Hyde et al. 2013).
Theoretical tools are still needed to address long-standing controversies in
experimental design that have occasionally engendered contentious academic
debate, including (1) the power of different genetic markers, (2) the relative utility
of taxon sampling versus gene sampling, (3) the differentiation between soft and
hard polytomies, and (4) the design of taxonomically dense phylogenetic studies
0.5
a
+
a
Phylogenetic Informativeness
+ 0.4
−
Mean rate
0.3
−
0.2
0.1
0
0.75 0.5 0.25 0
Time
b
1
0.9
Averaged support (PLRS)

0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
10 20 30 40 50
Cumulative loci sequenced
Fig. 3.1 Utility to phylogenetics of extraction of informative genes from genome sequence data.
(a) Phylogenetic informativeness can be estimated and compared among different genes for given
epochs (modified from López-Giráldez et al. 2013). Phylogenetic informativeness profiles for
simulated sequence alignments on a single molecular evolutionary unit. Each of the ten different
colors represents a different mean rate, from 0.0001 (slowest, bottom) to 0.001 (fastest, top) sub-
stitutions per site per time unit. Dashed lines are profiles from alignments simulated with gamma
rate heterogeneity (α = 0.5, 1, 2, and 3). (b) Cumulative proportionate likelihood-ratio support
(PLRS), averaged across nodes for simulated amino acid datasets. Genes are ranked by differential
phylogenetic informativeness encompassing all branches in the tree. The upper dashed line and the
lower dashed line separately represent cumulative PLRS when loci are prioritized, post hoc, from
highest to lowest PLRS values and from lowest to highest PLRS values. The intermediate dashed
line is the hypothetical average one would achieve sampling at random from loci available. The
solid line is the performance when genes are selected by their phylogenetic informativeness based
on inferred rates of sites only
38 Z. Wang et al.
optimized by taxonomically sparse genome-scale data (Lopez-Giraldez et al. 2013;

Moeller and Townsend 2013). A robust fungal phylogeny would provide a solid
framework for fungal systematics that would, in turn, be of increasing significance
in modern mycological research.
Fungal Systematics in the Future: Integration of Fungal

Systematics and Fungal Evolutionary Biology
Systematics is fundamental to organismal biology and is the discipline that synthe-

sizes achievements from all of biology and ultimately underlies all research in evo-
lutionary biology. Arising in part from systematics, the theory of evolution is the
basis of modern biology. A robust phylogeny and reliable classification is the first
step for the development of fungal systematics, and systematists should not be satis-
fied only with describing the evolutionary history of fungal lineages (Hibbett and
Taylor 2013). More importantly it is our responsibility to qualitatively and quanti-
tatively explain how this history led to the diversity we observe today, a question
that brings us to the integration of systematics and evolutionary biology. In fact,
from taxonomy, diversity, molecular phylogeny, to the tree of life, the study of
systematics of all organism groups has itself been evolving, and new contents from
evolutionary biology have been continually if controversially incorporated into
modern systematics (Losos et al. 2013).
Fungal systematics is critical for understanding the evolution of genes and their
functions in fungal genetics, and multigene analysis provides an opportunity to
avoid the pitfalls associated with assuming a single-gene phylogeny represents a
true species phylogeny. Genetics has long focused on gene behavior and function
within species, especially for model organisms, until recently the availability of
sequenced genomes and robust fungal phylogeny made data available to trace gene
ontology among different lineages within a long evolutionary history. Like many
other eukaryotic organisms, horizontal gene transfer and gene/genome duplication
are main contributors for new genes and gene functions in many fungal species
(Bruto et al. 2014; Cohen-Gihon et al. 2011; Fitzpatrick 2012; Wapinski et al.
2007), and horizontal transfer of toxic gene clusters among fungal species was dis-
covered based on sequenced fungal genomes across lineages of fungal tree of life
(Slot and Rokas 2011; Wisecaver et al. 2014). For many fungi, the dominant form
of their life history is haploid, and mitotic and meiotic recombination can happen
via parasexual and sexual reproductions in fungal species (Schoustra et al. 2007;
van de Vondervoort et al. 2007). Thus, the reconciliation of gene phylogeny and
species phylogeny in low-level species taxonomy in fungi could provide insights
into the modeling of speciation events (Taylor et al. 2000).
Fungal systematics and genome-enabled mycology are linked through evolu-
tionary biology. Sequenced genomes provide a huge amount of data that can be
brought to bear on all branches of fungal research. Recent progress has been espe-
cially interesting in efficiently addressing the genetic basis of various phenotypes

(Hibbett et al. 2013; Taylor and Berbee 2014). Genomic research based on fungal
models, such as S. cerevisiae, Neurospora, and Aspergillus species, has been
focused on fundamental biology with implications that extend toward many non-
fungal branches of the tree of life, including meiosis, cell cycle, and internal oscil-
lation (Galagan et al. 2005). In contrast, with an increase of released fungal genomes,
genome-enabled mycology has emerged: early studies have focused either on spe-
cific ecology or on metabolic pathways or functional gene families and their evolu-
tion (Spanu et al. 2010; Vogel and Moran 2013; O’Connell et al. 2012; Stajich et al.
2010; Pel et al. 2007; Martin et al. 2010; Morin et al. 2012). In most of these early
studies, fungal systematics generally serves not only as a guide for what taxa to
sample and study independently but also as a reference for tracking gene history.
With the expected robust phylogeny and well-sampled genomes that could come as
an outcome from the 1000 Fungal Genome project, a reliable gene ontology should
be inferred that would facilitate inference of how fungal morphologies and ecolo-
gies have evolved, knowledge of which is one of the overarching goals of fungal
systematics. For example, one-celled (yeast) stages are distributed throughout the
fungal kingdom, and comparative genomics has revealed that yeast forms arose
early and independently in multiple fungal clades via parallel diversification of a
fungal-specific transcription factor family involved in regulating yeast-filamentous
switches (Nagy et al. 2014). Reliable gene ontology is critical to the reconstruction
of gene networks and the assessment of gene functions, especially for systems biol-
ogy investigation that attempt to answer how complexity can be developed while
essential functions are maintained.
The importance of robust phylogenies to infer the evolution of fungal ecology is
clear, but fungal systematics is also an essential component of any complete under-
standing of fungal ecology. Inorganic and organic components of the environment
impose significant selection on fungal phenotypes (Tedersoo et al. 2014). Ecological
factors, such as host types, nutrient resources, or geographic distribution, have long
been considered characters that are important for fungal classification. With well-
resolved molecular phylogenies, we could evaluate the role of ecology in fungal
evolution, reconcile the ontology of specific gene function groups, and infer the
genetic basis of ecological success. Recent discoveries on the evolution of wood
decay among polypore species and mushroom-forming fungi have demonstrated
how this strategy can work (Binder et al. 2013; Floudas et al. 2012; Eastwood et al.
2011). Applying principles of systematics to metagenomics makes it possible to
monitor the dynamics of biological processes involving diverse fungal species on
both spatial and temporal scales to understand the contributions of those fungal spe-
cies to the process of interest. For instance, a study on global soil sampled by
(Tedersoo et al. 2014) demonstrates direct and indirect effects of climatic and
edaphic variables on plant and fungal richness. The National Science Foundation
has launched a program called Dimensions of Biodiversity, which “takes a broad
view of biodiversity and focuses on the intersection of genetic, phylogenetic, and
functional dimensions of biodiversity.”
40 Z. Wang et al.
Further extension of the broad impacts of fungal systematic research requires

experienced mycologists with broad training in traditional fungal classification,
molecular systematics, and bioinformatics/genomics. Mycologists have long been
considered as naturalists. Training of fungal systematics has been provided in many
institutes, especially in colleges or departments for plant pathology. Fungal classifi-
cation and taxonomy training usually via monographic work require a lot of time in
both field and laboratory, while molecular systematic training requires a decent
facility for sequencing and/or computation. Significant computational needs are
especially required for phylogenomics. Funding resources are heavily biased toward
molecular research, leading to a scarcity of high-quality training in traditional fun-
gal systematics, especially at the graduate level (Pearson et al. 2011). In the long
run, the lack of well-trained mycological systematists would be a problem not only
holding back the development of fungal systematics but also impeding many other
research fields that rely on knowledge of fungal biodiversity and evolutionary biol-
ogy. Well-trained mycologists are also critical for helping the public to understand
the gaps between the quickly developing “omics” sciences and the long-developed
traditional senses of fungi and fungal biology.
The greatest challenge for fungal systematics has always been to be able to
take disparate pieces of knowledge from diverse kinds of studies of fungi to make
synthetic biological inference, and only in this way will fungal systematics be of
maximum benefit to the whole community conducting research on fungi and the
scientific community at large.
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Chapter 4
Molecular Techniques in Mycological Studies
and Sequence Data Curating: Quality Control
and Challenges
R. Henrik Nilsson, Kessy Abarenkov, and Urmas Kõljalg
Introduction
Mycology is tasked with characterizing and cataloging the fungal kingdom.

Molecular (DNA-based) studies of substrates, such as soil and wood, have shown
this task to be enormous; each new study typically recovers a hundred or more spe-
cies of fungi that do not seem to be known from before and that do not produce
close matches to any fully identified reference sequence (Tedersoo et al. 2014).
Some 100,000 species are recognized formally, which is less than a tenth of the
estimated 1.5 million extant species of fungi (Hawksworth 2001). Given that only
about 1000 new fungi are described each year, it is not surprising that much of the
fungal diversity being unearthed through DNA sequencing is not easily fitted into
the framework of already described species (Hibbett et al. 2011). Incorporating also
these poorly known lineages into mycology and the fungal tree of life—often in
total absence of morphological structures and any data on ecology, trophic mode, or
other species traits—is perhaps the greatest challenge for the early twenty-first-
century mycology.
The nature of fungal life puts many obstacles in the way of expedient mycologi-
cal progress. Many species are subterranean or substrate dwelling and rarely—if
ever—manifest themselves through morphological structures such as fruiting bod-
ies (Stajich et al. 2009; Rosling et al. 2011). Morphological characters in fungi can
be deceptively similar or dissimilar, such that even expert observers may be easily
R.H. Nilsson, Ph.D. (*)

Department of Biological and Environmental Sciences, University of Gothenburg,
Box 461, 405 30 Göteborg, Sweden
e-mail: henrik.nilsson@bioenv.gu.se
K. Abarenkov, Ph.D. • U. Kõljalg, Ph.D.
Natural History Museum, University of Tartu, Vanemuise 46, Tartu 51014, Estonia
e-mail: kessy.abarenkov@ut.ee; urmas.koljalg@ut.ee

DOI 10.1007/978-3-319-29137-6_4
48 R.H. Nilsson et al.
lead astray (Yang 2011). Many fungi cannot readily be cultured in the laboratory,
which excludes them from the rich array of examination techniques available for
fungal cultures (Hawksworth 1991). These and other challenges have hampered
mycology for far too long, and it is critical that we adopt a different view of what
criteria must be fulfilled before a fungal lineage merits scientific attention.
Observable life stages and cultivability should not be among those criteria.
Fortunately, access to molecular data has done a lot to facilitate this transition in
thinking.
Molecular data gradually gained ground as a mycological research implement in
the 1990s; the first applications included inference of phylogenetic relationships
and taxonomic identification of mycorrhiza (Förster et al. 1990; Gardes and Bruns
1993). The low-throughput Sanger DNA sequencing approaches of those days soon
saw substantial improvement in speed, quality, and degree of automation, and they
were recently complemented by next-generation sequencing (NGS) methods with
the capacity to generate millions—even billions—of (typically short) sequence
reads over the course of a few hours to days (Liu et al. 2012). Today, molecular data
underpin much of our understanding of fungi and fungal diversity, and a molecular
thinking permeates most mycological research efforts. It is, however, a mistake to
believe that molecular data per se vouch for increased resolution in the results and
for objective answers to the research questions being pursued. Much like other
sources of data, DNA sequences will not give rise to appropriate, correct results
unless they are generated and analyzed in an appropriate, correct way.
The most popular genetic marker for research at the fungal species/genus level is
the nuclear ribosomal internal transcribed spacer (ITS) region. In addition to its
prominent role in phylogenetic inference, the ITS region is the formal fungal barcode
and the primary target for countless molecular ecology and environmental sequenc-
ing efforts of fungal communities (Schoch et al. 2012; Lindahl et al. 2013). To date,
some 500,000 (Sanger sequencing-derived) fungal ITS sequences have been gener-
ated by the scientific community and are available for reference in the International
Nucleotide Sequence Database Collaboration (INSDC: GenBank, ENA, and DDBJ;
Nakamura et al. 2013). The corpus of public ITS sequences is, however, not devoid
of complications. Only about half of the sequences are identified to species level,
the rest being given names of various degrees of informativeness, ranging from,
e.g., Cryptococcus sp. to “uncultured fungus.” Second, estimates show that more
than 10 % of the sequences annotated to species level may in fact carry incorrect
names (Bidartondo et al. 2008). Third, a nontrivial portion of the sequences suffers
from technical problems: some have reduced read quality/incorrect base-calling
(Hyde et al. 2013), some are chimeric (Nilsson et al. 2010), some represent other
genes and markers than the ITS region (Bengtsson-Palme et al. 2013), and some are
reverse complementary (given backward and with purines and pyrimidines trans-
posed; Hartmann et al. 2011). This makes uncritical use of public DNA sequences
for, e.g., molecular species identification prone to errors and suboptimal results.
Many users of those data may not be in a position to spot such errors, which
increases the pressure on the mycological community to provide ITS sequences of
high integrity and usefulness for the scientific community at large.
4 Molecular Techniques in Mycological Studies and Sequence… 49
The INSDC fills an invaluable function in providing long-term storage and

global access to DNA and protein sequences (and other data). The INSDC takes
many measures to highlight their data to researchers and to accommodate the wishes
of an increasingly molecular research community (Schoch et al. 2014). The INSDC
is to some degree interactive and gives users the opportunity to comment on certain
parts of the data (http://www.ncbi.nlm.nih.gov/pubmedcommons/). The balance
between long-term stability and immediate flexibility is however a hard one to navi-
gate. Users cannot change INSDC data directly even if they come across incorrect
or substandard information; such changes will have to go through the database cura-
tors. Certain changes—such as taxonomic re-annotation of incorrectly identified
sequences—require the explicit permission of the original sequence authors, whose
present contact information may or may not be available to the curators. Whereas
we have found the INSDC curators very helpful and knowledgeable, there is a limit
to the changes they can implement, particularly in the short-term perspective. The
field of molecular ecology, in contrast, sees continual bursts of technical progress
and new taxonomic and ecological reference data. This brings about the need for
frequent additions and changes in the reference data—changes that, ideally, should
take effect immediately.
The UNITE Database
The UNITE database (http://unite.ut.ee; Kõljalg et al. 2013) was devised to facili-
tate molecular identification of fungi. It mirrors the INDSC to offer all public fungal
ITS sequences as a reference corpus. This makes UNITE unusual; other reference
databases typically focus on select sequences known to be of particularly high tech-
nical quality, metadata richness, and taxonomic reliability. UNITE takes the posi-
tion that such a limited-taxon approach would not serve mycology well. The
majority of fungal operational taxonomic units (OTUs; Blaxter et al. 2005) known
from ITS sequences do not carry full species names, yet they are every bit as real as
those who do. Excluding the majority of the known species from molecular identi-
fication procedures would lead to massive losses of precision and scientific explana-
tory power. Instead, UNITE provides all public sequences and offers several ways
to highlight particularly reliable entries as well as to remove substandard ones. The
user is presented with several ways to interact with the sequences and modify them
in various regards. The inclusive taxonomic scope of the database means that a
limited number of database developers and curators cannot be expected to possess
the expertise to revise all taxonomic lineages equally well in the database. Thus, the
database comes with a web-based sequence management environment such that all
registered users can participate in data curation through a regular web browser.
Two times a year, all fungal ITS sequences are clustered to approximately the
genus level. A second round of clustering inside each such cluster seeks to produce
OTUs at approximately the species level. These OTUs are called species hypothe-
ses (SHs). By default, a 98.5 % similarity threshold (1.5 % distance to the closest
Fig. 4.1 A genus-level alignment of the basidiomycete genus Meira. The sequences are displayed
together with available metadata on country and host of collection. Sequences from type material
are used to fix the application of species names through reference sequences. Two examples of
taxonomic re-annotation are shown; for instance, sequence KF435880, originally deposited in the
INSDC as “fungal endophyte,” is now annotated as Meira. The filled, colored boxes indicate the
SHs and their scope under different similarity thresholds. The SHs point to the existence of hith-
erto unrecognized diversity in the genus
distinct SH) is used as proxy for the species level, but a user can reset the threshold
(in the interval 90 %, 95 %, and 97–100 % similarity in 0.5 % steps) to better reflect
the species level for individual SHs. A genus-level cluster of parts of the genus
Meira is shown in Fig. 4.1 together with the constituent sequences, SHs, and their
metadata on ecology and geography. The genus-level alignments form perhaps the
most straightforward arena for the user to navigate, explore, and interact with the
sequence data in UNITE. Tools such as BLAST (Altschul et al. 1997) allow users
to query newly generated sequences against the database. UNITE also serves as
primary or secondary data provider for a range of external applications and
resources, including the next-generation sequencing analysis pipelines QIIME
(Caporaso et al. 2010), mothur (Schloss et al. 2009), CREST (Lanzén et al. 2012),
UTAX (http://drive5.com/usearch/manual8/cmd_utax.html), and SCATA (http://
scata.mykopat.slu.se/) as well as the EUBOLD and ISHAM databases (http://www.
eubold.org/ and http://its.mycologylab.org/, respectively). This heterogeneous user
base highlights the need for high reliability and richness of the data in UNITE. As
a consequence, UNITE is the subject of intense sequence curation in various
regards. These curation and quality control processes are described below and are
given together with recommendations and ideas on how users can exercise quality
control also on personal, not-yet-public sequences.
Names and Taxonomic Re-annotation
Names of organisms and organism groups are central devices for communication in
biology. They allow precise and unambiguous reference and knowledge building
across studies and over time. Incorrectly applied names in sequence databases coun-
teract these processes and invite further misidentifications as users adopt and incor-
porate erroneous names from sequence similarity searches. Any user with a newly
generated set of DNA sequences should take steps to ensure that any hypothesized
taxonomic affiliations of the sequences check out—a BLAST search in UNITE or
the INSDC is usually enough to rule out contamination or severe cases of misiden-
tification. BLAST searches are often informative also on the reference sequences;
most searches will reveal at least one reference sequence that either seems incor-
rectly identified (such as cf. Puccinia sp. for an ascomycete) or that lack meaning
taxonomic annotation altogether (such as the common “uncultured fungus”). A
friendly email to the original sequence authors or the database curators may resolve
such problems, at least if the user provides sufficient data and detail to make the
validity of the claim easy to verify.
The original sequence authors are however not always known or available, which
makes it difficult for, e.g., the INDSC to seek permission to effectuate name changes.
This slows down the renaming process significantly. UNITE offers one-click taxo-
nomic re-annotation of public sequences using the names and classification of Index
Fungorum (http://www.indexfungorum.org/; Fig. 4.2). The changes take effect
immediately, and the user will be credited with the name change. Incremental name
changes are supported and encouraged; if a sequence cannot be assigned to species
level, a genus or order name will still be helpful. The same—or another—user can
then provide the full species or genus name if and when additional, explanatory data
emerge. All taxonomic re-annotations done in UNITE are made available to the
INSDC, and at least a subset of them are implemented there too.
Many species hypotheses cannot be given full species names for lack of refer-
ence data and/or taxonomic progress in those fungal lineages. Assigning them as far
as possible will be of great value to many users: Puccinia sp. is a great deal more
informative than “uncultured fungus.” Even so, these and similar progressional
names may be applied to hundreds or even thousands of other, non-conspecific
sequences. This makes precise reference to those species across publications and
datasets problematic and forms an obstacle to knowledge building and data assem-
bly for individual species. As a remedy, UNITE assigns names of the accession
number type to all species hypotheses. Whereas the shortest form of those names is
simply an accession number—such as SH203822.06FU—the longer form,
Hymenoscyphus pseudoalbidus | SH203822.06FU | 98.5 | GU586904, comprises
any name (Latin or otherwise) given to the sequence, the SH accession number, the
similarity threshold at which the SH was designated, and the INSDC accession
number of the sequence chosen to represent the SH in situations when only one
sequence per SH is used. (The latter includes the nonredundant BLAST databases
Fig. 4.2 Taxonomic re-annotation of INSDC entry FR731412, originally deposited under the low-
resolution name “uncultured ectomycorrhizal fungus.” Sequence analysis shows that it belongs to
the Thelephoraceae, and the according taxonomic annotation is a one-click process. Upon starting
to type the new name, the user is presented with a drop-down list of accepted names and ranks to
choose from
of many sequence analysis pipelines.) These SH names are implemented through-

out UNITE and are forwarded to the downstream resources and applications that
use UNITE. We hope that users will consider using these names for OTUs that
otherwise would lack an informative name (see, e.g., Morgado et al. 2014); this will
allow them and other users to trace those OTUs across publications and datasets,
much to the purpose of reproducibility and data harvesting.
Chimeras
Chimeras are artificial sequences composed of two or more sequence fragments that
do not belong together but that were joined in the PCR or sequence assembly steps
(Qiu et al. 2001). The typical chimera is produced when the PCR enzyme switches
template from one species to the other in mixed-template PCR reactions; any pres-
ence of a conserved sequence segment in the target marker—such as the 5.8S gene
in ITS sequences—increases the risk of chimeric unions (Fonseca et al. 2012).
Chimeras lack a meaningful biological interpretation and introduce noise and bias
to studies featuring them; molecular identification, richness estimations, multiple
sequence alignment, and phylogenetic inference are examples of processes that are
adversely affected by chimeric sequences. Steps can be taken to reduce the chances
of chimera formations in the PCR phase (Lindahl et al. 2013). Equally important is
to screen for chimeras in newly generated datasets; this should be done on a routine
basis for all new fungal ITS datasets.
Screening small-to-midsize, relatively homogeneous datasets for at least severe
cases of chimeras is relatively easy and amounts to a multiple sequence alignment
of the query sequences. Any sequence found to align well in the first half of the
alignment but to align poorly in the second half (or the other way around) merits
further scrutiny (Fig. 4.3; Nilsson et al. 2012). A BLAST search followed by man-
ual inspection of the results is another approach with a similar potential for chimera
discovery (Fig. 4.4). Separate BLAST searches of the two sequence halves are then
Fig. 4.3 A chimeric sequence showing (a) perfect alignment in the first half of the multiple
sequence alignment (the cursor indicates the start of the 5.8S gene) but (b) poor alignment in the
second half (the cursor marks the end of the 5.8S). Particularly conserved sequence elements—the
5.8S gene in this case—often serves as chimeric breakpoints. SeaView (Gouy et al. 2010) was used
to display the multiple sequence alignment
Fig. 4.4 A chimeric sequence often produces odd-looking BLAST results, such as the one shown
here. Either the last part of the sequence, or the first part of the sequence, but never both at the same
time, account for the alignment to the sequences in the reference database. Manual examination of
the query sequence and the BLAST results are needed in cases like this. Chimeras between closely
related species is unlikely to give rise to BLAST results as clear-cut as this one
usually enough to convince oneself of the chimeric—or authentic—nature of the

sequence. For users with larger datasets and/or some experience of the computer
command line, we recommend UCHIME (Edgar et al. 2011), which is a fast and
accurate software tool for chimera control. An automatically updated and reason-
ably chimera-free reference file for fungal ITS sequences is available from the
UNITE download page (http://unite.ut.ee/repository.php). (UCHIME also supports
de novo, reference-free chimera calling for NGS datasets.) Chimera detection is
nevertheless not a trivial undertaking, and elusive chimeras are certain to exist in
both the INSDC and UNITE. Users finding chimeras in UNITE are asked to mark
these as chimeric through a simple click, which will prevent their ulterior use as
representative sequences and in sequence identification efforts. The INSDC staff is
similarly very quick to remove chimeras once alerted to their presence. UNITE and
the INDSC exchange data on chimeras regularly.
Technical Quality of Sequences
Once sequences are deposited in public databases, quality control becomes hard.
The sequences are typically deposited without chromatograms and other auxiliary
data, leaving other researchers struggling to tell sequences of low technical quality
from sequences that differ from other sequences for the reason that they represent
biological novelties. DNA ambiguity codes, such as N, R, and S (Cornish-Bowden
1985), scattered across a sequence are a sure sign that the sequence should be
dropped from most research efforts. Lengthy homopolymer regions (such as
AAAAAAAAAA), particularly in the distal parts of a sequence, similarly hint at a
substandard entry. Distal ends are generally of lower read quality compared to inte-
rior parts of sequences (owing to the nature of the Sanger sequencing method), and
researchers should make it a habit to trim such noisy parts prior to sequence submis-
sion. A regular BLAST search coupled with manual inspection of the results is
usually enough to detect all the above problems (Fig. 4.5). It is often more or less
impossible to prove a sequence to be compromised, but for most purposes, it is
enough to establish that a compromised nature seems very likely. Such entries can
then be left out from analyses. To routinely exclude all sequences that differ from
known sequences is however not a good way to expand our understanding of the
world around us, and the user is probably best off excluding only sequences for
which a compromised nature is deemed beyond reasonable doubt.
Sequences can be of very high read quality and still suffer from technical prob-
lems. Sanger sequences are typically assembled from two or more primer reads.
Although (semi)automated, this process requires some understanding on part of the
user of the relative order and read direction of the primers. Failure to inspect—or
understand—the assembly results regularly leads to submission of chimera-like
sequences to the public databases. Sometimes the first half of the sequence is given
in the correct orientation, whereas the second half is given in the reverse comple-
mentary orientation. At other times, sequence fragments were assembled in the
wrong order or with a fragment missing. Insertion of fragments that do not belong
in the sequence to begin with has also been reported (Nilsson et al. 2012). These
sequences typically produce odd-looking results in BLAST. The alignment may be
divided into sections or cover only a part of the query sequence (Fig. 4.6); the
“strand” flag of the BLAST output is helpful in detecting reverse complementary
insertions. The technically inclined user will find that the software tools ITSx
(Bengtsson-Palme et al. 2013) and UCHIME (Edgar et al. 2011), although not
explicitly designed to find sequences of these types, work surprisingly well for the
Fig. 4.5 Failure to trim noisy ends of a sequence gives rise to BLAST results looking like this;
note how the distal ends of the sequence do not find matches in the counterpart sequences in the
reference database. The reader should keep in mind that a large proportion of public fungal ITS
sequences were submitted with noisy ends, such that it is not always straightforward to tell if it is
the query or the reference sequences that do not meet quality expectations
task. The INSDC is quick to take action when alerted to such substandard entries;
one of the actions available to their staff is to mark such sequences “UNVERIFIED.”
Users of UNITE similarly have the option to mark sequences as being of low techni-
cal quality. The INSDC and UNITE exchange data on substandard entries.
Fig. 4.6 The black vertical line in the BLAST results means that BLAST was unable to produce
a straightforward alignment involving the query sequences and the topmost reference sequences.
A segment was missing (or was extraneous) in the alignment; alternatively, the query sequence was
assembled incorrectly. Manual examination of the sequences involved is needed in cases like this
Non-ITS Sequences and Reverse Complementary Entries
Several hundred public sequences marked as fungal ITS sequences have been found
to represent other genes and markers than the ITS region. Mixed-up test tubes,
labels, and computer files are the presumed culprits. To include such non-ITS
sequences in ITS datasets is certain to lead to compromised results. Fortunately, it
is usually easy to find out whether sequences of at least some 300 bases indeed are
ITS sequences. Three hundred bases are normally enough to cover at least one end
of the very conserved 5.8S gene in the center of the ITS region. Thus, the query
sequence can be aligned to a random (diverse) set of ITS sequences known to be full
length. If the query sequence produces a match to the 5′ or 3′ ends of the 5.8S gene
(or the 3′ end of the SSU gene upstream of the ITS region, or the 5′ end of the LSU
gene downstream of the ITS region), then the user can be certain that the query
sequence indeed represents the ITS region (Fig. 4.7). A regular BLAST search in
the INDSC can also be used to the same effect: a list of reasonably close hits anno-
tated as fungal ITS sequences, preferably stemming from two or more different
studies, can be taken as tentative evidence that the query indeed is a fungal ITS
sequence. The technically inclined user is referred to the software tool ITSx, which
was designed to tell ITS sequences from others. (V-Xtractor (Hartmann et al. 2010)
and Metaxa2 (Bengtsson et al. 2011) are equivalents for SSU and LSU.) The above
solutions hinge on the presence of at least ~45 bp of one or more of the SSU, 5.8S,
or LSU as alignment anchor in the query sequence. For reads with shorter coverage
of these genes than this—partial ITS1 or ITS2 sequences—the user has little choice
but to do BLAST searches and examine the results.
Sequences can come from the ITS region and still appear to represent something
totally different when viewed in, e.g., a multiple sequence alignment. If the user
fails to take the read direction of the primers into account, the user may inadver-
tently export sequences in the reverse complementary orientation from the sequence
assembly step. Such sequences have no immediate resemblance to their true coun-
Fig. 4.7 Whether or not a sequence represents the ITS region can be established in several ways.
One of them is to focus on the very conserved 5.8S gene, particularly its 5′ end. Shown is the 5′
end (starting at alignment position 134, highlighted) of five random sequences from each of the
Basidiomycota, Ascomycota, Glomeromycota, and 5 the former Zygomycota. A sequence that pro-
duces a satisfactory alignment to the first part of such a 5.8S reference alignment is certain to
represent the ITS region. Strictly speaking, it does not necessarily indicate that the sequence is of
fungal origin though
Fig. 4.8 A reverse complementary sequence was computed from the entry highlighted in black. It
is shown immediately below that entry. Even though the two entries are identical in terms of infor-
mation content, there is little resemblance in the multiple sequence alignment. With the realization
that such odd-looking sequences may in fact be reverse complementary, their proper integration
into the alignment is easily overseen in modern alignment viewers
terpart (Fig. 4.8). Fortunately, BLAST supports reverse complementary sequences

by default and accounts for their nature in the alignment stage. A “strand” flag of
“plus/minus” or “minus/plus” in the BLAST output indicates opposing read direc-
tions of the query and the reference sequence. The user can reorient reverse comple-
mentary sequences in alignment editors such as AliView (Larsson 2014) or in any
of a number of online tools (e.g., http://www.bioinformatics.org/sms/rev_comp.
html). The tool ITSx can be used to track down reverse complementary entries in
ITS datasets of any size.
Metadata and Species Traits
Many sequences are submitted to sequence databases with little more extra data
than the name and address of the sequence authors. Tedersoo et al. (2011) showed,
for instance, that country of origin was provided for a modest 43 % of the public
fungal ITS sequences. Along the same line, Ryberg et al. (2009) found that less than
a quarter of the public ITS sequences were annotated with host of collection. This
is most unfortunate since data on geography and ecology are often needed to make
informed decisions in molecular identification and systematics. To be able to pro-
vide more metadata than those present in the original sequence entries, UNITE
offers the user the possibility to enter such data for entries lacking them. While the
two most important data items are probably country and host of collection, a long
range of parameters are available for specification. These include nutritional mode,
lifestyle, substrate type, mycorrhizal lineage, primers used, and voucher specimen/
culture. DarwinCore (http://rs.tdwg.org/dwc/) specifications are followed as appli-
cable. Associating individual sequences with voucher specimens and/or cultures is
straightforward and particularly valuable in the case of type material. The myco-
logical community has not always been consistent in the way specimen/culture data
are specified during the sequence submission process (and whether or not they are
provided at all), which makes subsequent searches difficult. Recent developments in

the INSDC show significant promise in this regard (Schoch et al. 2014).
Results and Data Sharing
UNITE has headed several annotation efforts to bring the public fungal ITS
sequences up to standards. Two efforts for mycorrhizal (Tedersoo et al. 2011) and
plant-pathogenic fungi (Nilsson et al. 2014) focused on improving taxonomic
names and affiliations given to sequences as well as providing ecological and geo-
graphical metadata that were missing. An annotation effort headed by GenBank
(Schoch et al. 2014) fixed the application or more than 3000 species names through-
out the fungal kingdom by associating sequences with type specimens/ex-type cul-
tures. Efforts focusing on specific technical problems of public ITS sequences
include chimera detection (Nilsson et al. 2014), reverse complementary entries
(Nilsson et al. 2011), and non-ITS sequences (Bengtsson-Palme et al. 2013). These
efforts, together with all changes contributed by UNITE users, translate into a full
31,593 taxonomic re-annotations, which corresponds to 7.4 % of the number of
public fungal ITS sequences as of December 2014. Data on ecology and geography
have been provided for 52,807 and 58,414 sequences (25,974 of these correspond-
ing to sequences for which both metadata on ecology and geography were added).
Specimen/culture associations have been established or clarified for 12,183
sequences, of which 3657 are related to type material. A full 7141 sequences have
been identified as being of low read/technical quality, and 2554 cases of chimeras
have been found and marked as such. Some 3034 sequences were originally depos-
ited in the reverse complementary orientation; these now appear in their correct
orientation.
These additions and improvements benefit anyone who uses UNITE for molecu-
lar identification or other analyses of fungal ITS sequences. UNITE furthermore
serves as data provider of fungal ITS sequences for a range of other databases and
sequence analysis tools; these resources, too, benefit from any improvements done
to the data in UNITE. The sharing of the species hypotheses and their unique, non-
ambiguous names with the INSDC and the largest NGS analysis pipelines further-
more solves a long-standing problem in fungal molecular ecology: how to refer to
specific, unidentified species in a standardized way across studies, datasets, and
software pipelines. This brings promise of an end to the large proportion of “uniden-
tified/fungi sp.” species recovered in NGS-based environmental sequencing efforts.
Any user deciding to dig a bit deeper on the taxonomic affiliation of such unidenti-
fied species is likely to uncover additional resolution; nearly all SHs we have exam-
ined, for example, can be assigned at least to the phylum—and often also order—level.
Once such a re-annotation is implemented in UNITE, the corresponding change in
the data export files of UNITE will reach the users of the NGS analysis pipelines.
UNITE also provides downloadable FASTA files of all sequences (http://unite.ut.ee/
repository.php) for the use in, e.g., local BLAST searches and sequence analysis
efforts. Various FASTA versions are available—it is our intention to suit all needs,
and we would be happy to consider requests for specific formats or data items to
support for regular exports. UNITE finally exchanges data—bidirectionally—with
the INSDC. This maintains some level of data synchronization among the datasets,
although UNITE typically lags behind in terms of the latest sequences and their
integration into BLAST searches. The species hypothesis platform is recomputed
twice a year, meaning that some six months may elapse before a sequence is assigned
to an SH.
Discussion
Fungi play significant ecological roles—notably as nutrient cyclers—and form key

players in most ecosystems. This makes fungi attractive research objects in a range
of scientific fields in addition to mycology, including forestry, soil biology, and
agriculture. Disturbingly, when researchers in these fields sequence their substrates
for fungal diversity, they often get results that are low in resolution (“uncultured
fungus”) or that are directly misleading. In our opinion, this makes mycology look
bad. There are, after all, several thousand mycologists worldwide, and their joint
knowledge and expertise are remarkable. And yet mycologists do not seem to have
found a way to communicate this knowledge to the scientific community at large—
at least not in such a way that it could be put to direct use. Although you could argue
that every environmental sequencing effort targeting fungal communities should
have at least one mycologist onboard, this will not always be the case. Thus, the
mycological community should see to it that analysis and interpretation of DNA
sequencing efforts of fungal communities are as straightforward and easy as possi-
ble also for non-mycologists. There are numerous ways in which mycologists can
contribute toward this goal. For instance, we feel that mycologists should provide a
DNA sequence (preferably from the ITS region) along with every new species they
describe. When mycologists describe species and deposit fungal sequence data in
public databases, they should make an effort to provide rich metadata and to estab-
lish the basic integrity of the sequence data (see guidelines in Seifert and Rossman
2010; Nilsson et al. 2012; Hyde et al. 2013; Schoch et al. 2014). Finally, as this
chapter has shown, a lot would be gained if individual mycologists made sure that
public sequence data from their particular fungal lineage of expertise were as cor-
rect and richly annotated as possible in the public sequence databases.
Another reason to ensure that your fungi of expertise are tidy and properly anno-
tated in the sequence databases is that your research is likely to benefit from it.
When other researchers recover those species in their samples, those species will
then be named and annotated correctly owing to your expert curation. This will
facilitate knowledge building for individual species and will prevent publication of
results attached to spurious names. The user is, furthermore, likely to find that
browsing the species hypothesis alignments in UNITE is a rewarding undertaking
in terms of data visualization and exploration. We often hear from users who have
found hitherto unrecognized patterns in the data as a result of the simultaneous

exploration of species hypothesis boundaries, sequence data, and information on
country and host of collection. These patterns are typically not apparent in regular
multiple sequence alignments, let alone in the lists of sequences in INSDC that
some researchers monitor to stay updated on developments in their fungal lineage
of interest. With this chapter, we welcome all researchers to examine the species
hypothesis alignments of their fungal groups of interest.
Most researchers would probably agree that you should try to review approxi-
mately as many manuscripts as you submit for review on an annual basis. That way,
some sort of balance is attained in that you both add to, and draw from, the collec-
tive expertise of the scientific community. The same line of thinking should be
applied to sequence data; if you plan to undertake a series of NGS studies of fungal
communities and thus draw from the pool of available reference sequences, then it
would be good if you could allot a small amount of money to sequence, say, a hand-
ful of previously unsequenced fungal type specimens/ex-type cultures in your local
herbarium/culture collection and make those sequences publicly available. Similarly,
when you find a public fungal sequence that is severely compromised, it would be
very helpful if you could take action. To do nothing, which sadly is all too common,
is an indefensible position in the long run. It makes mycology look bad and invites
further mistakes and suboptimal results based on that particular compromised entry.
Mycology, we feel, is not a field whose name should conjure up visions of subopti-
mal results and failure to resolve research hypotheses in the mind of the general
scientific audience. Indeed, mycology struggles for funding in competition with
fields that are often deemed larger or more fashionable, and we simply cannot afford
public fungal DNA sequences to remain in a suboptimal state. Fortunately, at least
part of the remedy comes at no cost and is no further away than the nearest Internet
connection.
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Chapter 5
Challenges and Future Perspectives
in the Systematics of Kickxellomycotina,
Mortierellomycotina, Mucoromycotina,
and Zoopagomycotina
Gerald L. Benny, Matthew E. Smith, Paul M. Kirk, Eric D. Tretter,

and Merlin M. White
Introduction
The basal lineages or early-diverging fungi contain the aquatic (zoosporic) chytrids
(Chytridiomycota (including Neocallimastigomycetes), Blastocladiomycota) and the
terrestrial (aplanosporic) zygomycetes (Entomophthoromycota, Glomeromycota,
Kickxellomycotina, Mortierellomycotina, Mucoromycotina, Zoopagomycotina)
(reviewed by Shelest and Voigt 2014). The members of the final four subphyla,
Kickxellomycotina, Mortierellomycotina, Mucoromycotina, and Zoopagomycotina,
which constitute four of the six predominantly terrestrial clades of the early-diverging
fungi, have been discussed elsewhere (Benjamin 1979; Benny 2001, 2012; Benny
et al. 2001, 2014; Voigt 2012; Voigt and Kirk 2014). The fifth member of the terres-
trial early-diverging fungi, the phylum Entomophthoromycota, is discussed by
(Gryganskyi et al. 2012, 2013; Humber 2016) and in the following chapter (Humber
2016). These fungi often are presented only in ordinal discussions or more recently
as subphyla. The Mortierellales were previously combined with Mucoromycotina
before the description of the Mortierellomycotina (Hoffmann et al. 2011). Information
on the Internet for the Asellariales and Harpellales can be found at this URL that
G.L. Benny, Ph.D. (*) • M.E. Smith Ph.D.

Department of Plant Pathology, University of Florida, PO Box 110680,
Gainesville, FL 32611-0680, USA
e-mail: gbenny@ufl.edu
P.M. Kirk, Ph.D.
Mycology Section, Kew Gardens, Royal Botanic Gardens, Kew, Richmond,
Surrey TW9 3AB, UK
e-mail: p.kirk@kew.org
E.D. Tretter, M.S. • M.M. White, Ph.D.
Department of Biological Sciences, Boise State University, Boise, ID 83725-1515, USA
e-mail: merlinwhite@boisestate.edu

DOI 10.1007/978-3-319-29137-6_5
66 G.L. Benny et al.
contains Lucid keys (http://keys.lucidcentral.org/key-server/data/0b08020c-0f0c-

4908-8807-030c020a0002/media/Html/home.htm/), and the remaining zygomycetes
exclusive of the Asellariales, Harpellales, and Entomophthoromycota can be located
at the following URL (http://www.zygomycetes.org/).
A few taxa of the Mortierellomycotina and Mucoromycotina are economically
important as the causative agents of plant diseases, including Choanephora blight
on cucurbits and a few other vegetables [Choanephora cucurbitarum], postharvest
diseases on a variety of fruits (Gilbertella persicaria, Mucor piriformis, Rhizopus
stolonifer), and mucormycosis on humans and other mammals (Michailides and
Spotts 1990; Ribes et al. 2000; Gomes et al. 2011; Benny et al. 2014)]. Other taxa
are used in industry for biotechnology, biodegradation, biosorption, bioremedia-
tion, and biotransformation. Some Mucorales are used in the production of fer-
mented Asian foods and for laboratory studies on photobiology and physiology,
including zygospore formation, the production of polyunsaturated fatty acids and
β-carotene, as well as subsequent derivative products such as lycopene and various
sterols (Benny 2012; Benny et al. 2014).
Many species of the Kickxellomycotina, Mortierellomycotina, Mucoromycotina,
and Zoopagomycotina are deposited in culture collections around the world,
although their ability to grow and persist in culture is highly variable among taxa.
The most complete representations are in CBS (Centraalbureau voor
Schimmelcultures, Utrecht, The Netherlands), IMI (CABI Europe UK, Bakeham
Lane, Egham, Surrey, United Kingdom), and NRRL (USDA, ARS, NCAUR,
Peoria, Illinois, USA). Other taxa or isolates may be obtained from soil, dung, and
other organic material or host organisms using the methods described below. Some
methods are widely useful for many taxa, whereas others are specialized for specific
species or lineages within these subphyla.
The phylogenetic relationships of these fungi have been elucidated using molecu-
lar techniques, initially, using sequences from one or a few genes for: (1) Mucorales
(O’Donnell et al. 2001; Voigt and Wöstemeyer 2001), (2) Harpellales and Kickxellales
(O’Donnell et al. 1998), (3) Dimargaritales and Zoopagales (Tanabe et al. 2000),
(4) Harpellales (White 2006), and (5) Zygomycota sensu lato (White et al. 2006a).
James et al. (2006) included six genes from approximately 200 taxa analyzed by
Bayesian analysis to reveal the early evolution of fungi. The phylogeny of James
et al. (2006) and other multilocus analyses of the fungi have presented a relatively
stable classification at the ordinal level and above (Hibbett et al. 2007).
Karpov et al. (2014) and James et al. (2013) analyzed phylogenetic and phyloge-
nomic data to determine that members of the Cryptomycota (synonym =
Rozellomycota; Corsaro et al. 2014) such as Amoeboaphelidium protococcarum and
Rozella allomyces belong to a clade that also includes the Microsporidia. Their
results also suggest that this lineage is the sister clade of all other fungi. The branch-
ing order of the zoosporic chytrid lineages (Chytridiomycota, Neocallimastigomycetes,
Blastocladiomycota) has not yet been confidently determined. In this chapter, we
focus on isolation, culture preservation, detection in the environment, and evolution
of those zygomycotan early-diverging fungi in the Kickxellomycotina,
Mortierellomycotina, Mucoromycotina, and Zoopagomycotina.
5 Challenges and Future Perspectives in the Systematics of Kickxellomycotina… 67
Subphylum Kickxellomycotina (Hibbett et al. 2007; Tretter

et al. 2013, 2014) (Table 5.1)
Fungi produce hyphae that are regularly septate; each septum possesses a single,
more or less central lenticular cavity containing a plug. Asexual reproduction by the
formation of arthrospores, one- or two-spored merosporangia bearing
Table 5.1 Classification of Kickxellomycotina Benny

the four orders and four
Asellariales Manier ex Manier & Lichtw.
clades of the
Kickxellomycotina Asellariaceae Manier ex Manier & Lichtw.
Asellaria R.A. Poiss.
Orchesellaria Manier ex Manier & Lichtw.
Genus of unknown affinity
Baltomyces Cafaro emend. Oman and M.M. White
Dimargaritales R.K. Benj.
Dimargaritaceae R.K. Benj.
Dimargaris Tiegh.
Dispira Tiegh.
Tieghemiomyces R.K. Benj.
Spinalia Vuil.
Harpellales Lichtw. & Manier
Harpellaceae L. Léger & Duboscq ex P.M. Kirk &
P.F. Cannon
Carouxella Manier, J.-A. Rioux & Whisler ex Manier,
J.-A. Roux & Lichtw.
Harpella L. Léger & O. Duboscq
Harpellomyces Lichtw. & S.T. Moss emend. Lichtw.,
M.M. White and Colbo
Stachylina L. Léger & M. Gauthier
Stachylinoides Lichtw. & López-Lastra
Legeriomycetaceae Pouzar
= Genestellaceae L. Léger & M. Gauthier
Allantomyces M.C. Williams & Lichtw.
Austrosmittium Lichtw. & M.C. Williams
Bactromyces R.T. William & Strongman
Baetimyces L.G. Valle & Santam.
Barbatospora M.M. White, Siri & Lichtw.
Bojamyces Longcore emend. L.G. Valle & Santam.
Capniomyces S.W. Peterson & Lichtw.
Caudomyces Lichtw., Kobayasi & Inhoh
Coleopteromyces Ferrington, Lichtw. & López-Lastra
Dacryodiomyces Lichtw.
Ejectosporus S.W. Peterson, Lichtw. & M.C. Williams
emend. Strongman
(continued)
Table 5.1 (continued) Ephemerellomyces M.M. White & Lichtw.

Furculomyces Lichtw. & M.C. Williams
Gauthieromyces Lichtw.
Genistelloides S.W. Peterson, Lichtw. & B.W. Horn
Genistellospora Lichtw.
Glotzia M. Gauthier ex Manier & Lichtw.
Graminella L. Léger & M. Gauthier ex Manier
Graminelloides Lichtw.
Klastostachys Lichtw., M.C. Williams & M.M. White
Laculus R.T. William & Strongman
Lancisporomyces Santam.
Legeriodes M.M. White
Legerioides M.M. White
Legeriomyces Pouzar
= Genistella L. Léger & M. Gauthier
Legeriosimilis M.C. Williams, Lichtw., M.M. White &
J.K. Misra
Orphella L. Léger & M. Gauthier emend. Santam. & Girbal
Pennella Manier ex Manier
Plecopteromyces Lichtw., Ferrington & López-Lastra
Pseudoharpella Ferrington, M.M. White & Lichtw.
Pteromaktron Whisler
Simuliomyces Lichtw.
Sinotrichium Juan Wang, S.Q. Xu & Strongman
Smittium R.A. Poiss.
Spartiella Tuzet & Manier ex Manier
Stipella L. Léger & M. Gauthier
Tectimyces L.G. Valle & Santam.
Trichozygospora Lichtw.
Trifoliellum Strongman & M.M. White
Zancudomyces Yan Wang, Tretter, Lichtw. & M.M. White
Zygopolaris S.T. Moss, Lichtw. & Manier
Kickxellales Kreisel ex R.K. Benj.
Kickxellaceae Linder
Coemansia Tiegh. & G. Le Monn.
Dipsacomyces R.K. Benj.
Kickxella Coem.
Linderina Raper & Fennell
Martensella Coem.
Martensiomyces Meyer
Mycoëmilia Kurihara, Degawa & Tokum.
Myconymphaea Kurihara, Degawa & Tokum.
Pinnaticoemansia Kurihara & Degawa
Ramicandelaber Y. Ogawa, S. Hayashi, Degawa & Yaguchi
Spirodactylon R.K. Benj.
(continued)
Table 5.1 (continued) Spiromyces R.K. Benj.

From the Harpellales
Clade 1. Barbatospora M.M. White, Siri & Lichtw.
Clade 2. Orphella L. Léger & M. Gauthier emend.
Santam. & Girbal
From the Kickxellales
Clade 3. Mycoëmilia Kurihara, Degawa & Tokum. and
Spiromyces R.K. Benj.
Clade 4. Ramicandelaber Y. Ogawa, S. Hayashi, Degawa
& Yaguchi
Possible members of the Kickxellomycotina
Ballocephala Drechsler
Zygnemomyces Miura
sporangiospores, or trichospores. Sexual reproduction by zygospores that are more

or less globose, fusiform lanceolate, or long cylindrical. Suspensors either: (1)
somewhat differentiated and with only one produced or (2) hyphoid, with two or
three produced in the substratum. Mostly saprobes with the exception of a putative
parasite, Martensella (Kickxellales), haustorial parasites of fungi (Dimargaritales),
or symbiotic and attached to the gut lining of an arthropod by a cellular or noncel-
lular holdfast (Asellariales, Harpellales).
Four orders, Asellariales, Dimargaritales, Harpellales, and Kickxellales, and an
additional four distinct clades may deserve formal recognition in the future. Two
other genera may belong in this subphylum based on septal ultrastructure.
The Kickxellomycotina contained four orders when the subphylum was proposed:
Asellariales, Dimargaritales, Harpellales, and Kickxellales (Hibbett et al. 2007). The
cardinal feature of the subphylum is the formation of regularly septate hyphae that have
a lenticular cavity containing a variously shaped plug, depending on the species.
Previous studies have suggested that three genera of the Kickxellales s.l. likely deserve
to be recognized as separate orders: (1) Mycoëmilia + Spiromyces and (2)
Ramicandelaber (Kurihara 2002; Kurihara et al. 2005; Ogawa et al. 2005). A phyloge-
netic construction by Tretter et al. (2013, 2014) recovered the Mycoëmilia + Spiromyces
clade and the Ramicandelaber clade as well as two additional groups (the Barbatospora
clade and the Orphella clade). Two genera, Ballocephala and Zygnemomyces, were
transferred to the Kickxellomycotina from the Meristacraceae (Entomophthoromycota).
Humber (2016) made this transfer because one member of each genus produces septal
plugs in lenticular cavities (Saikawa 1989; Saikawa et al. 1997b).
Order Asellariales (Lichtwardt 1986; Benny 2001; Valle

and Cafaro 2008; Benny et al. 2014)
Mycelium branched, composed of regularly formed, plugged septa. Asexual repro-

duction by arthrospores. Globose, intercalary, or terminal chlamydospores formed
by some species. Sexual reproduction by formation of conjugation tubes that result
in the formation of more or less globose, thick-walled zygospores. Thalli are

attached to the hindgut by a cellular holdfast or cells secreting a holdfast. Symbionts
in the gut of either Collembola or Isopoda.
One family: Asellariaceae with two or three genera.
Members of the Asellariales are attached to the hindgut of their hosts by a cellular
or acellular holdfast and species identification for some species hinges on holdfast
morphology (Valle 2006; Valle and Cafaro 2008). Taxa of Asellaria are associated
only with Isopoda (Arthropoda), whereas species of Orchesellaria (Valle 2006) are
specific to Collembola (Insecta). Baltomyces styrax, a putative member of the order,
also is associated with isopods (Oman and White 2012). These fungi, along with the
Harpellales, are often referred to collectively as the trichomycete fungi.
Morphological evidence for including Asellariales in the Kickxellomycotina
includes the presence of septa containing plugs in a lenticular cavity (Manier 1973;
Saikawa et al. 1997a, b) and the discovery of zygospores in Asellaria jatibonicua
(Valle and Cafaro 2008). Chlamydospores bearing some resemblance to the zygo-
spores of Asellaria (Valle and Cafaro 2008) have been reported in Orchesellaria
(Lichtwardt and Moss 1984; Degawa 2009). Sequence data also support the inclu-
sion of the Asellariales in the Kickxellomycotina (Tretter et al. 2014).
Entomological forceps have been recommended for collecting isopods under
moist organic substrata and rocks for terrestrial species (Valle 2006). Common sub-
urban “roly-polies” or pill bugs and woodlice from gardens or compost piles are
hosts that are easy to collect. Other than handpicking with forceps, some freshwater
hosts may otherwise be dislodged and swept up with an aquatic net using a kick
sampling technique (Oman and White 2012). Marine hosts may be collected in tide
pools or near the shoreline using a net. These hosts are typically placed in a jar
containing ocean water and native vegetation and then supplemented with an oxy-
gen tablet. “Essentially terrestrial” marine hosts, such as rock lice, might be found
near the high tide zone on rocks or among shoreline debris. All samples should be
placed in a cooler for transport to the laboratory.
Trichomycetologists are increasingly paying attention to fungi outside of actual
animal guts. Because the digestive tract serves as conduit for the attachment and
establishment of the thallus, spores will be dispersing and cycling from inside the
gut to the environment and back again. In many cases, critical life history stages of
the fungus might be found in the molted exoskeleton. This is especially true for
hindgut-dwelling fungi, as the gut lining is ectodermal in origin and is shed with the
exuvium at each stage of host development. This might include individual parts of
the thallus or spores, including sexual spores, which can be key to some species
identifications. Additionally, fungal development can parallel that of the host, and
therefore the most mature specimens are often found with mature hosts or those
nearing the end of an instar stage.
Some isopods are large enough to dissect by eye, but typically a microscope and
fine tools are needed for further manipulation and dissection. Most hyaline fungi are
best spotted with a dissecting scope equipped with an indirect backlighting. As soon as
possible after collecting, candidate hosts are dissected to remove the digestive tract.
Different regions of the gut might be specifically targeted before opening to look for
endobionts attached along the inside. The gut is opened by slicing along the length
or tearing smaller portions of it open. Depending on content, the gut may need to be
rinsed with distilled water to flush it and subsequently reveal the fungi. It is common
to dissect larger hosts in a Petri dish before moving specific sections of the digestive
tract to glass slides as wet mounts. Dissecting needles aid final opening and teasing
of gut sections of interest as well as orienting the microfungi (with practice these
can be seen at 40–60x). A coverslip is then added to scan the specimen for tricho-
mycete fungi using a compound light microscope. Thalli are preserved by replacing
the water with lactophenol cotton blue, which is infiltrated from under the edge of
the coverslip that has minimal distilled water (i.e., after excess has evaporated or
been wicked away gently with a tissue). Infiltration can be in as little as 20 min or
slides can be left 24–48 h before double sealing with clear fingernail polish. Wet-
mounted specimens may be imaged before (living) or after fixation and staining
(with lactophenol cotton blue). Many such “semipermanent” vouchers have been
revisited and imaged decades later. Some slides will dry over time if not sealed well,
so care and diligence with sealing coverslips is important. Drawings and measure-
ments can be performed later (Valle 2006; Valle and Cafaro 2008), although some
gut fungi suffer from distortion due to storage on slides (Kandel and White 2012).
Using similar methods, fungi may be obtained from collections of Collembola.
Springtails offer a challenge due to their small size but they can occur in large num-
bers and varied habitats. Many could be obtained with modifications of a Baermann
funnel approach, but most collections have been obtained during larger surveys
from aquatic habitats where they can be present as clusters on the surface of water
near the edges of creeks, streams, or pools. The use of a small aspirator can aid their
collection as well, but in many cases, springtails can occur in large enough numbers
to be swept by hand into a net, bag, or jar. Except for their occasional mention in
survey-based reports, perhaps no group of trichomycete hosts has been more over-
looked in recent years. Degawa (2009) extended our understanding of their devel-
opmental potential, which may play into possible future attempts to culture
Orchesellaria species and understand their ecology. The phylogenetic placement of
this understudied group is clearly in need of further research (Tretter et al. 2014).
Baltomyces is one of the smallest trichomycetes endobionts, but has been
observed across multiple states in the USA and over extended sampling occasions
in Idaho (Oman and White 2012). Spores have been observed with appendage-like
filaments. They are also present in freshwater isopods (Oman and White 2012).
There is no report of any member of the Asellariales being successfully isolated
in axenic culture, nor is there any known commercial value for species in this group.
However, it seems likely that some species could be cultivated under the proper
conditions.
Order Dimargaritales (Benjamin 1979)
Vegetative hyphae septate, simple, or branched, giving rise to simple or branched

merosporangiophores. Septa with a lenticular cavity containing a biconvex plug
bearing polar protuberances; plugs dissolving in dilute KOH or NaOH. Asexual
reproduction by two-spored merosporangia borne directly on a fertile vesicle or on

fertile branchlets arising from vesiculate or avesiculate merosporangiophore
branches. Sexual reproduction by more or less globose zygospores borne on sexual
hyphae. In nature obligate, haustorial mycoparasites. Haustorial parasites of species
of Chaetomium (Ascomycota), Mortierella (Mortierellales, Mortierellomycotina),
and Mucorales (Mucoromycotina).
One family: Dimargaritaceae with three or four genera.
Members of the Dimargaritales are typically isolated from dung and rarely from
soil. These fungi are regularly septate and produce a septal plug bearing a protuber-
ance that extends from each surface into the cytoplasm of the cells on each side of
the septum (Benjamin 1959; Benny 1972; Brain et al. 1982). The merosporangia are
two-spored and the spores are either dry or wet at maturity, depending on the spe-
cies. The zygospores are formed directly in the substratum and are more or less
globose with a hyaline wall and with typical hyphoid suspensors.
Members of the Dimargaritales are not common but appear to be worldwide in
distribution. The Dimargaritales are all haustorial mycoparasites of Chaetomium
(Dispira implicata, D. simplex; Benjamin 1959, 1961; Misra and Lata 1979),
Mortierellales (Mehrotra and Baijal 1963), or Mucorales (Benjamin 1959, 1961,
1963, 1965; Misra and Lata 1979). All species of Dimargaris and Tieghemiomyces
and both of the other species of Dispira (D. cornuta, D. parvispora) parasitize spe-
cies of Mortierella or the Mucorales in nature. In the laboratory, Benjamin (1959,
1961, 1963, 1965) cultivated Dispira simplex on cultures of Chaetomium botrych-
odes. Normal development of both host and parasite occurred when grown on
MEYE agar, PDA, and YpSs agar, but the parasite could not be grown without the
host (Benjamin 1961). The remaining species were grown on YpSs agar using
Cokeromyces recurvatus as the host (Shanor et al. 1950; Benny and Benjamin
1976). These fungi grow and sporulate well from 21 °C to room temperature (23–24
°C), but Dimargaris cristalligena grows optimally at 18 °C.
Culture of Dimargaritales has been reported several times in the literature.
Ayers (1933) grew D. cornuta in culture without a host on high protein content
culture medium (beef, egg, swordfish), but the egg medium proved to be the best
for D. cornuta. Benjamin (1959, p. 384) was able to grow D. cornuta in pure cul-
ture through four transfers on MEYE agar. As with D. simplex, Dispira parvispora
cannot grow or sporulate in culture without a host (Benjamin 1961, 1963).
Dimargaris cristalligena, D. bacillispora, and D. verticillata grow saprobically on
PAB-DEX agar, PDA, and YpSs agar, but optimal growth occurs on MEYE agar
(Benjamin 1959, p. 368).
Benjamin reported that species of Tieghemiomyces can be grown on MEYE agar
without host fungi (Benjamin 1959, p 392; Benjamin 1961, p. 11). Some members
of the Dimargaritales can be grown on a culture medium (YGCH agar) emended
with vitamins (biotin, pyridoxine, thiamine) using glycerol as the carbon source
(O’Donnell et al. 1998; see also Barnett 1970). Axenic cultures of these taxa are
extremely slow growing, and only five of the taxa (Dimargaris verticillata
[RSA 527], an undescribed species of Dimargaris [RSA 2174], Dispira cornuta
[RSA 632], Tieghemiomyces californicus [RSA 1194], T. parasiticus [RSA 2429B])
sporulate under these conditions. Methods of isolation and cultivation can be found
in Benjamin (1959) and Benny (2008). The fastest recovery of members of the
Dimargaritales from lyophils occurs on V8 juice agar and possibly also on clarified
V8 (cV8) juice agar.
The best dishes to observe maximum sporophore height of Dimargaris cris-
talligena with Cokeromyces recurvatus are 7 cm high and contain YpSs agar
incubated at 21 °C. When D. cristalligena is grown in test tubes containing
unslanted YpSs agar, the sporophores are shorter (5–8 mm long), but zygospores
are formed abundantly.
Spinalia radians (Vuillemin 1904; Benjamin 1959; Wrzosek and Gajowniczek
1998) may also be a member of the Dimargaritales, but its final placement will depend
on obtaining an axenic culture and sequencing informative DNA regions. Spinalia
radians was reported from France growing on Mucor fragilis (Vuillemin 1904) and
Poland as a parasite of Mucor hiemalis (Wrzosek and Gajowniczek 1998).
There is no known economic use for species of Dimargaritales although as
mycoparasites they have potential uses as biocontrol agents of spoilage fungi.
Order Harpellales (Lichtwardt 1986; Benny 2001; Lichtwardt

et al. 2001)
Vegetative hyphae simple or branched, septate. Each septum with a lenticular cav-
ity containing a biconvex plug. Asexual reproduction by the formation, in basipetal
succession, of unispored trichospores with one or more long, thin appendages or
appendages lacking. Sexual reproduction by variously attached biconical or fusi-
form zygospores, lanceolate zygospores attached basally, or spores that are
elongate-cylindrical or somewhat coiled at one end. Symbionts typically attached
with an acellular holdfast to the digestive tract lining of larval aquatic insects or,
more rarely, isopods.
Two families: Harpellaceae with six genera and Legeriomycetaceae with
44 genera.
Harpellales are found in guts of their hosts using the methods described by
Lichtwardt (1986). Members of several genera of the Legeriomycetaceae have been
isolated and cultured on 10 % BHIv overlaid with a thin layer of sterile, distilled
water (Benny 2001; Benjamin et al. 2004; Benny et al. 2014; Lichtwardt 1986;
Lichtwardt et al. 2001). A second culture medium (TGv) also has been used for
many years (Benjamin et al. 2004; Lichtwardt 1986). More recently, success has
been found by mixing these two media equally (BHIGTv) as an agar layer under a
distilled water overlay. The key with this method, regardless of the agar medium, is
to include an antibiotic rinse at the time of dissection and/or with the addition of the
water overlay, especially during the initial or earliest attempts to move the gut fungi
(and invariably minimal gut content or other host tissue) to the Petri dish. Petri
dishes can be stored with or without parafilm around the lid, but care should be
taken to not splash the water overlay near the lid.
Most of these fungi grow well at room temperature (20–22 °C), but certain taxa
do better at specific and/or lower temperatures (e.g., many Genistelloides isolates
do best at 18 °C). It is likely that our understanding of optimal culture conditions
and temperatures will continue to evolve. For example, should we not be attempting
isolations of fungi from winter-emerging insects in streams where the hosts and
their fungi are at 1–5 °C in dishes that are held in a 4 °C fridge? Or with reduced
oxygen? The latter do not represent typical approaches or modifications but are sug-
gestions worthy of further consideration.
Daily monitoring of fungal growth is extremely important. Once growth of the
fungus is noted, it should be monitored for thallus colony enlargement and spore
production. As soon as possible, the colony should be partitioned into other dishes,
eventually without antibiotic. Once growth is established axenically, some fungi
(e.g., many Smittium species) will produce trichospores that extrude their sporan-
giospores in vitro, with each one potentially serving to produce a branching mass of
typically asexually fertile thalli. Depending on the genus or species, the degree of
vegetative growth versus sporulation will vary by culture medium employed.
Current trends suggest that 10 % BHIv offers more spore production per colony
compared to enhanced vegetative growth with TGv. Thus, once cultures are estab-
lished, some attention to the degree of sporulation, ease of thallus fragmentation
(either naturally with plate or slant agitation or by actively breaking up “tougher”
thalli), and whether the spores extrude in vitro versus those that must be vegeta-
tively propagated via thallus fragmentation are important details to consider. Some
cultures of Smittium were first isolated in the early 1960s and maintained (at the
laboratory of Robert Lichtwardt, University of Kansas) for years at 4 °C in living
stock culture collections (as test tube slants with a distilled water overlay) and/or
under liquid nitrogen. These collections are invaluable resources, deserving of the
time, diligence, and efforts to maintain them.
Several new genera have been described in the Legeriomycetaceae since the
recent list in Benny (2012): Bactromyces, Dacrodiomyces, Laculus, Sinotrichum,
Trifoliellum, and Zancudomyces (Wang et al. 2010, 2013; Lichtwardt 2011;
Strongman and White 2011; William and Strongman 2012). One new genus,
Klastostachys (Lichtwardt et al. 2011), was recently described in the Harpellaceae.
It is noteworthy that the disproportionate success and resultant bias in our attempts
to culture gut fungi have been among members of the branched family, the
Legeriomycetaceae, from the hindguts of aquatic insects. Historically, fungi were
isolated first from larval Diptera (mosquitoes, midges, black flies, etc.) and then
from stoneflies. Lesser known successes have been from mayflies and caddis
worms (White, unpublished). The number of possible host groups worthy of con-
sideration as candidates that could host putative culturable taxa of gut fungi will
undoubtedly broaden with future attempts. All of these have been dissected from
hindguts; the pH of the media used for these successful isolation attempts has
always been slightly acidic and therefore approximating the conditions of the hind-
gut in this broad range of hosts.
Why has the Harpellaceae not been isolated under similar conditions? Is it just
that fewer attempts have been made, in the pursuit of the “more likely” success
among the hindgut-dwelling taxa, or could it be that just adjusting the in vitro
conditions to better match the midgut (where unbranched members of the
Harpellaceae reside) would improve success? Undoubtedly the key is triggering the
release of the sporangiospores from the sporangium or trichospore (or the zygo-
spore for that matter), and these fungi have adapted to specific gut triggers such as
pH and specific ions. Once in culture, they exhibit growth patterns and rates that
rival fungi in the Dikarya. Future efforts to culture members of both families of
these fungi are worthwhile.
Zygospores are not observed in culture, but have been described for several of
the genera. Four zygospore types are currently recognized (Moss et al. 1975;
Lichtwardt 1986), but there are several genera where zygospore formation has not
been observed. The zygospores of species of Orphella (Valle and Santamaria 2005)
are unique in that they are long, cylindrical, and coiled rather than fusiform or
lanceolate as is characteristic of the other taxa where sexual reproduction is known
(Lichtwardt 1986).
An undescribed stage (ovarian cysts) in the Harpellales life cycle was first
revealed by Moss and Descals (1986), and the life cycle of Harpellales in infected
blackfly larvae with ovary cysts was published based on material from New York,
USA (Labeyrie et al. 1996). Overnight incubations of cysts as wet mounts on slides
can promote germ tube development and even spore production. Clearly there is
potential for consideration of these stages as sources of material for culture isolation
as well—most studies of gut fungi in insects focus first on immature stages, but the
adult “dispersive” stage should not be overlooked. For example, infected adult
blackflies swarm and can be field collected with healthy blackflies, but in the lab
and to the trained eye, slight discoloration and distention of their abdomen can be
used as a clue to the thousands of fungal cysts inside infected individuals (White
unpublished and see White et al. 2006b).
White et al. (2006b) used molecular techniques to identify the Harpellales that
are the causative agents of ovary cysts of blackfly larvae, including some of the
same specimens from eastern North America. It is tempting to consider if future
methods might be devised to induce ovarian cysts of Harpellales in blackfly larvae
as a means of biocontrol.
Lipid content has been studied in Smittium culisetae (Patrick et al. 1973). There
is also a wealth of possible comparative physiological studies that could be under-
taken, especially as more cultures become available.
Order Kickxellales (Benjamin 1979; Benny 2012;

Benny et al. 2014)
Vegetative hyphae septate, simple, or branched, giving rise to simple or branched

merosporangiophores. Septa with a lenticular cavity containing a biconvex plug;
plugs do not dissolve in dilute KOH or NaOH. Asexual reproduction by unispored
merosporangia arising from pseudophialides borne on uni- or multicelled
sporocladia or directly from sporocladia and pseudophialides not formed. Sexual

reproduction by more or less globose zygospores borne on sexual hyphae that is
often hyphoid. Most species saprobes but one genus (Martensella) may be parasitic
on Corticium radiosum, a resupinate member of the Basidiomycota.
One family: Kickxellaceae with 12 genera.
Four new monotypic genera (Mycoëmilia [M. scoparia], Myconymphaea [M.
yatsukahoi], Pinnaticoemansia [P. coronantispora], Ramicandelaber [R. longispo-
rus]) have been added to the Kickxellales since 2001 (Ogawa et al. 2001; Kurihara
et al. 2001, 2004; Kurihara and Degawa 2006). Myconymphaea yatsukahoi was
isolated from earwig dung in Japan (Kurihara and Degawa 2006; Kasuhiro and
Degawa 2013). Kasuhiro and Degawa (2013) described another unnamed member
of the Kickxellales with an unusual spore, a stalked sporulating head with several
long sterile spines, and hyphae that appear to enclose the fertile head. This is likely
an undescribed species but no other information is given, and the illustrations are
extremely small and not diagnostic (Kasuhiro and Degawa 2013).
Ramicandelaber is currently the second largest genus in the order with four spe-
cies (Ogawa et al. 2001; Kurihara et al. 2004; Chuang et al. 2013).
Three new species have been added to Coemansia in the last 15 years (C. asiat-
ica, C. furcata, C. javanensis) (Kurihara et al. 2000, 2008). Kwaśna et al. (2002)
renamed three species of Coemansia with spiral sporangiophores.
Both species of Linderina were reported from Indonesia (Kurihara et al. 2008)
and Taiwan (Ho et al. 2007; Chuang and Ho 2009). These fungi (L. macrospora, L.
pennispora) can be found in soil from the tropics to temperate regions with high
humidity.
The majority of the taxa in the Kickxellales release spores in a droplet of fluid at
maturity, including all of the species in the newly described genera Mycoëmilia,
Myconymphaea, Pinnaticoemansia, and Ramicandelaber (Kurihara et al. 2001,
2004; Kurihara and Degawa 2006). Wet-spored species that have been studied by
electron microscopy have spines embedded in the wall (Benny and Aldrich 1975;
Young 1968; Zain et al. 2012). Taxa with spores that are dry at maturity are species
of Spirodacylon (S. aureum) and Spiromyces (S. aspiralis, S. minutus) (Benjamin
1963; O’Donnell et al. 1998). These fungi produce spore walls that are covered with
spines or warts (O’Donnell et al. 1998; Young 1968), but spines do not appear to be
embedded in the spore wall.
The asexual apparatus of the Asellariales, Harpellales, and Kickxellales was dis-
cussed by Moss and Young (1978). Young (1999) reviewed the radiation of the
asexual apparatus in Kickxellales, sporocladia and sporangiophores, and the mor-
phology of the merosporangiospores of the eight genera treated by Benjamin (1958,
1959, 1961, 1963, 1965, 1966).
Konova et al. (2002) found that the fatty acid cis-9-hexadecenoic acid consti-
tuted 37 % of the total fatty acids produced by Linderina pennispora, and Konova
et al. (2005) found that this compound was also present in Kickxella alabastrina.
This and other fatty acids can be used as precursors for biofuel production. The
sterol most frequently isolated from Kickxellales, Dimargaritales, and Harpellales
is 22-dihydroergosterol (Weete et al. 2010).
Isolation and Culture of Kickxellales
The most commonly encountered genus of the Kickxellales is Coemansia with 21

described taxa. The species of Coemansia can be isolated from both dung and soil.
Coemansia cannot be found in all dung or soil collections, but sometimes one or
two species can be isolated from a single soil sample. Ramicandelaber occasionally
can be isolated from soil. The other taxa are rarely encountered or are only known
from the original descriptions.
Kickxellales also can be isolated from soil that has been placed in Petri dishes (60
or 100 mm; glass may be better than plastic). The soil is moistened using a water
mist. Other soil can be enriched with a sterile nutrient-poor broth (5 % yeast extract
or 2.5 % peptone/2.5 % soytone). These soil plates can be baited with dried and
broken edible shrimp, dried mealworm larvae, dried krill (food for birds or pet
amphibians), dead aphids, or Drosophila. Soil plates are typically kept for approxi-
mately 30 days and examined for growth of Kickxellales or Mortierella using a
dissecting microscope. Spores are then transferred to a nutrient-rich solid medium
for isolation such as MEYE agar.
Members of the Kickxellales can be isolated from dung, soil, and other organic
substrata. Isolation and culture of these fungi can be conducted using the methods
listed in Kurihara and Degawa (2006), Kurihara et al. (2001, 2004), Krug (2004),
Krug et al. (2004), and Benny (2008). Many taxa described in these publications
were isolated from soil using crustacean baiting or enrichment (Kurihara et al.
2008). Benjamin (1959) recommended the use of MEYE or YpSs agars for growth
and sporulation of many Kickxellales. Benjamin (1959) used a variety of media
formulations to obtain isolates of Coemansia, depending on the species and isolate,
including: carrot, CM, CM-S, 2 % ME, MED, ME-P, MEYE, OMS, PDA, PG,
PYED, PYEDS, SDY, STA, Wg, Wg-DD, Wg-S, V8, WSH-DD, YGCH, and YpSs
(Benny et al. 2008 and see Supplement 1).
Wg10 agar supplemented with antibiotics and benomyl is also excellent for iso-
lating Coemansia (G. Benny, unpublished). The use of benomyl is critical to retard
the growth of Trichoderma, Penicillium, and other rapidly sporulating Ascomycota.
Subphylum Mortierellomycotina (Hoffmann et al. 2011)

(Table 5.2)
Fungi that produce coenocytic hyphae, septa are formed to wall-off reproductive
structures or old or damaged hyphae. Sporangiophores not cylindrical, base usually
wider than apex, somewhat constricted basally. Asexual reproduction by chlamydo-
spores or unicelled sporangiospores produced in uni-, few-, or mutispored sporan-
gia. Columella not well developed, convex, or septoid. Sexual reproduction, where
known, by zygospores formed in the substratum on apposed, heterogamous, or
more or less isogamous suspensors. Saprobes, colony may appear to have irregular
Table 5.2 Classification of Mortierellomycotina Kerst. Hoffm., K. Voigt & P.M. Kirk
the taxa of the
Mortierellales Caval.-Sm.
Mortierellomycotina
Mortierellaceae A. Fischer
Aquamortierella Embree & Indoh
Dissophora Thaxt.
Echinochlamydosporium X.Z. Jiang, X.Y. Liu, Xing
Z. Liu
Gamsiella (R.K. Benj.) Benny & M. Blackw.
Lobosporangium M. Blackw. & Benny
= Echinosporangium Malloch, non Echinosporangium
Kylin
Modicella Kanouse
Mortierella Coem.
= Haplosporangium Thaxt.
= Azygozygum Chesters
= Actinomortierella Chalab.
Nothadelphia Degawa & W. Gams
zones and undulate margins on nutrient-rich media and may produce a garlic- or
onion-like odor.
One order, Mortierellales, and one family Mortierellaceae (Benjamin 1978,
1979; Benny 2012; Benny et al. 2014) with eight or nine genera.
Members of the genus Mortierella are among the most common zygomycete
fungi encountered in soil. These fungi also can be found on dung and other organic
substrata. Procedures for the isolation and culture of Mortierella, and some of the
other members of Mortierellales, can be found in Kuhlman (1972), Benny and
Blackwell (2004), and Benny (2008). Gams (1976) recommended growing species
of Mortierella at 18–22 °C for at least 7 days. Colony growth rate and macroscopic
characters are determined by culture on MEA (2 % malt extract agar), and the
microscopic structures (hyphae, sporophore morphology and branching, sporangiola,
sporangiospores) are observed after growth on PCA (potato-carrot agar, CBS
formulation) (http://www.cbs.knaw.nl/index.php/food-mycology/101-mycological-
media-for-food-and-indoor-fungi) or SEA (soil extract agar). Some species of
Mortierella do not readily sporulate in culture. However, these isolates can some-
times be induced to sporulate by transferring an agar plug from MEA containing the
Mortierella colony to 2 % water agar. Often the Mortierella will then sporulate on
the edges of the MEA plug.
Degawa and Tokumasu (1997) recommended a method for isolating species of
Mortierella. They suggest collecting soil where the isopod Armadillidium vulgare
was plentiful, placing it in 101 × 44 mm plastic sample cups, keeping it moist, and
then placing a piece of sterile, dry shrimp on the soil surface. Spores of Mortierella
capitata that formed on the shrimp were then transferred to Miura agar (LcA) for
isolation (Sugiyama et al. 2003). Two disks of agar (5 mm diam.) that contain
Mortierella hyphae from different colonies were placed 10 mm apart on 0.3 %

shrimp agar (ShA, Supplement 1) plates, incubated at various temperatures. When
cultures were found to represent the opposite mating types, plugs of each isolate
were placed 20 mm apart on Czapek’s agar (CZA, Supplement 1) and a dead, sterile
isopod placed in the center. The temperature optimum for M. capitata zygospore
formation is 15–20 °C on Czapek’s agar amended with an isopod (approximately 1
week) or ShA (approximately 2 weeks). These methods, with slight modification,
were used to isolate and induce zygospore formation in Mortierella cogitans,
M. microzygospora, and M. umbellata (Degawa and Tokumasu 1998a, b). Degawa
and Gams (2004), however, placed pieces of bat dung on the surface of ShA and
found that Mortierella hypsicladia was a good host for a new monotypic genus of
unknown zygomycete affinity, Nothadelphia mortierellicola. Mortierella indohii
also was a good host for N. mortierellicola, but other species of Mortierella that
were tested did not function as hosts.
The other genera are known from only a single isolate or a few reports in the
literature, including Gamsiella multidivaricata, Lobosporangium transversale, and
Modicella malleola (Smith et al. 2013). Many of these taxa may be more common
than currently thought because of exacting requirements for sporulation.
Lobosporangium transversale will only sporulate on selected culture media, includ-
ing CMA, CZA, HAS, LA, MEA, PAB, ShA, and TSM. In contrast, little or no
sporulation is induced on AM, MAM, MEYE, PDA, PGA, V8, Wg, and YpSs when
incubated at 25 °C, either in continuous light or 12 h light/12 h dark cycle (Benny
and Blackwell 2004). Modicella malleola was isolated in pure culture on water, soil
extract, or other media (Walker 1923). The spores readily germinate in water but
cornmeal agar was used for cultural studies (Walker 1923).
Echinochlamydosporium variable (Jiang et al. 2011) was originally described as
a member of the Mortierellales, but it is the sister species of a taxon in the
Mucoromycotina (Hirose et al. 2014).
The culture of Dissophora decumbens (Thaxter 1914; Benny 1995a) was lost
when Thaxter was away from his lab on an extended collecting trip. No culture
was available when the description of D. decumbens was prepared. This fungus is
a psychrophile (Carreiro and Koske 1992), and it was isolated from leaf litter
using MYP-ps agar incubated at 0 °C when grown for 1–4 weeks. Sporulation
occurred on PCA (CBS formulation; http://www.cbs.knaw.nl/index.php/food-
mycology/101-mycological-media-for-food-and-indoor-fungi) incubated at
15–20 °C. Dissophora decumbens also was found in cloned sequences from bee-
tle guts (Zhang et al. 2003); it is now in culture (Gams and Carreiro 1989).
Aquamortierella (Embree and Indoh 1967) has not knowingly been cultured and
is rarely collected or reported.
In a phylogenetic study published by Petkovits et al. (2011), the Mortierellaceae
are distributed among 12 clades. Species of Dissophora, Gamsiella, and
Lobosporangium are dispersed among species of Mortierella, making the latter
genus paraphyletic. The sectional classification of Mortierella (Gams 1977), which
is based on morphology, was not supported. Additional data that included ITS and
LSU sequences from 400 cultures was added to the analysis of the Mortierellomycotina
published by Wagner et al. (2013), and only seven clades were revealed. This study
also demonstrated that the Mortierella sections were not maintained and that
morphology of these fungi was variable depending on culture criteria.
Mortierella wolfii is the only species in the Mortierellomycotina that is the caus-
ative agent of zygomycosis and the only taxon that can grow at 37 °C (48 °C maxi-
mum). This species usually is known only as an animal pathogen, especially of
cattle (Papp et al. 2011), but recently it was reported as the causative agent of myco-
sis in a human (Layios et al. 2014).
Crude glycerol, a major biodiesel production by-product, can be used as a carbon
source instead of glucose, to produce arachidonic acid (AA) by Mortierella alpina.
Several other species of Mortierella can use bioglycerol for the production of both
AA and dihomo-γ-linolenic acid (Hou 2008). Münchberg et al. (2013) found that
the hyphal oil composition is variable in the first 600 μm of the hyphae in Mortierella
alpina and M. elongata.
Weete et al. (2010) reported that ergosterol is the major sterol of Mucorales. One
of the following or combinations of 24-methyl-cholesterol, 24,25-methylene-
cholesterol, and desmosterol are formed by Mortierellales (Weete et al. 2010).
Sterol composition of members of the Mortierellales and Mucorales further sup-
ports the separation of these orders into different subphyla.
Mortierellomycotina (Mortierellales) and Mucoromycotina (Mucorales) grow
readily, and at least one isolate of most genera in these subphyla is currently in
culture collections. These fungi can be isolated from many substrata including dung
(using a moist chamber) and soil sprinkled on agar (using nutrient-poor culture
media emended with antibiotics and benomyl). Other procedures can be used to
isolate these fungi from soil. The largest number of taxa of the Mortierellales is
present in the Centraalbureau voor Schimmelcultures (CBS) filamentous fungi col-
lection although other culture collections (ATCC, IMI, NRRL) also contain isolates
of Mortierella and other members of the Mortierellales.
Subphylum Mucoromycotina (Hibbett et al. 2007) (Table 5.3)
structures or old or injured hyphae. Asexual reproduction by unicelled conidia arising
from conidiogenous cells or unicelled sporangiospores produced in sporangia, spo-
rangiola, merosporangia, or spores lacking or less commonly by arthrospores, chla-
mydospores, or yeast cells. Columella usually well defined and easily observed using
the light microscope, hemispherical or obovoid to obpyriform, difficult to observe, or
lacking in some unispored taxa. Sexual reproduction unknown or by the formation of
zygospores produced in the aerial hyphae on opposed suspensors or in the substratum
or in sporocarps on apposed suspensors. Saprobes, facultative gall-forming parasites,
or ectomycorrhizal.
Two orders: Endogonales and Mucorales (as well as two additional unnamed
clades).
Table 5.3 Synopsis of the Mucoromycotina Benny

classification of the two
Endogonales Moreau ex R.K. Benj. emend. Morton & Benny
orders and two clades of
Mucoromycotina to family Endogonaceae Paoletti emend. J.B. Morton & Benny
and subfamily Endogone Link
Peridiospora C.G. Wu & J. Lin
Sclerogone Warcup
Youngiomyces Y.J. Yao
Densospora McGee
Mucorales Fr.
Backusellaceae K. Voigt & P.M. Kirk
Backusella Hesselt. & J.J. Ellis
Choanephoraceae J. Schröt.
= Gilbertellaceae Benny
Choanephoroideae K. Voigt & P.M. Kirk
Blakeslea Thaxt.
Choanephora Curr.
Poitrasia P.M. Kirk
= Abradeosporangium Subrahm. & Swathi Sri
Gilbertelloideae K. Voigt & P.M. Kirk
Gilbertella Hesselt.
Cunninghamellaceae R.K. Benj. emend. Benny, R.K. Benj.
& P.M. Kirk
≡ Absidiaceae Arx
Cunninghamelloideae K. Voigt & P.M. Kirk
Cunninghamella Matr.
Absidioideae K. Voigt & P.M. Kirk
Absidia Tiegh. s.s.
= Tieghemella Berl. & De Toni
= Proabsidia Vuill.
Chlamydoabsidia Hesselt. & J.J. Ellis
Gongronella Ribaldi
Halteromyces Shipton & Schipper
Hesseltinella H.P. Upadhyay
Lentamycetaceae K. Voigt & P.M. Kirk
Lentamyces Kerst. Hoffm. & K. Voigt
?Siepmannia Kwaśna & Nirenberg ex Nirenberg &
Kwaśna
Lichtheimiaceae Kerst. Hoffm., G. Walter & K. Voigt
Dichotomocladioideae K. Voigt & P.M. Kirk
Dichotomocladium Benny & R.K. Benj.
Lichtheimioideae K. Voigt & P.M. Kirk
Lichtheimia Vuill.
Rhizomucoroideae K. Voigt & P.M. Kirk
Rhizomucor Lucet & Costanin
(continued)
Table 5.3 (continued) Thermomucor Subrahm., B.S. Mehrotra & Thirum.

Mucoraceae Dumort.
= Chaetocladiaceae A. Fisch.
= Thamnidiaceae Fitzp.
= Dicranophoraceae J.H. Mirza
Dicranophoroideae K. Voigt & P.M. Kirk
Dicranophora J. Schröt.
Chaetocladioideae K. Voigt & P.M. Kirk
Chaetocladium Fresen.
Mucoroideae K. Voigt & P.M. Kirk
Actinomucor Schostak.
= Glomerula Bainier
Ambomucor R.Y. Zheng & X.Y. Liu
Circinomucor Arx
= Mucor Fresen.
Hyphomucor Schipper & Lunn
?Isomucor J.I. Souza, Pires-Zottar. & Harakava
Kirkiana L.S. Loh, Kuthub. & Nawawi
Mucor Fresen.
= ?Zygorhynchus Vuill.
Nawawiella L.S. Loh & Kuthub.
Parasitella Bainier
Pilaira Tiegh.
Tortumyces L.S. Loh
?Zygorhynchus Vuill
Zygambella S. & A. Subrahm.
Thamnidioideae K. Voigt & P.M. Kirk
Ellisomyces Benny & R.K. Benj.
Helicostylum Corda emend. Benny
Pirella Bainier
Thamnidium Link, non Thamnidium Tuck. ex Schwend
Mycocladaceae Kerst. Hoffmann, S. Discher & K. Voigt
Mycocladus Beauverie
Mycotyphaceae Benny & R.K. Benj.
Cokeromycetoideae K. Voigt & P.M. Kirk
Benjaminiella Arx
Cokeromyces Shanor
Kirkomycetoideae K. Voigt
Kirkomyces Benny
= Kirkia Benny, non Kirkia Oliv.
Mycotyphoideae K. Voigt & P.M. Kirk
Mycotypha Fenner
Phycomycetaceae Arx
Phycomyces Kunze
Spinellus Tiegh.
(continued)
Table 5.3 (continued) Pilobolaceae Corda

Pilobolus Tode
Utharomyces Boedijn
Radiomycetaceae Hesseltine & J.J. Ellis
Radiomyces Embree
Rhizopodaceae K. Schum.
Amylomyces Calmette
Rhizopus Ehrenb.
Sporodiniella Boedijn
Syzygites Ehrenb.
Saksenaeaceae Hesselt. & J.J Ellis
Apophysomyces P.C. Misra
Saksenaea S.B. Saksena
Syncephalastraceae Naumov ex R.K. Benj.
Circinella Tiegh. & G. Le Monn.
Fennellomyces Benny & R.K. Benj.
Phascolomyces Boedijn
Protomycocladus Schipper & Samson
Syncephalastrum J. Schröt.
Thamnostylum Arx & H.P. Upadhyay
Zychaea Benny & R.K. Benj.
Umbelopsidaceae W Gams & W. Meyer
Umbelopsis R.E. Amos & H.L. Barnett
= Micromucor (W. Gams) Arx
Clade 1. Calcarisporiella de Hoog
Clade 2. Sphaerocreas Sacc. & Ellis
Genera of unknown family affiliations
?Isomucor J.I. Souza, Pires-Zottar. & Harakava
Rhizopodopsis Boedijn
?Siepmannia Kwaśna & Nirenberg ex Nirenberg &
Kwaśna
Unnamed Clade 1
Hyphae fragile, relatively wide and more or less undulating, aerial hyphae forming
one or two conidiogenous cells that are undifferentiated. Conidiogenous cell swol-
len slightly above the base, two to several conidia-bearing denticles formed api-
cally. Each denticle more or less cylindrical giving rise to conidia sympodially.
Conidia ellipsoid to ovoid with a basal hilum and hyaline and walls smooth and
thin. Sexual reproduction unknown.
A family, not described, contains only a monotypic genus, Calcarisporiella ther-
mophila, although several unnamed taxa have been differentiated based on molecu-
lar data (Hirose et al. 2012).
The growth temperature range of C. thermophila is 25–40 °C. Isolation is

possible when the plates are incubated at 35 °C. Sporulation was good at 25–30 °C.
The temperature range for the unnamed species of Calcarisporiella (NBRC 105922)
is 15–35 °C with the best growth occurring between 20 °C and 30 °C (Hirose et al.
2012). These isolates of Calcarisporiella grow on several ordinary fungal culture
media when incubated in the optimal temperature range. This monotypic genus, C.
thermophila, was originally thought to be an anamorphic member of the
Pezizomycotina [Evans 1971; de Hoog 1974; Index Fungorum (http://www.index-
fungorum.org/Names/Names.asp) accessed 11 October 2014]. A phylogenetic anal-
ysis of 18S rDNA sequences, however, demonstrated that C. thermophila is separate
from the Endogonales and Mucorales clades. Another study of the relationship of
C. thermophila (Hirose et al. 2014) revealed an unnamed Calcarisporiella that is
the sister species to Echinochlamydosporium variable (Jiang et al. 2011; described
as a member of the Mortierellales). Petkovits et al. (2011) and Wagner et al. (2013)
did not have a culture or sequences of E. variable to include in their analyses of the
Mortierellales.
Unnamed Clade 2
Sporocarps, 0.2–2 mm in diameter, formed on dead plant material (branches, leaves,

twigs), covered with a light-colored hyphal tomentum 60–100 μm long, borne in
tightly attached bundles, 12–30 μm wide basally, and tapering to 2 μm apically.
Sporocarps contain branched refractive hyphae ca. 2 μm in diameter, which give rise
to numerous, randomly arranged chlamydospores, broadly ellipsoid to subspherical,
18 × 15 to 25 × 22 μm, walls yellowish, 1.5–2.5 μm thick, contents hyaline, with many
granules and one or more oil droplets. Sporocarps hard when dry (Thaxter 1922).
A family, not named, contains only a monotypic genus: Sphaerocreas pubescens.
Sphaerocreas pubescens has been included in several genera by previous authors,
including Endogone, Glomus, and Sclerocystis. Various sources believe that the cur-
rent name should be Glomus pubescens (or Sclerocystis pubescens (Glomeraceae
[Thaxter 1922; Index Fungorum (http://www.indexfungorum.org/Names/Names.asp)
accessed 11 October 2014; Schüßler and Walker 2010; Hirose et al. 2014]).
Sphaerocreas pubescens may be a saprobe but the formation of sporocarps being
produced in culture was not mentioned. The species was cultured on MNC (Modified
Norkrans’ C medium). The fungus was grown in the dark on MNC agar with added
thiamine (100 mg) according to Hirose et al. (2014) before DNA extraction.
A phylogenetic analysis (Hirose et al. 2014) revealed that S. pubescens is the
sister taxon to those fungi in Mucoromycotina that are symbionts of hornworts and
liverworts but are known only from environmental DNA sequences (Bidartono
et al. 2011). These latter sister taxa form a clade that is separate from the members
of the Endogonales and Mucorales and a third clade composed of
Echinochlamydosporium and Sphaerocreas (Hirose et al. 2014); Calcarisporiella
thermophila is the fourth clade in this subphylum.
Order Endogonales (Benjamin 1979)
Hyphae coenocytic, with few septa. Asexual reproduction unknown. Sexual repro-
duction usually by many zygospores with apposed suspensors that are formed in a
sporocarp. Saprobes or ectomycorrhizal.
One family: Endogonaceae with four genera.
The majority of the Endogonales must be collected as sporocarps that are found
on or in the substrate. One species, Endogone pisiformis Link, has been grown
(Endogone agar) and forms sporocarps in culture (Berch and Fortin 1983; Berch
and Castellano 1986; Dalpé 1990). A culture of Endogone pisiformis produces
hyphal swellings and chlamydospore-like structures; mature zygospores were not
produced. Optimal growth was attained when thiamine was added to the growth
medium (Dalpé 1990). Sporocarps of Endogone pisiformis were produced in axenic
cultures in vitro (Berch and Castellano 1986).
Endogonales all form sporocarps that contain only zygospores. The sporocarps
can be collected intact in the field on leaves or other organic substrata or in soil.
Some sporocarps are eaten and dispersed by rodents. These zygospores may be
found in dung of these rodents. Most species are apparently saprobes but a few taxa
are ectomycorrhizal.
Descriptions and illustrations for the four genera in the Endogonales—Endogone,
Pteridiospora, Sclerogone, and Youngiomyces—can be found in Gerdemann and
Trappe (1974), Yao et al. (1996), and Wu and Lin (1997). A species of Endogone,
E. maritima, was described later (Błaszkowski et al. 1998).
Bidartono et al. (2011) discussed the possibility that a member of the
Mucoromycotina, and not the Glomeromycota (Schüssler et al. 2001), was the first
organism to form a symbiotic association with hornworts and liverworts. Desirò
et al. (2013a), after conducting a worldwide survey, discovered that fungi from the
Glomeromycota and Mucoromycotina both formed symbioses with hornworts.
Mollicutes-related endobacteria (Mre) were found in almost 45 % (13 of 29) of the
Endogone isolates examined by Desirò et al. (2014a); they also are known in
Glomeromycota (Desirò et al. 2013b). Several isolates of Gigaspora margarita
from soil collected in Cameroon (Desirò et al. 2014b) contained both Mre and
Candidatus Glomeribacter gigasporarum (CaGg).
A fossil was recently described from the Middle Triassic of Antarctica that
resembles a member of the Endogonaceae (Jimwhitea circumtecta). Another fossil
species of Endogonaceae, Mycocarpon asterinum, also was described from Triassic
sediments in Antarctica (Krings et al. 2012, 2013).
Jabaji-Hare et al. (1988) examined the fatty acid profile in Endogone pisiformis.
The composition varied with the time in culture. Several fatty acids were produced
with oleic acid being the most abundant, and both isomers (ω3, ω6) of linolenic
acid were also formed, among others. Although it is possible to use E. pisiformis
for fatty acid production, the isolates of this species are difficult to maintain in
culture, and obtaining new isolates can be challenging because fresh sporocarps
are difficult to find.
Order Mucorales (Benjamin 1979)
structures or old or damaged hyphae. Asexual reproduction by unicelled sporangio-
spores produced in sporangia, sporangiola, merosporangia, or spores lacking or less
commonly by arthrospores, chlamydospores, or yeast cells. Columella usually well
defined and easily observed using the light microscope, hemispherical of obovoid to
obpyriform, difficult to observe, or lacking in some unispored taxa. Sexual repro-
duction by the formation of zygospores produced in the aerial hyphae on opposed
suspensors or in the substratum on apposed suspensors. Saprobes or facultative
parasites that form galls on the host.
Fifteen families, Backusellaceae, Choanephoraceae, Cunninghamellaceae,
Lentamycetaceae, Lichtheimiaceae, Mucoraceae, Mycocladiaceae, Mycotyphaceae,
Phycomycetaceae, Pilobolaceae, Radiomycetaceae, Rhizopodaceae, Saksenaeaceae,
Syncephalastraceae, and Umbelopsidaceae, and at least 57 genera.
The families of the Mucorales are listed because some taxa require special
conditions for culture. The abbreviations for the media are listed below and can be
found in the Appendix.
Families
Backusellaceae The sporangiophore is initially curved immediately below the spo-

rangium but is erect at maturity. Sporangiola may be produced. One genus is
included and the species are isolated from soil and other organic debris. Several
species were transferred to Backusella (Walther et al. 2013), but three—Backusella
circina, B. lamprospora, and B. recurva—were included by Hoffmann et al. (2013).
Asexual growth and sporulation and zygospore formation of all species of Backusella
occur at 26 °C on 2 % ME, MEYE, MSMA, and YpSs (Benny and Benjamin 1975).
MSMA and other nutrient-poor media are optimal for observing branching patterns.
One species, B. ctenidia (syn. = Mucor ctenidia), grows and sporulates asexually on
most media, but zygospores form only on LYE agar.
Choanephoraceae As currently accepted, the Choanephoraceae is composed of

two subfamilies (Voigt and Kirk 2012), Choanephoroideae (Blakeslea,
Choanephora, Poitrasia; Kirk 1984) and Gilbertelloideae (Gilbertella; Benny
1991). All members form sporangiospores bearing several hyaline appendages at
each end, and the sporangia have a dark, persistent, spinose wall that separates into
two or more parts at maturity (Kirk 1984; Benny 1991). Choanephoroideae form
zygospores in the substratum with apposed (parallel), coiled suspensors, whereas
the Gilbertelloideae produce zygospores in the aerial hyphae with opposed (grow
toward one another) suspensors. Nutrient-poor culture media, such as CHA
(Choanephora agar), PCA, and Wg10, can be used to both isolate and culture mem-
bers of the Choanephoroideae. Gilbertella persicaria grows and sporulates on any
ordinary culture media used in the laboratory, including PDA, but observations of
reproductive structures are best on nutrient-poor media.
Cunninghamellaceae The genera in this family are dispersed in two subfamilies

(Voigt and Kirk 2012): Cunninghamelloideae (Cunninghamella) and Absidioideae
(Absidia s.s., Chlamydoabsidia, Gongronella, Halteromyces, Hesseltinella). There
are no morphological characters that are cardinal features of the genera in the fam-
ily. Cunninghamella (Zheng and Chen 2001) produces pedicellate, unispored spo-
rangiola on the surface of fertile vesicles. The Absidia-like taxa (Absidia s.s.,
Chlamydoabsidia, Halteromyces, Gongronella—Hesseltine and Ellis 1961, 1964,
1966; Ellis and Hesseltine 1965) produce apophysate, columellate sporangia on
stolons but not opposite a rhizoid; a septum is formed in the sporangiophore. The
zygospores, where known, have appendaged (Absidia s.s.) or non-appendaged sus-
pensors (Gongronella). Hesseltinella (Benny and Benjamin 1991) is similar mor-
phologically to Gongronella (the subsporangial vesicle of Hesseltinella resembles
the constricted apophysis of Gongronella).
These taxa all grow and sporulate on most culture media, but V8 and cV8 agars
are excellent for studying branching, spore formation, and other morphological
characteristics. Zygospore formation can be induced on both nutrient-rich and
nutrient-poor media. Zygospore ontogeny can be observed optimally on nutrient-
poor agar media. A few species of Absidia s.s. are homothallic, but Gongronella
butleri and other taxa of Absidia s.s. are heterothallic.
Lentamycetaceae Members of Lentamycetaceae (Lentamyces) are Absidia-like

and homothallic. Zygospores are produced that have opposed suspensors that lack
appendages (Hoffmann and Voigt 2009). The upper limit for growth temperature is
30 °C for both species (Lentamyces parricida, L. zychae). These taxa were dis-
cussed by Ellis and Hesseltine (1966) and Hoffmann and Voigt (2009). Siepmannia
(Kwaśna and Nirenberg 2008a, b) may be transferred eventually to the
Lentamycetaceae if supported by molecular data. Hoffmann and Voigt (2009),
however, transferred two of the four species of Siepmannia to Lentamyces. Species
of Lentamyces were crossed on SUP medium at 20 °C (Hoffmann and Voigt 2009).
Lichtheimiaceae The Lichtheimiaceae (Kirk 2012) is divided into three subfami-

lies (Voigt and Kirk 2012), Dichotomocladioideae (Dichotomocladium; Benny and
Benjamin 1975, 1993), Lichtheimioideae (Lichtheimia; Alastruey-Izquierdo et al.
2010), and Rhizomucoroideae (Rhizomucor, Thermomucor; Schipper 1978b, 1979).
Dichotomocladium initially was treated as a member of the Thamnidiaceae.
The former genus resembles Chaetocladium because members of both genera
form sterile spines and unispored sporangiola. Dichotomocladium was includedin
the Chaetocladiaceae by Benny and Benjamin (1993); both of the aforementioned
families are now considered synonyms of Mucoraceae (Hoffmann et al. 2013).
All known species of Dichotomocladium, except D. hesseltinei, have been iso-
lated from dung (Benny and Benjamin 1975, 1993). The descriptions and draw-
ings of all species of Dichotomocladium were made on MSMA. However, good
fertile head formation occurs on LYE, TPO, and WSH but not on MEYE. Sexual
reproduction is optimal on TPO, WSH, and whey. All species of Dichotomocladium
are mesophiles (optimal growth at 26 °C). Zygospores of D. hesseltinei were
observed when cultures were crossed on TPO and grown at 26 °C in the dark
(Benny and Benjamin 1993).
Lichtheimia (Hoffmann et al. 2009; Santiago et al. 2014) is an Absidia-like genus
in which five of six species are thermotolerant whereas Absidia s.s. is mesophilic
(Hoffmann et al. 2007; Hoffmann 2010). There are six species in Lichtheimia and
three (L. corymbifera, L. ornata, L. ramosa) are the causative agents of mucormy-
cosis (Alastruey-Izquierdo et al. 2010; Schwartz et al. 2014).
Rhizomucor and Thermomucor (Schipper 1978b, 1979) are thermophilic
Mucorales that comprise the remaining genera of the Lichtheimiaceae. Rhizomucor,
R. pusillus, and Thermomucor indicae-seudaticae can cause mucormycosis.
Thermomucor, however, is rarely reported as the causative agent of mycosis.
Species of Lichtheimia, Rhizomucor, and Thermomucor can be grown and will
sporulate on almost any culture medium discussed above under Dichotomocladium.
Mucoraceae The Mucoraceae is, by a number of genera and species, the largest
family of the Mucorales. Currently, three families (Chaetocladiaceae,
Dicranophoraceae, Thamnidiaceae) are treated as synonyms of the Mucoraceae
(Hoffmann et al. 2013). The Mucoraceae is divided into three subfamilies (Voigt
and Kirk 2012): Chaetocladioideae (Chaetocladium), Dicranophoroideae
(Dicranophora), and Mucoroideae (Actinomucor, Ellisomyces, Helicostylum,
Hyphomucor, Mucor, Parasitella, Pilaira, Pirella, Thamnidium, Zygorhynchus, and
possibly Ambomucor, Isomucor, Kirkiana, Nawawiella, Tortumyces, and
Zygambella) (Benjamin and Hesseltine 1957; Benny and Benjamin 1975, 1976;
Schipper 1975, 1978a, b, 1986; Benny and Schipper 1992; Benny 1992, 1995c;
Volgmayr and Krisai-Greilhuber 1996; Loh et al. 2001; Nagalakhsmi et al. 2008;
Zheng and Liu 2009, 2014; de Souza et al. 2012). A subfamily for the thamnidia-
ceous Mucoraceae, Ellisomyces, Helicostylum, Pirella, and Thamnidium currently
in the Mucoroideae, may be justified.
The type species for the family and order is Mucor mucedo (Benny and Benjamin
1975; Benny and Schipper 1992; Benny 1992, 1995c). De Bary (1865) treated
Mucor mucedo and Thamnidium elegans as the same species. Some taxa in the
Mucoraceae are isolated from soil (Ambomucor, Isomucor, Hyphomucor,
Zygorhynchus). In contrast, the thamnidiaceous Mucoraceae are best isolated from
rodent dung: Chaetocladium and Ellisomyces (asexual reproduction on MSMA,
zygospore formation on MSMA and YpSs), Helicostylum (asexual reproduction is
optimal on MSMA at 18 °C, zygospore formation on whey in the dark at 7 °C for
60 days), Pilaira (asexual reproduction on MEYE and YpSs, zygospore formation
on PDA at 25 °C), Pirella (asexual reproduction on MSMA at 22 °C, optimal zygo-
spore formation on TPO at 15 °C), and Thamnidium (optimal asexual reproduction
on MSMA at 18–22 °C, zygospore formation on MEYE, YPD, or YpSs when incu-
bated at 7–10 °C for 3–5 weeks). Three genera are mycoparasites in nature
(Dicranophora on mushrooms, Chaetocladium and Parasitella on Mucorales).
There are two genera (Actinomucor, Mucor) that can be isolated from dung, soil,
and other substrates. Dicranophora fulva, a rare mushroom parasite, must be grown
at 19 °C or less and optimal sporulation occurs on V8. Three other species
(Chaetocladium jonesii, Helicostylum elegans, H. pulchrum) are psychrotolerant.
Chaetocladium brefeldii and C. jonesii are gall-forming facultative parasites; both
grow and sporulate well on MEYE. Zygospore formation of C. brefeldii occurs on
MEYE and YpSs. Several taxa are most commonly encountered when it is cool
(more northern latitude, higher elevation, or during winter in warm regions)
including Ambomucor, Chaetocladium, Dicranophora, Helicostylum, Pirella,
Thamnidium, and some species of Mucor and Zygorhynchus. Except for the taxa
where optimal conditions are mentioned for asexual and sexual reproduction, the
majority of the other species will grow and sporulate on almost any culture medium
at 25 °C.
Pilaira has been included with several classical genera of thamnidiaceous fungi
including Helicostylum, Pirella, and Thamnidium based on the analysis of sequence
data (Hoffmann et al. 2013; Walther et al. 2013). Pilaira lacks a subsporangial
vesicle and trophocyst, but it has been classically treated in the Pilobolaceae based
on other morphological criteria (dark, persistent sporangium wall and a circumscis-
sile zone of dehiscence between the sporangial wall and the columella; Grove 1934;
Zheng and Liu 2009). All species of Pilaira (Benny and O’Donnell 1978; Zheng
and Liu 2009), however, are morphologically distinct from Helicostylum, Pirella,
and Thamnidium (Benny 1992, 1995b; Benny and Schipper 1992).
Mycocladiaceae The family was described by Hoffmann et al. (2007) and later
discussed by Hoffmann et al. 2009). The type and only species in the Mycocladiaceae
is Mycocladus verticellatus (Beauverie 1900). This species was probably a mixture
of two fungi, Lentamyces parricida (Lentamycetaceae) and a host fungus, a species
of Absidia (Hoffmann et al. 2009). A name based on a mixed type (http://www.iapt-
taxon.org/nomen/main.php?page=art9#9.11) is not invalid. The fungus is not
known in culture and, therefore, no recommendations for media, temperature, etc.
can be given.
Mycotyphaceae Mycotyphaceae has four genera (Benjaminiella, Cokeromyces,

Kirkomyces, Mycotypha) in three subfamilies: (1) Cokeromycetoideae
(Benjaminiella, Cokeromyces), (2) Kirkomycetoideae (Kirkomyces), and (3)
Mycotyphoideae (Mycotypha). Members of Benjaminiella, Cokeromyces, and
Mycotypha grow and sporulate readily on MEYE and YpSs agars although other
media will suffice (LYE, MSMA, PDA, TPO, V8, whey, YpD). Benjaminiella,
Cokeromyces, and Mycotypha are mesophilic or thermotolerant. In nature these
three taxa are usually found on dung but they are not commonly encountered.
Members of the Mycotyphaceae, except Kirkomyces, form a culture with relatively
short sporangiophores that is easily overlooked and probably readily overgrown by
other fungi. They can all form yeast cells on the surface of MEYE agar (Benny and
Benjamin 1976; Benny et al. 1985) at 26 °C. Cokeromyces recurvatus can cause
mucormycosis in humans and other animals but it is rarely reported. Kirkomyces
cordense was isolated from soil collected in India (Benny 1995b, c). The only
known culture of K. cordense is a mesophile that will readily grow and sporulate on
all of media listed above, but the morphological features are best observed on
MSMA or another nutrient-poor culture medium.
Phycomycetaceae Phycomycetaceae has no subfamilies and two genera:

Phycomyces and Spinellus. These fungi are found in nature on organic substrata
(Phycomyces, dung, pressed seedcakes for birds, mushrooms, etc.; Spinellus, on
fungi, especially Collybia or Mycena). Phycomyces is usually found when the
weather is cool (winter in Florida) although it can grow at room temperature; zygo-
spores form best at 15–20 °C on PDA, TPO agar, or YpSs agar (Benjamin and
Hesseltine 1959). Phycomyces blakesleeanus is the most common of the three
known species in nature and often can be found on dung. Spinellus contains five
species (Zycha et al. 1969) but only S. fusiger is in culture collections. Spinellus
fusiger is a psychrophile and must be grown on MEYE agar at 19 °C or below.
Pilobolaceae Pilobolaceae has no subfamilies and two genera. This family has
traditionally contained two genera, Pilaria and Pilobolus, with Utharomyces added
later. Molecular evidence has resulted in the transfer of Pilaira to the Mucoraceae
(Hoffmann et al. 2013; Benny et al. 2014). The remaining genera, Pilobolus and
Utharomyces (Grove 1934; Kirk and Benny 1980; Hu et al. 1989), are characterized
by the production of a trophocyst that forms a simple sporophore bearing an apical
or subapical vesicle with an apical columellate, multispored sporangium having a
persistent black wall. Pilaira does not produce a trophocyst or a subsporangial ves-
icle. These genera, Pilobolus and Utharomyces, are coprophilous. Pilobolus is an
obligate coprophile that forcibly disperses the sporangium. Culture of Utharomyces
is best on a nutrient-rich medium such as YpSs agar or 2 % malt agar (Kirk and
Benny 1980); dung extract is not required. Most species of Pilobolus, however,
requires a culture medium that contains dung extract or hemin (SHM, Levetin and
Caroselli 1976). Transfer the entire sporangium of Pilobolus with sterile forceps,
preferably a pair of watchmaker’s forceps, before being forcibly dispersed. This
sporangium is then placed on the surface of an agar plate emended with dung extract
or hemin (SHM). Place the inoculated plate in a 37 °C incubator overnight.
Germination of at least some spores will occur as a result of the overnight 37 °C
incubation. Optimal sporulation is probably best observed if the culture is trans-
ferred to sterile dung, but observation of the trophocyst and zygospore formation, if
crosses are made, will be impaired.
Radiomycetaceae Radiomycetaceae has no subfamilies and one genus. Initially the

Radiomycetaceae included two genera, Hesseltinella and Radiomyces (Ellis and
Hesseltine 1974). This pair of genera also was the subject of three papers (Benny
and Benjamin 1991; Benny and Khan 1988; Benny and Samson 1989). Molecular
analysis reveals, however, that these taxa belong in different mucoralean families
(O’Donnell et al. 2001; White et al. 2006a). Based on current molecular studies,
Hesseltinella belongs in the Cunninghamellaceae, whereas Radiomyces is the only
genus in the Radiomycetaceae.
The three known species of Radiomyces usually are found on dung (Benny and
Benjamin 1991) but Ranzoni (1968) made two isolations of R. embreei from soil
collected in Arizona and California (USA). None of the species of Radiomyces are
common in nature. Sporulating head and sporangiolar formation are promoted by
growth on nutrient-poor culture media (V8, Wg), whereas zygospores are formed
optimally on MEYE agar. YpSs agar produces variable results for both asexual and
sexual reproduction depending on the species and probably the isolate (Benny and
Benjamin 1991).
Rhizopodaceae Rhizopodaceae has no subfamilies. The Rhizopodaceae

(Schumacher 1894) includes four genera: Amylomyces, Rhizopus, Sporodiniella,
and Syzygites (Ellis et al. 1976; Ekpo and Young 1979; Schipper 1984; Schipper
and Stalpers 1984; Gbaja and Young 1985; Zheng et al. 2007). These fungi, except
Amylomyces, produce ornamented spores, the sporangia are apophysate, and the
sporangial wall is fugacious or deliquescent; sporangia and spores are not produced
by Amylomyces rouxii (Ellis et al. 1976). Sporodiniella umbellata is only found on
insect larvae, mature insects, and spiders in nature; Syzygites megalocarpus is a
facultative parasite of mushrooms in nature. Amylomyces and some species of
Rhizopus are used in the production of Asian foods or indigenous fermented prod-
ucts in Guyana (South America) and can be isolated from these substrates (Hesseltine
1965, 1985; Henkel 2005). Many other species of Rhizopus can be isolated from
dung, soil, and other organic materials. Rhizopus stolonifer can cause postharvest
disease of strawberries, sweet potatoes, and other crops. A few species of Rhizopus
are the causative agents of mucormycosis. Observation of Rhizopus and possibly
Amylomyces branching patterns is best made on nutrient-poor media.
Syzygites megalocarpus can be cultured on CM+ (Kovacs and Sundberg 1999),
GYP (Kaplan and Goos 1982a), MEA (Baker 1931), MEYE [the same as yeast-malt
agar, Difco] (Davis 1967), and PDA (Hesseltine 1957; Benny and O’Donnell 1978).
The formation of sporangia versus zygospores can be manipulated depending on the
temperature, media, and light. Idnurm (2011) found that some isolates of S. megalo-
carpus do not produce zygospores, whereas others will reproduce sexually.
Zygospores may or may not germinate to produce germspores. However, one culture
(CBS 108947) produces germ sporangia containing germspores that do germinate.
Sporodiniella umbellata is a monotypic genus that is found in nature on insects,
larvae of Homoptera and Lepidoptera, and spiders (Evans and Samson 1977).
Growth and sporulation can be induced on CA (Chien and Hwang 1997) and CMA
(after plates solidify place a sterile mealworm larvae on the agar surface; Chien and
Hwang 1997) with isolation conducted in the dark at 20 °C and the artificial infec-
tion at 24 °C. Evans and Samson (1977) used MEA and MWA (Samson 1974) in
order to isolate, and for growth and sporulation of S. umbellata at 25 °C in diffuse
light or a light/dark period, but sporulation was not observed on any of the afore-
mentioned culture media. Sporodiniella umbellata is heterothallic (Degawa 2006).
Saksenaeaceae Saksenaeaceae has no subfamilies and two genera. When originally

described, Saksenaeaceae (Ellis and Hesseltine 1974) contained two unusual but
unrelated monotypic genera, Saksenaea and Echinosporangium (now
Lobosporangium (Benny and Blackwell 2004), Mortierellales). Currently,

Saksenaeaceae contains Apophysomyces and Saksenaea. Additional species were
described in both genera (Alvarez et al. 2010a, b; Bonifaz et al. 2014). The
pathogenic species are found in both genera (Guarro et al. 2011; Hospenthal et al.
2011; Austin et al. 2013).
Members of both Apophysomyces and Saksenaea do not sporulate readily when
grown using ordinary culture methods. Padhyay and Ajello (1988) reported a simple
method that can be used to induce sporulation. This method involved growing the
fungus on a nutrient-rich culture medium, such as Sabouraud dextrose agar (SDA),
and then transferring a piece of the culture to 20 ml of sterile distilled water supple-
mented with 0.2 ml of filter-sterilized 10 % yeast extract solution. These latter cul-
tures were then incubated for 7–10 days at 37 °C; sporulation is abundant and
reliable.
Syncephalastraceae There are seven genera (Circinella, Fennellomyces,

Phascolomyces, Protomycocladus, Syncephalastrum, Thamnostylum, Zychaea:
Hesseltine and Fennell 1955; Hesseltine and Ellis 1961; Benny and Benjamin 1975,
1976; Schipper and Stalpers 1983; Zheng et al. 1988; Schipper and Samson 1994)
in the family. Syncephalastraceae can be divided into three clades: (1)
Protomycocladus (2 % MEA for both asexual and sexual reproduction); (2)
Circinella (SMA (asexual reproduction), steep agar (zygospore formation)),
Fennellomyces (MSMA (asexual reproduction, no zygospores produced)),
Phascolomyces (MSMA, MEYE, YpSs (asexual reproduction varies among iso-
lates, zygospores unknown)), Thamnostylum (MSMA [Wort + 3.5] for T. repens
sporangial formation, whereas zygospores are produced on MEYE, TPO, YPD, and
YpSs), and Zychaea reproduces asexually on MSMA but zygospores are unknown;
and (3) Syncephalastrum produces merosporangia on PDA and YpS, but sexual
reproduction is optimal on YpSs when compatible cultures are grown at 25 °C.
According to Hoffmann et al. (2013), these genera were all maintained in the latter
family because of low branch support. The final distribution of the aforementioned
members of the Syncephalastraceae will depend on additional sequencing and phy-
logenetic analysis.
Members of the genera of Syncephalastraceae can all be isolated from dung,
soil, and other organic substrata. Initial isolation probably is best done on a clear,
nutrient-rich culture medium such as MEYE in order to observe spore germination
and also to check for contamination. Sporulating cultures can then be transferred to
any one of several nutrient-poor culture media in order to observe branching, sporu-
lating structures, etc.
Umbelopsidaceae This family (Meyer and Gams 2003) contains only the type of
genus, Umbelopsis, with 14 species (Meyer and Gams 2003; Sugiyama et al.
2003; Mahoney et al. 2004; Wang et al. 2014). The majority of species of
Umbelopsis are isolated from leaf litter and soil (Meyer and Gams 2003). Culture
can be done on 2 % MEA, CMA, CZA, Miura agar (LcA, same as MA), PCA, and
PDA (Sugiyama et al. 2003; Mahoney et al. 2004; Wang et al. 2014). Zygospores
have never been reported.
A synopsis of the isolation and culture of species of the Mucorales from dung,
soil, and any other organic substratum is presented in the preceding section.
Additional methods for isolation and culture of Mucorales are detailed in Krug et al.
(2004) and Benny (2008).
Subphylum Zoopagomycotina (Hibbett et al. 2007) (Table 5.4)
Fungi produce hyphae that are coenocytic or septate. Asexual reproduction by arthro-
spores, chlamydospores, conidia, or sporangiospores in multispored merosporangia.
Sexual reproduction, where known, by zygospores borne on apposed suspensors.
Obligate haustorial, endo- or ectoparasites of amoebae, fungi, nematodes, and rotifers.
One order, Zoopagales, and five families with 19 or 20 genera.
Table 5.4 Synopsis of the Zoopagomycotina Benny

classification of the
Zoopagales Bessey ex R.K. Benj.
Zoopagomycotina
Cochlonemataceae Dudd.
Amoebophilus P.A. Dang.
Aplectosoma Drechsler
Bdellospora Drechsler
Cochlonema Drechsler
Endocochlus Drechsler
Euryancale Drechsler
Possible member of the family
Aenigmatomyces R.F. Castañeda & W.B. Kendr.
Helicocephalidaceae Boedijn
Brachymyces G.L. Barron
Helicocephalum Thaxt.
Rhopalomyces Corda
Verrucocephalum Degawa
Piptocephalidaceae J Schröt.
Kuzuhaea R.K. Benj.
Piptocephalis de Bary
Syncephalis Tiegh. & G. Le Monn.
Sigmoideomycetaceae Benny, R.K. Benj. & P.M. Kirk
Reticulocephalis Benny, R.K. Benj. & P.M. Kirk
Sigmoideomyces Thaxt.
Thamnocephalis Blakeslee
Zoopagaceae Drechsler emend. Duddington
Acaulopage Drechsler
Cystopage Drechsler
Stylopage Drechsler
Zoopage Drechsler
Zoophagus Sommerst. emend. M.W. Dick
Possible member of the subphylum
Basidiolum Cienk
Order Zoopagales (Benjamin 1979)
With the description of the subphylum.

Five families: Cochlonemataceae, Helicocephalidaceae, Piptocephalidaceae,
Sigmoideomycetaceae, and Zoopagaceae.
Members of the Zoopagomycotina can be isolated from any organic substrata
that support the hosts because all taxa are obligate parasites of small animals or
other fungi in nature. Only the members of the Piptocephalidaceae, especially
Piptocephalis and Syncephalis, are usually found in nature and grow readily in
culture (Benjamin 1959, 1961, 1963, 1966) as mycoparasites, usually on a member
of the Mortierellales or Mucorales.
Two of these families, Cochlonemataceae and Zoopagaceae, will be pre-
sented together because the methods of isolation and culture are similar.
Members of the remaining families may be encountered when looking for the
aforementioned taxa.
In the culture of species of the Cochlonemataceae and Zoopagaceae, members
of these two families parasitize small animals (amoebae, nematodes). Drechsler
(1936) recommended making media (CMA?) with 1.5–2.0 % agar to keep the hosts
on the surface of the substratum. The agar must be kept moist by adding a small
amount of sterile water to each plate and then placing the culture in containers that
prevent water loss to promote both bacterial growth and host migration. When the
hosts have reached a critical population, then the zoopagalean parasites also will be
more readily observed (Drechsler 1936). They first appear after 2 weeks but must be
kept and observed for a few months.
Conditions must be promoted to maximize the growth of these host animals on
agar in a Petri dish of 2 % water agar or NN-agar emended with Page’s amoeba
saline solution. Enterobacter cloacae has been used to feed amoebae (Michel et al.
2014) although it is possible that other bacteria could be used instead. Incubation of
plates is probably best at 18–20 °C. Higher or lower temperature may stop growth
or induce changes in the cytoplasm of either the host or parasite (Drechsler 1946).
Stylopage cephalote (Drechsler 1938) can appear at higher temperatures. Conidia
are produced when the host dies on the agar surface, but zygospores form when the
dead host is in the agar (Drechsler 1935a).
The presence of many members of the Cochlonemataceae and Zoopagaceae ini-
tially can be detected by shinning a light laterally over its surface. Taxa with many
spore chains, such as Cochlonema agamum (Drechsler 1946), C. eryblastum
(Drechsler 1942), and C. symplocum (Drechsler 1941), will be noted because of the
white, scattered conidial tufts. The conidial tufts of other species may appear thin
and scattered (Zoopage pachyblasta, Drechsler 1947) or restricted to a 15 mm circle
(Z. mitospora; Drechsler 1938). Cystopage lateralis (Drechsler 1941) sporulates on
or below the agar surface but not aerially. Single spores formed by some species
(Acaulopage rhicnospora (Drechsler 1935b), Endocochlus asteroides (Drechsler
1935b)) can be located in the plate with a low or medium power light microscope
objective. Many of Drechsler’s zoopagalean fungi are probably easier to find after
the first one or two have been observed (see references in Lumsden 1987).
Drechsler (1929) inoculated cornmeal agar (CMA) with a species of Pythium

and then added decaying vegetable material to the plate (Drechsler 1936). His later
cultures often were inoculated a second time with compost, leaf mulch, moss, etc.
The details of Drechsler’s techniques of both isolation and observation are discussed
in Benny (2008). Members of the Cochlonemataceae and Zoopagaceae are not in
culture collections. Drechsler also reported the appearance of species of
Helicocephalum and Rhopalomyces (Helicocephalidaceae; see below) using these
same methods.
Cochlonemataceae Cochlonemataceae (Duddington 1973) was described for the
endo- and ectoparasitic members of the order. These microfungi are obligate para-
sites of amoebae or nematodes. Cochlonemataceae contains six genera: two that are
ectoparasites (Amoebophilus, Bdellospora) and four endoparasites (Aplectosoma,
Cochlonema, Endocochlus, Euryancale). The thallus can arise from the infecting
conidium that produces a haustorium in the host and chains of conidia or zygo-
spores (Amoebophilus, Bdellospora) and is internal and coiled (Cochlonema),
cushion-shaped (Aplectosoma), or fertile hyphae forming single conidia per hypha
(Endocochlus, Euryancale). Asexual reproduction is by conidia formed singly or in
chains of spores. Sexual reproduction via zygospores formed on apposed
suspensors.
Amoebophilus simplex (Barron 1983) has been reported several times recently,
both on the Internet and in the literature (see Benny et al. 2014). Siemensma (2012)
believes that Mayorella penardi may be the only host for A. simplex. Leidy (1879)
may have illustrated A. simplex as Ouramoeba botulicauda.
Saikawa and Kadowaki (2002) illustrated amoebae capture by two species of
Acaulopage, A. dichotoma and A. tetraceros (Cochlonemataceae). These two spe-
cies of Acaulopage were isolated from debris collected from a fire reservoir. Some
of the debris was placed in a Petri dish containing 10 ml of water, the Acaulopage
sporulated on the water surface, was transferred to the surface of 2 % water agar
with amoebae that was cultured on SA agar (formula in Benny 2008). Cochlonema
euryblastum (Drechsler 1942) was cultured on a host, Thecamoeba quadrilineata,
and 18S rRNA was sequenced by Koehsler et al. (2007). The phylogenetic analysis
demonstrated that C. euryblastum is the sister species of, but basal to, Kuzuhaea
moniliformis (Benjamin 1985) and Piptocephalis corymbifera (Piptocephalidaceae,
Zoopagales).
Zoopagaceae The Zoopagaceae as defined by Duddington (1973) was reserved for

the taxa that are predaceous. Hyphae are coenocytic and formed externally. Conidia
are not produced (Cystopage), formed singly (Acaulopage, Stylopage, Zoophagus)
or in chains (Zoopage). Zoopagaceae are haustorial parasites of amoebae, nema-
todes, and rotifers. Sexual reproduction is by zygospore formation.
Zoophagus is a genus formerly thought to be a zoosporic fungus (Dick 1990).
Zygospores were reported in Acaulopage pectospora, and it was transferred to the
zygomycetes as Zoophagus pectosporus (Dick 1990). This transfer was verified by
the phylogenetic study, based on an 18S rDNA data set, by Tanabe et al. (2000).
The host for Zoophagus spp., in water, is usually a rotifer but nematodes also can be
hosts (Karling 1936; Saikawa et al. 1988; Glocking 1997; Liu et al. 1998). Cultures
of a species of Zoophagus may be in one or more culture collections. Shimada and
Saikawa (2006) observed nematode capture and chlamydospore germination in
Cystopage cladospora.
A review of the literature on the ultrastructure of members of Cochlonemataceae
and Zoopagaceae has been published (Saikawa 2011b). Other publications (Saikawa
2011a, 2012) reviewed the morphology and presented keys to taxa in the latter two
families. The first color illustrations are included for several taxa (Saikawa 2012).
The techniques described by Drechsler below for finding Cochlonemataceae and
Zoopagaceae also were efficacious for isolating some members of the
Entomophthoromycota (Meristacrum, Drechsler 1940), Kickxellomycotina
(Ballocephala, Drechsler 1951), and other Zoopagomycotina (Helicocephalum,
Rhopalomyces, Syncephalis; Drechsler 1934, 1943, 1961).
Cochlonemataceae and Zoopagaceae—information that applies to both families.
Hirotane-Akane and Sakawa (2010) studied zygospore morphology and germi-
nation in Cochlonema cerasphorum, C. megalosomum (Cochlonemataceae), and
Acaulopage lophospora (Zoopagaceae). One year later, Saikawa (2011) reviewed
his ultrastructural studies of selected taxa in the Cochlonemataceae (Acaulopage
dichotoma, A. tetraceros, Stylopage cephalote, Zoophagus insidians, Z. tenticulum)
and Zoopagaceae (Cochlonema odontosperma, Endocochlus gigas).
Helicocephalidaceae This family is reserved for four genera (Brachymyces,
Helicocephalum, Rhopalomyces, Verrucocephalum; Thaxter 1891a, b; Barron 1975,
1980a, b; Drechsler 1943; Ellis 1963; Cano et al. 1989; Roux 1996; Degawa 2014)
that are parasites of nematodes or their eggs. The sporangiophores are erect, have
basal rhizoids, and either form spores blastosporically on pedicels directly from the
sporophores or pedicellate sporangiola on fertile vesicles or arthrospores resulting
from disarticulation of the sporophore apex. The spores are relatively large and
pigmented. Sexual reproduction has not been reported. The members of the
Helicocephalidaceae are very rarely reported in the literature. Rhopalomyces ele-
gans (Corda 1839, 1840; Ellis 1963) probably is the most common member of the
family.
Ellis (1963) reported that the cultures of R. elegans were initially isolated on hay
extract agar (HEA) or 2 % water agar plates, inoculated with soil-plant debris, and
incubated at 25 °C for 5 days to 6 weeks. Ellis (1963) found three varieties of R.
elegans (apiculatus, crassus, elegans). He described the first two varieties as new;
they were cultured on his Rhopalomyces medium (RM) that consisted of two media:
(1) TKY and (2) LFK.
His studies of R. elegans var. apiculatus revealed that spore germination was 10
% when the medium (TKY—see under RM) alone was used. Bacillus cereus “wild
B” NRRL B509 was inoculated on the TKY agar in a glass plate and incubated at
25 °C for 20 h. These plates were autoclaved for 20 min at 15 psi. When cooled and
solidified, the plate containing the sterilized Bacillus culture was inoculated with
the R. elegans var. apiculatus spores and placed in a 25 °C incubator. Another cul-
ture medium (LFK) needs to be prepared, also in a glass Petri dish (100 × 15 mm).
This culture medium contains 2 % agar with the addition of K2HPO4 0.4 % before
being autoclaved. The glass dish contained a 5 mm3 piece of baby beef liver, washed
in three changes of distilled water, and placed in the middle of the dish and the dish
autoclaved. The hot agar (ca. 25 ml) is added to the dish with liver, two drops of
10 % KOH are added to one side of the liver, and lamb fat is heated until liquefied,
and two small drops are added using a hot Pasteur pipet, and then gently mix to
distribute the lamb fat droplets. Let the LFK (see RM in Supplement) agar solidify
and cool and then inoculate one side of the liver with the R. elegans spores that
germinated on the TKY agar; incubate at 20–25 °C. The R. elegans hyphae will fill
the dish in 4 days, and then the sporophores form near the fat droplets in 5–6 days
(Ellis and Hesseltine 1962; Ellis 1963).
Piptocephalidaceae Aerial hyphae not produced or relatively thin, giving rise to

appressoria and haustoria. Sporophores are unbranched or if branched, then branch-
ing is usually dichotomous. Merosporangia uni- or multispored. Spores remaining
dry or released in a droplet of fluid at maturity. Sexual reproduction by zygospores
usually formed in the substratum on apposed suspensors. Three genera are currently
in the Piptocephalidaceae (Kuzuhaea, Piptocephalis, Syncephalis; Benjamin 1959,
1985a, b; Kirk 1978; Jeffries 1979; Ho and Kirk 2009; Santiago et al. 2011).
These fungi are all haustorial mycoparasites in nature. Two species are parasites
of the Ascomycota [Piptocephalis xenophila on Penicillium waksmanii, Syncephalis
wynneae on Wynnea macrotis (Dobbs and English 1954; Thaxter 1897)], but the
remainder grow on hosts in Mortierellales (Mortierellomycotina) and Mucorales
(Mucoromycotina). Many species are found in nature on dung, humus, soil, and
other organic substrates. Both the host and parasite usually grow well in culture and
these taxa are relatively large and readily recognized. The Piptocephalidaceae are
the most common members of the Zoopagales recognized in nature. A phylogenetic
study, based only SSU rDNA, indicates that Syncephalis may not be closely related
to Piptocephalis and Kuzuhaea moniliformis may be a species of Piptocephalis
(Hou 2011; White et al. 2006a).
Isolation of Piptocephalidaceae differs for Piptocephalis and Syncephalis so
they are discussed separately: (1) Syncephalis can be isolated on sterile Wg10 that
was then cooled to 55 °C and emended with sterile chlortetracycline hydrochloride
(50 ppm), streptomycin sulfate (100 ppm), and benomyl (10 ppm). After the plates
are solidified, soil was sprinkled on each plate and then incubated at room tempera-
ture (ca. 21–22 °C) on a laboratory bench near a south-facing window. The benomyl
inhibits some species of Mortierella (Strauss et al. 2000) but also soil-inhabiting
members of Penicillium and Trichoderma that could overgrow the plate before the
parasites (and species Coemansia) normally appear (6–14 days). The Syncephalis is
transferred to the host that it initially parasitized, a species of Absidia,
Cunninghamella, Mortierella, Mucor, Zygorhynchus, etc. Some species of
Syncephalis also can be transferred to Cokeromyces recurvatus or another slow-
growing host. Growth on C. recurvatus or on Ellis’ medium (Ellis 1966) without a
host could be used to collect Syncephalis hyphae and sporangiophores for molecu-
lar studies. Syncephalis can be transferred, with the appropriate host, to Wg10 with
or without benomyl, and then zygospores also can be studied, if they are present.
When more than one culture of a species is available, then they can be crossed on
Wg10 or YpSs/5. As many as four species of Syncephalis can occur in each soil
sample, based on examination of over 500 soil samples (Benny et al. 2016). (2)
Piptocephalis can be isolated from dung, soil, or other organic debris. After isola-
tion cultures should be incubated at 18 °C because some species of Piptocephalis
may not sporulate above this temperature (R.K. Benjamin, pers. comm.). Cultures
can be transferred to Cokeromyces recurvatus, if available, or to the original or
another suitable host. The majority of known species of Piptocephalis will grow
and sporulate on C. recurvatus, but P. minuta (Kuzuha 1976) only parasitizes a spe-
cies of Mortierella, P. cruciata on Mycotypha microspora, and an undescribed spe-
cies (literature name P. digitata) on Umbelopsis ramanniana (Gräfenhan 1998).
Isolation of Piptocephalis from soil can be performed on 2 % WA emended with the
antibiotics mentioned above. These plates then are inoculated with C. recurvatus,
either pieces of colony culture from the agar, sporangiola scraped from the colony
surface, or using the technique and broth (PG) recommended to produce Y-phase
yeast cells by Jeffries and Kirk (1976). When the isolates of Piptocephalis are in
culture, then different media can be used to study the anamorph and teleomorph.
Gräfenhan (1998) made species descriptions on MEA for Piptocephalis parasitizing
C. recurvatus and M. microspora, PCA for Mortierella (P. minuta), YpSs for
Umbelopsis, and PDA for Penicillium waksmanii (P. xenophila). Benjamin (1959)
used MEYE and YpSs for routine culture of many parasites including Piptocephalis.
Appearance of the parasite is relatively slow on MEYE or YpSs (2–3 weeks), ver-
sus on cV8 and 50 % PCA (7–12 days). The appearance of zygospores, when pro-
duced, often is obscured by the luxuriant growth of the host on MEA, MEYE, and
YpSs. Zygospores, if formed, were readily observed when the Piptocephalis culture
was grown on cV8 and PCA. One isolate of Piptocephalis tieghemiana (NRRL
A-19043) produced zygospores on 2 % WA.
The third genus, Kuzuhaea, is known only in culture from the initial isolation
made from soil in Japan (Benjamin 1985a). Kuzuhaea moniliformis was cultured on
Umbelopsis ramanniana on YpSs agar at 25 °C. Kuzuhaea moniliformis will grow
over the host culture and then sporulate on the surface of the colony. Zygospore
formation was studied on YpSs/5 agar. Kuzuhaea moniliformis (11.2 % of the
fungal community) has been reported from Alaska permafrost soil using 454 pyro-
sequencing (Penton et al. 2013).
Sigmoideomycetaceae The family contains three genera (Reticulocephalis,

Sigmoideomyces, Thamnocephalis; Benny et al. 1992). The fertile hyphae are
dichotomously branched and coenocytic when young but multiseptate in age. Pairs
of stalked fertile vesicles are produced from the cell at the branching point of the
fertile hyphae. Fertile vesicles are more or less globose and covered with unispored
sporangiola borne on small denticles. Sexual reproduction has never been observed.
The hosts for these fungi are generally unknown because they have only been
studied as herbarium specimens (Benny et al. 1992), whereas those in culture are
mycoparasites (hosts Basidiobolus ranarum, Cokeromyces recurvatus; Benny and
Benjamin 1992; Chien 2000). Basidiobolus ranarum was grown on 2 % cornmeal

agar and then transferred to MEA (2 % ME agar?) and then inoculated with the
spores from a growing colony of Thamnocephalis quadrupedata (Blakeslee 1905).
Infection of B. ranarum was observed within 48 h (Chien 2000).
Thamnocephalis sphaerospora was isolated from frog dung; Basidiobolus rana-
rum might have been the original host. This Thamnocephalis then was transferred
to Cokeromyces recurvatus. It also grew on a culture of Microascus singularis
(Malloch and Cain 1971) with lunate ascospores that did not produce conidia. The
T. sphaerospora cultures grown on C. recurvatus were placed in a lighted incubator
set at 26 °C with a light/dark (12 h/12 h) cycle. Those cultures grown on YpSs agar
produced fruiting heads and very little aerial mycelium, whereas when on MEYE or
2 % ME + 0.5 % YE agars, the aerial hyphae were abundant and yellow orange,
reached the Petri dish lid, and contained many fertile heads. Recently, one of us
(GLB) grew the T. sphaerospora/C. recurvatus coculture on V8 juice agar at 24 °C
on the laboratory bench near a south-facing window, and the formation of both
fertile heads and aerial hyphae was better than that observed on the media contain-
ing malt extract.
Suyama and Degawa (2013) reported that there were two members of the
Sigmoideomycetaceae found in Japan Thamnocephalis sphaerospora and
Sphondylocephalum verticillatum (Stalpers 1974). The latter species was previ-
ously an anamorphic member of the Pezizomycotina. It was described originally as
Oedocephalum verticillatum (Thaxter 1891a).
There is no known commercial value for the Zoopagales. In the future, however,
species of Syncephalis may be utilized as biocontrol agents for those members of
the Mucorales that are plant pathogens (Choanephora cucurbitarum) or that cause
storage rots (Gilbertella persicaria, Mucor piriformis, Rhizopus stolonifer) in the
USA. Species of Syncephalis and not Piptocephalis should be used because some
taxa of the former species can completely overgrow or inhibit sporulation of the
host or both. Selected species of Syncephalis can reduce asexual reproduction and,
as a result, could delay or prevent the plant disease or postharvest diseases caused
by C. cucurbitarum, G. persicaria, M. piriformis, and R. stolonifer.
Other Zoopagales can be isolated from bat dung (Verrucocephalum), frog dung
(species of Thamnocephalis), or nematode eggs (Rhopalomyces elegans); these
fungi are not commonly encountered in nature. One possible member of the
Zoopagomycotina is Basidiolum fimbriatum (Cienkowski 1863; White 2003) but its
affinities are unknown at this time.
Discussion
The early-diverging fungi include all of the fungi with the exception of the Dikarya
(Grigoriev et al. 2011). These early-diverging fungi can be either aquatic
(Blastocladiomycota, Chytridiomycota, Monoblepharidiomycota, Neocallimastigo-
mycetes, Olpidium) or terrestrial (Glomeromycota, Entomophthoromycota and
Kickxellomycotina, Mortierellomycotina, Mucoromycotina, Zoopagomycotina) (Benny

et al. 2014; Corsaro et al. 2014; Didier et al. 2014; James and Berbee 2011; James
et al. 2014; Powell and Letcher 2014; Redecker et al. 2013; Sekimoto et al. 2011).
DNA Barcoding and Environmental Sampling
DNA barcoding using the internal transcribed spacer ribosomal DNA (the ITS1-
5.8S-ITS2 region, known most frequently as simply ITS) has become an invaluable
approach to facilitate comparisons among fungal species and to enhance identifica-
tion of fungi from environmental samples (Schoch et al. 2012; Nilsson et al. 2011;
Tsui et al. 2011). Barcodes are available for most members of the Mortierellales
(Mortierellomycotina) and Mucorales (Mucoromycotina) that are currently in cul-
ture (Wagner et al. 2013; Walther et al. 2013). Unfortunately, the ITS region is often
too divergent to be aligned across zygomycete orders or sometimes even within
genera and, therefore, is not phylogenetically informative (Smith and Benny, unpub-
lished). Practically, this means that even for taxa for which other informative loci
such as RPB1, RPB2, 18S and 28S rDNA, and EF-1α are available, the ITS
sequences are quite often missing from public DNA sequence databases. For exam-
ple, two major papers on the Kickxellomycotina (Asellariales, Dimargaritales,
Harpellales, Kickxellales) were recently published, but ITS was not included in
these analyses (Tretter et al. 2013, 2014).
Samples should be collected using the techniques presented by Barron (2004),
Krug et al. (2004), and Benny (2008). Dung and other larger samples can be incu-
bated in moist chambers (large samples, Krug 2004; small samples, Benny 2008).
Many specialized techniques have been devised to find and culture fungi of the
Kickxellomycotina, Mortierellomycotina, Mucoromycotina, and Zoopagomycotina
(Benny 2008).
A paper with complete coverage of the techniques used to study Zoopagales
(Zoopagomycotina ) has not been published because many of the organisms
are parasites of small animals (amoebae, rotifers, nematodes, water bears) and
the hosts require specialized isolation and culture techniques. This includes fungi
in the Cochlonemataceae, Helicocephalidaceae, Sigmoideomycetaceae, and
Zoopagaceae. One species of Helicocephalidaceae (Rhopalomyces elegans) and
one species of Sigmoideomycetaceae (Thamnocephalis sphaerospora) have been
successfully cultured. The apparent scarcity of these species may be due more to
lack of optimal modes of collecting and culturing rather their true rarity in nature.
Members of the Piptocephalidaceae (Kuzuhaea moniliformis and many species of
both Piptocephalis and Syncephalis) are common in culture collections and can be
isolated from nature. Members of both genera can be readily isolated and grown in
culture on appropriate host fungi, but only a handful of sequences are available for
these taxa due to the difficulty of obtaining pure cultures and/or pure DNA (e.g.,
without contamination from the host fungi). In contrast, Kuzuhaea moniliformis is
known only from the original isolate from soil in Japan (Benjamin 1985a). However,
28S rDNA sequences similar to that of the type strain of Kuzuhaea were found in
Alaska permafrost soil, suggesting that this genus may be more widespread than
previously thought (Penton et al. 2013). A few species of Zoopagales do have ITS
sequences deposited in GenBank, but many more DNA barcodes are needed to
enhance our understanding of their ecology based on environmental sequencing.
The lack of DNA barcode information for the zygomycete fungi is a serious limi-
tation for studies that seek to use environmental DNA sequences to examine fungal
communities. Molecular fungal community studies are only as accurate as the
curated and correctly identified sequences in GenBank that are used as a library for
identification. In addition to an incomplete database of fungal sequences, prelimi-
nary data from our studies of the genus Syncephalis (Zoopagomycotina) suggest
that “fungal-specific” primers such as ITS1F may also be problematic (Smith and
Benny, unpublished). PCR primers such as ITS1F were often designed based on a
small number of DNA sequences from a biased pool of species (e.g., Gardes and
Bruns 1993) and therefore may be inappropriate to use in targeting all fungi. Our
data suggest that the ITS1F primer site is altered in some species of zygomycetes
and therefore that PCR with mixed fungal templates is likely to preferentially
amplify sequences of Dikarya and not those of zygomycete targets. This problem is
further enhanced for some zygomycete taxa that may have long ITS rDNA sequences
(Lazarus et al., unpublished). Future molecular approaches that use specific primers
to focus on zygomycetes and/or account for the longer sequence length in some
zygomycetes are likely to find a much greater diversity of these fungi than studies
that attempt to document all fungi from complex substrata. We suspect that a more
focused approach is likely to be effective when combined with next-generation
DNA sequencing (e.g., 454, MiSeq, PacBio).
Genome Sequencing
Resolving evolutionary relationships among the early-diverging fungi will be

dependent on genome sequencing and phylogenetic comparison of available repre-
sentatives as discussed for the 1000 Fungal Genomes project in the MycoCosm
portal (Grigoriev et al. 2013; http://genome.jgi-psf.org/programs/fungi/index.jsf).
Kuo et al. (2014) discussed the procedures used to sequence and annotate fungal
genomes. These procedures are based on the platforms used for next-generation
sequencing (Quail et al. 2012). A long list of fungal genomes has been sequenced
or nominated for sequencing (http://genome.jgi.doe.gov/pages/fungi-1000-projects.
jsf/), including species in Kickxellomycotina, Mortierellomycotina, Mucoromycotina,
and Zoopagomycotina (Table 5.5). Hopefully, more species will be sequenced
before the Fungi-1000 project is complete.
Phylogenetically informative genes will be extracted from these genomes and
the sequences aligned and then analyzed using phylogenetic methods. A data set
composed of the genes from the terrestrial early-diverging fungi can also be
Table 5.5 Taxa of zygomycota with genomes sequenced or to be sequenced as part of the 1000
Fungal Genomes project (F1000)
Kickxellomycotina
Asellariales
None nominated
Dimargaritales
Dimargaritaceae
Dimargaris cristalligena Tiegh.
Dispira cornuta Tiegh.
Tieghemiomyces californicus R.K. Benj.
Tieghemiomyces parasiticus R.K. Benj.
Harpellales
Harpellaceae
None nominated
Legeriomycetaceae
Capniomyces stellatus S.W. Peterson & Lichtw.
Furculomyces boomerangus (M.C. Williams & Lichtw.) M.C. Williams & Lichtw.
Smittium angustum M.C. Williams & Lichtw.
Smittium annulatum Licht.
Smittium culicis Tuzet & Manier ex Kobayasi
Smittium culicioides Licht.
Smittium fecundum Lichtw. & M.C. Williams
Smittium megazygosporum Manier & F. Coste
Smittium simulii Licht.
Smittium simulatum Licht. & Arenas
Zancuomyces culisetae (Lichtw.) Yan Wang, Tretter, Lichtw. & M.M. White
Kickxellales
Kickxellaceae
Coemansia mojavensis R.K. Benj.
Coemansia pectinata (Coem.) Bainier
Coemansia reversa Tiegh. & G. Le Monn.
Coemansia spiralis Eidam
Dipsacomyces acuminosporus R.K. Benj.
Kickxella alabastrina Coem.
Linderina pennispora Raper & Fennell
Martensiomyces pterosporus J.A. Mey.
Myconymphaea yatsukahoi Kurihara, Degawa & Tokum.
Pinnaticoemansia coronantispora Kurihara & Degawa
Spirodactylon aureum R.K. Benj.
Clade 1 (Barbatospora sp.)
Clade 2 (Orphella spp.)
Clade 3 (Mycoemilia scoparia, Spiromyces spp.)
Spiromyces aspiralis Benny & R.K. Benj.
Clade 4 (Ramicandelaber spp.)
(continued)
Table 5.5 (continued)

Ramicandelaber brevisporus Kurihara, Degawa & Tokum.
Mortierellomycotina
Mortierellales
Mortierellaceae
Gamsiella multidivaricata (R.K. Benj.) Benny & M. Blackw.
Lobosporangium transversale (Malloch) M. Blackw. & Benny
Mortierella alpina Peyronel
Mortierella elongata Linnem.
Mortierella verticillata Linnem.
Nothadelphia mortierellicola Degawa & W. Gams
Mucoromycotina
Clade 1
Calcarisporiella thermophile (H.C. Evans) de Hoog
Clade 2
None nominated
Endogonales
Endogonaceae
Endogone flammicorona Trappe & Gerd.
Endogone lactiflua Berk.
Endogone pisiformis Link
Endogone sp.
Mucorales
Backusellaceae
Backusella circina J.J. Ellis & Hesselt.
Choanephoraceae
C hoanephoroideae
Choanephora cucurbitarum (Berk. & Ravenel) Thaxt.
Blakeslea trispora Thaxt.
G ilbertelloideae
Gilbertella hainanensis J.Y. Cheng & F.M. Hu
Gilbertella persicaria var. persicaria (E.D. Eddy) Hesselt.
Cunninghamellaceae
A bsidioideae
Absidia repens Tiegh.
Chamydoabsidia padenii Hesselt. & J.J. Ellis
Gongronella butleri (Lendn.) Peyronel & Dal Vesco
Halteromyces radiatus Shipton & Schipper
Hesseltinella vesiculosa H.P. Upadhyay
C unninghamelloideae
Cunninghamella bertholletiae Stadel
Cunninghamella echinulata (Thaxt.) Thaxt.
Cunninghamella elegans Lendn.
Lentamycetaceae
(continued)

Lentamyces parricida (Renner & Muskat ex Hesselt. & J.J. Ellis) Kerst. Hoffm. & K. Voigt
Lichtheimiaceae
D ichotomomycetoideae
Dichotomocladium elegans Benny & R.K. Benj.
Dichotomocladium robustum Benny & R.K. Benj.
L ichtheimioideae
Lichtheimia corymbifera (Cohn) Vuill.
Lichtheimia hyalospora (Saito) Kerst. Hoffm., Walther & K. Voigt
Lichtheimia ramosa (Zopf) Vuill.
Rhizomycetoideae
Rhitzomucor miehei (Cooney & R. Emers.) Schipper
Rhizomucor pusillus (Lindt) Schipper
Rhizomucor variabilis R.Y. Zheng & G.Q. Chen (see Mucor irregularis)
Mucoraceae
C haetocladioideae
Chaetocladium jonesii (Berk. & Broome) Fresen.
D icranophoroideae
Dicranophora fulva J. Schröt.
M ucoroideae
Mucor circinelloides
Mucor indicus Lendn.
Mucor irregularis Stchigel, Cano, Guarro & Ed. Álvarez
Mucor racemosus Fresen.
Mucor ramosissimus Samouts.
Mucor sp.
Mucor velutinosus Ed. Álvarez, Stchigel, Cano, Deanna A. Sutton & Guarro
Parasitella parasitica (Bainier) Syd.
Zygorhynchus heterogamous (Vuill.) Vuill.
T hamnidioideae
Ellisomyces anomalus (Hesselt. & P. Anderson) Benny & R.K. Benj.
Helicostylum pulchrum (Preuss) Pidopl. & Milko
Pilaira anomala (Ces.) J. Schröt.
Pirella circinans Bainier
Thamnidium elegans Link
Mycotyphaceae
C okeromycetoideae
Benjaminiella poitrasii (R.K. Benj.) Arx
Cokeromyces recurvatus Poitras
K irkomycetoideae
Kirkomyces cordense (B.S. Mehrotra & B.R. Mehrotra) Benny
M ycotyphoideae
Mycotypha africana R.O. Novak & Backus
Phycomycetaceae
(continued)

Phycomyces blakesleeanus Burgeff
Spinellus fusiger (Link) Tiegh.
Pilobolaceae
Pilobolus umbonatus Buller
Utharomyces epallocaulus Boedijn
Radiomycetaceae
Radiomyces spectabilis Embree
Rhizopodaceae
Rhizopus delemar Boidin ex Wehmer & Hanzawa
Rhizopus microsporus var. chinensis (Saito) Schipper & Stalpers
Rhizopus microsporus var. microsporus Tiegh.
Rhizopus microsporus var. oligosporus (Saito) Schipper & Stalpers
Rhizopus microsporus var. rhizopodiformis (Cohn) Schipper & Stalpers
Rhizopus oryzae Went & Prins. Geerl.
Rhizopus stolonifer (Ehrenb.) Vuill.
Syzygites megalocarpus Ehrenb.
Saksenaeaceae
Apophysomyces elegans P.C. Misra, K.J. Srivast. & Lata
Apophysomyces trapeziformis E. Álvarez, Stchigel, Cano, Deanna A. Sutton & Guarro
Saksenaea oblongispora E. Álvarez, Stchigel, Cano & Guarro
Saksenaea vasiformis S.B. Saksena
Syncephalastraceae
Clade 1
Syncephalastrum monosporum R.Y. Zheng, G.Q. Chen & F.M. Hu
Syncephalastrum racemosum Cohn ex J. Schröt.
Clade 2
Protomycocladus faisalabadensis (J.H. Mirza, S.M. Khan, S. Begum & Shagufta) Schipper &
Samson
Clade 3
Circinella umbellate Tiegh. & G. Le Monn.
Fennellomyces linderi (Hesselt. & Fennell) Benny & R.K. Benj.
Phascolomyces articulosus Boedijn
Thamnostylum lucknowense (J.N. Rai, J.P. Tewari & Mukerji) Arx & H.P. Upadhyay
Zychaea mexicana Benny & R.K. Benj.
Umbelopsidaceae
Umbelopsis isabellina (Oudem.) W. Gams
Umbelopsis ramanniana (Möller) W. Gams
Zoopagomycotina
Zoopagales
Cochlonemataceae
None nominated
Helicocephalidaceae
Rhopalomyces elegans Corda
(continued)

Piptocephalidaceae
Piptocephalis corymbifera Vuill.
Syncephalis fuscata Indoh
Syncephalis plumigaleata Embree
Syncephalis pseudoplumigaleata Benny & H.M. Ho
Sigmoideomycetaceae
Thamnocephalis sphaerospora R.K. Benj. & Benny
Zoopagaceae
None nominated
supplemented with data from zoosporic early-diverging fungi and Dikarya, aligned
and analyzed to provide a phylogeny of the fungi. As aptly pointed out by Stajich
(2015), phylogenomics will undoubtedly revolutionize our view of the fungal tree
of life. We suspect that the most exciting discoveries in the fungal tree of life during
the coming decade will be centered on the zygomycete subphyla Kickxellomycotina,
Mortierellomycotina, Mucoromycotina, and Zoopagomycotina.
Supplement 1: Culture Media Other sources for media can

be found in Bills and Foster (2004) and Benny (2008)
2 % ME (same as 2 % MEA (2 % malt extract agar): malt extract, 20 g; agar, 20 g;

distilled water, 1 l (Benny and Benjamin 1975).
2 % ME + 0.5 % YE—malt-yeast agar: malt extract, 20 g; yeast extract, 5 g; agar,
15 g; distilled water, 1 l (Benny et al. 1992; Benny and Schipper 1992).
2 % WA—2 % water agar: agar, 20 g; distilled water, 1 l.
BHIv/10—10 % brain-heart infusion agar + vitamins: brain-heart infusion agar
(Difco), 3.7 g; thiamine-HCl, 200 μg; biotin, 50 μg; 50 agar, 15 g; glass-distilled
water, 1 l (Lichtwardt 1986).
CA—carrot agar: carrots, 200 g, blended (boil 20 min in 500 ml distilled water,
filter, make volume to 1 l); agar, 16 g (Chien and Hwang 1997).
CHA—Choanephora agar: dextrose, 3 g; casamino acids [Difco], 2 g; KH2PO4, 1
g; MgSO4•7H2O, 0.5 g; thiamine-HCl, 25 mg; agar, 20 g; distilled water, 1 l; pH
6.0 (Benny 2008—as CH).
CM or CMA—cornmeal agar: yellow cornmeal, 20 g (boil 10 min in 700 ml dis-
tilled water, filter, and add distilled water to make 1 l); dextrose, 10 g; agar, 15 g;
adjust pH to 6.0 (Benjamin 1958, 1959).
CM+—cornmeal agar+: cornmeal agar (Difco), 17 g; yeast extract, 1.0 g; malt
extract, 1.0 g; distilled water, 1 l (Kovacs and Sundberg 1999).
CM-S—cornmeal-steep agar: CM + corn steep liquor (Sigma), 5 ml [adjust pH to
6.0 with 1N NaOH] (Benny et al. 1992).
cV8—clarified V8 juice agar—filter v8 juice [regular] through MiraclothⓇ to remove

pulp; add CaCO3 [powdered], 3 g/l; mix, decant off supernatant, make volume to
original amount with distilled water, add ca. 45–50 ml plastic tubes, and freeze;
when media is needed, thaw and dilute one 45 ml tube to 1 l, agar 20 g.
CDY—Czapek-Dox agar [CZA, Difco] + yeast extract: CZA + yeast extract, 6.5 g;
pH 5.0; adjust with HCl (Gams and Williams 1963).
CZA—Czapek’s solution agar: NaNO3, 3.0 g; K2HPO4, 1.0 g; MgSO4•7H2O, 0.5 g;
KCl, 0.5 g; FeSO4•7H2O, 0.1 g; sucrose, 30 g; agar, 15 g; distilled water, 1 l.
DD—dung decoction: horse dung [moist], 125 g; tap water, 1 l (soak 2–3 h, filter,
make volume to 1 l); pH 6.0 or above 7.0 for Pilobolus (use in place of water for
media needing DD) (Benny and Schipper 1992).
GYP—glucose-yeast-peptone agar: glucose, 10 g; yeast extract, 1 g; peptone, 2 g;
agar, 15 g; distilled water, 1 l (Kaplan and Goos 1982b).
LA—Leonian’s agar: malt extract, 6.25 g; peptone, 0.625 g; maltose, 6.25 g;
KH2PO4, 1.25 g; MgSO4•7H2O, 1.25 g; agar, 15 g; distilled water, 1 l (Leonian
1924; Benny and Blackwell 2004).
LcA—Miura agar (see “MA” below).
LYE—Leonian’s agar + yeast extract: malt extract, 6 g; yeast extract, 1 g; peptone,
0.6 g; maltose, 6 g; KH2PO4, 1.2 g; MgSO4•7H2O, 1.2 g; agar, 15 g; distilled
water, 1 l (Malloch and Cain 1971; Benny and Benjamin 1975).
MED—2 % MEA + 5 g dextrose per liter.
ME-P—malt extract-peptone agar: malt extract, 20 g; peptone, 5 g; agar, 15 g; dis-
tilled water, 1 l (Richard K. Benjamin—pers. comm. 1988).
MEYE—malt extract-yeast extract agar: malt extract, 3 g; yeast extract, 3 g; pep-
tone, 5 g; dextrose, 10 g; agar, 15 or 20 g; distilled water, 1 l (Benjamin 1958,
1959; Benny and Benjamin 1975).
MA—Miura agar: glucose, 1 g; KH2PO4, 1 g; MgSO4•7H2O, 0.2 g; KCl, 0.2 g;
NaNO3, 2 g; yeast extract, 0.2 g; agar, 15 g; distilled water 1 l (Sugiyama et al.
2003—no KH2PO4; yeast extract, 2 g; agar, 13 g).
MWA—mealworm agar: dried mealworms (ground), 200 g (boil in tap water 3 h,
filter, add tap water to make supernatant to 1 l); sucrose, 20 g; agar, 20 g (Samson
1974).
MEYE/2—malt extract-yeast extract agar one-half strength: malt extract, 1.5 g;
yeast extract, 1.5 g; peptone, 2.5 g; dextrose, 5 g; agar, 15 g; distilled water, 1 l
(Kurihara et al. 2001).
MNC+T—modified Norkrans’ C medium + thiamine: K2HPO4, 1.0 g; MgSO4•7H2O,
0.5 g; ZnSO4, 0.5 ml 2 % sol; NH4-tartrate, 0.5 g; Fe-tartrate, 0.5 ml 1.0 % sol;
thiamine, 0.5 ml of 100 ppm stock sol; casein hydrolysate, 0.23 g; yeast extract,
0.5 g; dextrose, 10 g; agar, 15 g; distilled water, 1 l; streptomycin, 100 ppm; tet-
racycline, 50 ppm + thiamine 100 mg (Hirose et al. 2014).
MSMA—modified synthetic Mucor agar: dextrose, 10 g; NaNO3, 4 g; K2HPO4, 0.5
g; MgSO4•7H2O, 0.5 g; thiamine-HCl, 0.5 mg; agar, 15 g; distilled water, 1 l
(Benny and Benjamin 1975).
MRN + thiamine—Endogone agar + thiamine: saccharose, 5 g [replace dextrose,
10.7 g]; NH4(SO4)2, 1.15 g; CaCl2.4H2O, 55.5 mg; MgSO4•7H2O, 0.5 g; KCl,
0.67 g; ferric citrate, 5 mg; ZnSO4•4H2O, 4.4 mg; MnSO4, 5 mg; CaCl2•4H2O,
55.5 mg; ferric or aluminum phytate, 200 mg; sodium inositol hexaphosphate,
550 mg; thiamine, 100 mg; distilled water, 1 l; agar, 15 g (Dalpé 1990; Mitchell
and Read 1981).
MWA—mealworm agar: dried mealworms (ground), 200 g (boil in tap water 3 h, filter,
add tap water to make supernatant to 1 l); sucrose, 20 g; agar, 20 g (Samson 1974).
MYP-ps—malt extract-yeast extract-peptone agar with antibiotics: malt extract, 7 g;
yeast extract, 0.5 g; peptone, 1.0 g; penicillin G, 0.5 g; streptomycin sulfate, 0.5
g; agar, 15 g; distilled water, 1 l; autoclave medium, cool to 43 °C, and add anti-
biotics aseptically using a Millipore syringe filter (0.22 μm)—used for both dilu-
tion and direct inoculation on chilled plates [Carreiro and Koske 1992])).
NN-agar + PAS—non-nutrient agar + Page’s amoeba saline (PAS) solution: agar
(Oxoid L11), 15 g; plus PAS (stock 1 (NaCl, 12 g; MgSO4•7H2O, 0.40 g;
CaCl2•6H2O, 0.60 g; distilled water, 500 ml) and stock 2 (Na2HPO4, 14.20 g;
KH2PO4, 13.60 g; distilled water, 500 ml)); combine stocks 1 and 2, 5.0 ml each;
distilled water, 990 ml. Mix agar, 15 g; with PAS 1 l, slowly bring to a boil; auto-
clave 15 min (from Culture Collection of Algae and Protozoa—accessed 6
January 2015, http://www.ccap.ac.uk/pdfrecipes.htm/).
OMA—oatmeal agar: rolled oats, 30 g; distilled water, 1 l (heat to boiling and sim-
mer 2 h, filter, and bring volume to 1 l); agar, 15 g (Gams et al. 1975).
PAB—pablum agar: pablum, 50 g (boil in 700 ml distilled water, filter, adjust final
volume to 1 l); agar, 15 g (Benjamin 1959).
PAB-DEX—pablum agar: pablum, 50 g (boil in 700 ml distilled water, filter, adjust
final volume to 1 l); dextrose, 10 g; agar, 15 g (Benjamin 1959).
PCA—potato-carrot agar: potato (peeled and diced), 20 g; carrots (peeled and
diced), 20 g (boil carrots and potatoes in 300 ml of tap water, filter, and add water
to adjust volume to 1 l); agar, 20 g (Bawcutt 1983).
PDA—potato dextrose agar: potatoes (peeled and cut), 200 g (boil extract 10 min in
700 ml distilled water, filter, adjust final volume to 1 l); dextrose, 20 g; agar, 15
g (Schipper 1969—pH 6.6; Benjamin 1958, 1959—pH not mentioned).
PG—peptone-glucose agar: peptone [Difco], 10 g; dextrose, 20 g; agar, 20 g; dis-
tilled water, 1 l (Gauger 1961).
PYED—peptone-yeast extract-dextrose agar: peptone, 1 g; yeast extract, 1 g; dex-
trose, 0.5 g; agar, 15 g; distilled water, 1 l; adjust pH to 6.5 (Benjamin 1978).
PYEDS—peptone-yeast extract-dextrose-corn steep agar: PYED + corn steep
liquor, 5 ml; adjust pH to 6.0 with 1N NaOH.
RM—Rhopalomyces medium:
(1) TKY (tryptone, 0.5 %; yeast extract, 0.5 %; K2HPO4•H2O, 0.4 %; agar, 2
%; pH 9 with KOH). Bacillus cereus Frankland & Frankland “wild B”
NRRL B509 was inoculated on the TKY agar in a glass plate and incubated
at 25 °C for 20 h. These plates were autoclaved for 20 min at 15 psi. When
cooled and solidified, the plate containing the sterilized Bacillus culture
was inoculated with the R. elegans var. apiculatus spores and placed in a
25 °C incubator.
(2) LFK needs to be prepared, also in a glass Petri dish (100 × 15 mm). This cul-
ture medium contains 2 % agar with the addition of K2HPO4 (0.4 %) before
being autoclaved. The glass dish contained a 5 mm3 piece of baby beef liver,
washed in three changes of distilled water, and placed in the middle of the dish
and the dish autoclaved. The hot agar (ca. 25 ml) is added to the dish with liver,
two drops of 10 % KOH are added to one side of the liver, and lamb fat is
heated until liquefied, and two small drops are added using a hot Pasteur pipet,
and then gently mix to distribute the lamb fat droplets. Let the LFK agar solid-
ify and cool and then inoculate one side of the liver with the R. elegans spores
that germinated on the TKY agar; incubate at 20–25 °C. The R. elegans hyphae
will fill the dish in 4–6 days, and then the sporophores form near the fat
droplets in 5–6 days (Ellis and Hesseltine 1962; Ellis 1963).
SA—Sato and Aoki’s agar: KNO3, 0.2 g; MgSO4•7H2O, 0.02 g; KH2PO4, 0.1 g;
KHPO4, 0.3 g; NaHCO3, 0.02 g; Na2SiO3, 0.02 g; agar, 20 g; distilled water,
make volume to 1 l; unadjusted pH 7.8 (Saikawa and Kadowaki 2002).
SDA—Sabouraud dextrose agar (BBLTM or DifcoTM): peptic digest of animal tissue, 5 g;
pancreatic digest of casein, 5 g; dextrose, 40 g; agar, 15 g; distilled water, 1 l.
SDA+YE—Sabouraud dextrose agar + yeast extract: 20 ml sterile distilled water,
cool and add 0.2 ml filter-sterilized, 10 % yeast extract solution; transfer a small
piece of growing culture of a species of Apophysomyces or Saksenaea to the
latter solution and incubate at 37 °C for 7–10 days, and the fungus will reliably
sporulate (Padhyay and Ajello 1988).
SDY—soil-dextrose-yeast extract agar: loam soil extract, 100 g (boil 10 min in
800 ml distilled water, filter, make volume to 1 l); dextrose, 2 g; yeast extract, 1
g; K2HPO4, 1 g; MgSO4•7H2O, 0.5 g; agar, 15 g.
SEA—soil extract agar: garden soil and distilled water, equal weights; autoclave
30 min, let soil settle to the bottom of flask, and filter; agar, 15 g; soil extract, 1 l
(Gams et al. 1975).
ShA—shrimp agar: dried ground edible shrimp, 3 g; agar, 15 g; distilled water, 1 l
(Degawa and Tokumasu 1997, 1998a, b).
SHM—simplified hemin medium for Pilobolus: hemin, 10 mg (dissolved in 37.5 ml
of 0.1N NaOH); sodium acetate (CH3COONa•3H2O), 10 g; thiamine-HCl, 10
mg; (NH4)2SO4, 0.66 g; K2HPO4, 1.0 g; MgS04•7H2O, 0.5 g; deionized distilled
water to bring the volume to one liter; agar, 15 g (Levetin and Caroselli 1976).
SMA—synthetic Mucor agar: dextrose, 40 g; asparagine, 2 g; KH2PO4, 0.5 g;
MgSO4•7H2O, 0.25 g; thiamine HCl, 0.5 mg; agar, 15 g; distilled water, 1 L
(Benny 2008).
STA—steep agar (Czapek’s solution agar [DifcoTM, CZA, or CZB broth DifcoTM] +
corn steep liquor): CZA, 49 g, or CZ broth, 35 g; corn steep liquor [SigmaⒸ
C4648-500G], 10 ml; adjust pH to 7.0 with NaOH before autoclaving; agar, 15 g
[if needed]; distilled water, 1 l (Raper and Thom 1949).
SUP—SUP medium: glucose, 50 mg; potassium dihydrogen phosphate, 30 mg;
ammonium chloride, 20 mg; dipotassium hydrogen phosphate, 5 mg; magnesium
sulfate, 1 mg; calcium chloride, 100 mg (Hoffmann and Voigt 2009), or dextrose
10 g; yeast extract, 5 g; KH2PO4, 4 g; K2HPO4, 0.9 g; NH4Cl, 1.0 g; MgSO4•7H2O,
0.25 g; agar, 15 g; distilled water, 1 l (Wöstemeyer 1985).
TGv—tryptone-glucose medium + salts and vitamins: tryptone (Difco), 20 g; glu-

cose, 5 g; KH2PO4, 0.28 g; K2HPO4, 0.35 g; (NH4)2SO4, 0.26 g; MgCl2•6H2O,
0.10 g; CaCl2•2H2O, 0.07 g; thiamine-HCl, 200 μg; biotin, 50 μg; 50 g agar, 15
g; glass-distilled water, 1 l (Lichtwardt 1986).
TPO—tomato paste-oatmeal agar: tomato paste, 20 g; instant baby oatmeal, 20 g;
agar, 15 g; distilled water, 1 l (Hesseltine 1960; Benny and Benjamin 1975).
TSM—Thorton’s standardized medium: K2HPO4, 1 g; MgSO4•7H2O, 0.2 g; CaCl2,
0.1 g; NaCl, 0.1 g; FeCl3, 0.002 g; KNO3, 0.5 g; asparagine, 0.5 g; mannitol, 1 g;
agar, 15 g; distilled water, 1 l (Thorton 1922; Benny and Blackwell 2004).
V8—V8 juice agar [modified]: V8Ⓡ juice [original], 5.5 fluid oz (163 ml) can; adjust
volume to 1 l with distilled water; CaCO3 [powdered], 3 g; agar, 15 g (Benny and
Benjamin 1991).
Wg—wheat germ agar: wheat germ, 15 g (boil 10 min in 700 ml distilled water,
filter, and adjust volume to 1 l); dextrose, 5 g; agar, 15 g (Benny 1972).
Wg10—one-tenth strength wheat germ agar: wheat germ, 1.5 g; heat in a micro-
wave oven for 3 min in 300 ml of distilled water, and then filter through
Miracloth® (Calbiochem 475855) or cheese cloth, take the supernatant, and add
distilled water to adjust volume to 1 l; dextrose, 0.5 g; agar, 15 g.
WgDD—wheat germ-dung decoction agar: Wg broth, 500 ml, + DD made with
distilled water, 500 ml.
Wg-S—wheat germ-steep agar: Wg + corn steep liquor, 5 ml; adjust pH to 6.0 with
1N NaOH.
Whey—whey agar: powdered whey, 20 g; dextrose, 10 g; agar, 15 g; distilled water,
1 l (Schipper 1969; Benny et al. 1985).
WSH—Weitzman and Silva-Hutner medium: Alpha-Cel [powdered cellulose], 20 g;
MgSO4•7H2O, 1 g; KH2PO4, 1.5 g; NaNO3, 1 g; tomato paste, 10 g; baby oatmeal,
10 g; agar, 18 g; distilled water, 1 l; pH 5.6 (Weitzman and Silva-Hutner 1967).
WSH-DD—Weitzman and Silva-Hutner medium (WSH) in distilled water 500 ml
+ dung decoction (DD) 500 ml.
YpSs—Emerson’s yeast-phosphate-soluble starch agar: soluble starch, 15 g; yeast
extract, 4 g; K2HPO4, 1.0 g; MgSO4•7H2O, 0.5 g; agar, 20 g [15 g used later;
Benny and Benjamin 1975]; distilled water, 1 l (Benjamin 1959).
YpSs/5—one-fifth strength YpSs agar: soluble starch, 3 g; yeast extract, 0.8 g;
MgSO4•7H2O, 0.5 g; KH2PO4, 0.5 g; K2HPO4, 0.5 g; agar, 15 g; distilled water,
1 l (Benjamin 1985a, b).
YGCH—yeast extract-glycerol-casein hydrolysate agar: yeast extract, 10 g; glycerol,
15 ml; casein hydrolysate, 15 g; K2HPO4, 1.0 g; MgSO4•7H2O, 0.5 g; agar, 15 g;
distilled water, 1 l (O’Donnell et al. 1998).
Supplement 2: Isolation and Transfer
Members of the four subphyla discussed here all have asexual reproductive struc-
tures that are either dry- or wet-spored when mature (Ingold and Zoberi 1963).
Dry-spored taxa can be isolated using watchmakers forceps, whereas wet-spored
species can be collected using stainless steel, minuten insect pins (0.20 mm in diam-
eter; Carolina #654370) in one of several pin-vise handles or with forceps (Benny
2008). Transfer the spores to a clear, nutrient-rich culture medium such as MEYE
and observe spore germination. For best results be sure that the bottom of the plate
is pre-marked with several small circles, and additionally be sure to note which are
inoculated first. The zygomycotan fungi, especially Mucorales, will germinate rap-
idly, and the mycelium will be very robust; members of the Mortierellales are much
small in diameter. Contaminated cultures will grow relatively slow and sporulation
will be indicated by the color of the colony. A species of Penicillium is one of the
most common contaminants, but other imperfect fungi such as Aspergillus and
Trichoderma also can appear instead.
Supplement 3: Microscope Slides
Benjamin (1959) recommended that the zygomycotan fungi be mounted in KOH-

phloxine or 2 % KOH for the observation of vegetative and both asexual and sexual
reproductive structures. An aqueous solution of phloxine is used if the fungi are
hyaline and they are examined using a microscope not equipped with interference-
phase contrast. Young portions of an actively growing colony are used to make the
slide mounts for taking photographs, making drawings and observation of the char-
acteristic structures of each species. The process can be accomplished using the
following steps:
1. Take a small amount of the fungus from the colony.
2. Transfer the material to the surface of a clean microscope slide.
3. Add a drop of 95 % ethyl alcohol to wet the specimen and minimize air bubbles.
4. Tilt the slide on a paper towel or filter paper to blot away excess alcohol.
5. Add a drop of 2.0 % KOH to the slide before the alcohol dries and a drop of
distilled water or phloxine to dilute the KOH to ca. 1.0 %.
6. The specimen can be manipulated at this stage to reveal the best material for
documentation.
7. Carefully add an 18 mm2 coverslip by placing it on one side at a 30–45° angle
and slowly lowering it over the specimen without migrating it to the edge if
excess mounting agent has been used.
8. Blot away excess mounting agent using bibulous or filter paper.
9. Seal the slide with paraffin (I use canning paraffin melted in a baby food jar)
using a large paper clamp opened to expose one straight side.
10. Heat the paper clamp above warm enough to melt the paraffin, touch it to the
paraffin, and transfer it to one side of the slide, while still warm, so that paraffin
covers both the edge of the coverslip and adjacent slide.
11. Repeat until all four edges of the coverslip are sealed with paraffin; reheat the
paper clamp, if necessary, to seal any gaps in the paraffin coating.
12. Seal the slide again using clear fingernail polish so that a slight band of polish
extends from the coverslip, completely covers the paraffin, and also reaches to
the subtending slide.
13. This slide should last 3–7 days if no gaps are present in the fingernail polish
coating.
14. Any photographs should be taken as soon as possible to avoid recording
anomalies in the specimen due to preservation or heating. This is especially
true for the cytoplasm, which could be altered during preservation or storage.
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Chapter 6
Entomophthoromycota: A New Overview
of Some of the Oldest Terrestrial Fungi
Richard A. Humber
Phylogenetic Reclassifications
The All-Fungal Tree of Life (AFTOL) project (James et al. 2006) confirmed several
pertinent points about the traditional classification of the Entomophthorales: The
phylum Zygomycota is unsupportable and was subsequently divided by Hibbett
et al. (2007) into five phylogenetically distinct subphyla not assigned to any specific
phylum or phyla. Entomophthoroid fungi were shown to include Basidiobolus
(whose controversial placement is discussed below) and to occupy a unique and
special position at the bottom of the vastly diverse tree of terrestrial (nonflagellate)
fungi immediately adjacent to the flagellate fungal phyla Chytridiomycota,
Neocallimastigomycota, and Blastocladiomycota (Hibbett et al. 2007) that form the
“roots” of the fungal tree. It should be expected that entomophthoroid fungi, because
of their age and phylogenetic position, show some biological traits not shared with
the more recent and biologically homogeneous basidiomycetes and ascomycetes in
the subkingdom Dikarya in the “canopy” of this phylogenetic tree.
The most extensive phylogenetic studies of entomophthoroid fungi (Gryganskyi
et al. 2012, 2013a) analyzed more genes and taxa than any previous studies, vali-
dated the place of Basidiobolus in a monophyletic lineage within the traditionally
classified Entomophthorales (Humber 1989), and showed the entomophthoroid
fungi to be sufficiently divergent from all other fungi to merit their placement in a
new phylum (Table 6.1) (Humber 2012). While some poorly resolved taxa still
require more study, the six-family classification of these fungi (Humber 1989) was
R.A. Humber, Ph.D. (*)

USDA-ARS Emerging Pests and Pathogens Research Unit, Robert W. Holley Center for
Agriculture and Health, 538 Tower Road, Ithaca, NY 14853-2901, USA
e-mail: richard.humber@ars.usda.gov; rah3@cornell.edu

DOI 10.1007/978-3-319-29137-6_6
128 R.A. Humber
Table 6.1 Classification of fungi in the phylum Entomophthoromycota (Humber 2012)

Class Basidiobolomycetes
Order Basidiobololales
Family Basidiobolaceae Basidiobolus, plus two undescribed genera
Class Neozygitomycetes
Order Neozygitales
Family Neozygitaceae Neozygites, Apterivorax, Thaxterosporium
Class Entomophthoromycetes
Order Entomophthorales
Family Ancylistaceae Ancylistes, Conidiobolusa, Macrobiotophthora
Family Completoriaceae Completoria complens (monotypic)
Family Entomophthoraceae
Subfam. Entomophthoroideae Batkoa, Entomophaga, Entomophthora, Massosporab
Subfam. Erynioideae Erynia, Eryniopsis, Furia, Orthomyces, Pandora,
Strongwellsea, Zoophthorac
Family Meristacraceaed Meristacrum, Tabanomyces
a
Still requiring phylogenetic revision that will result in recognition of more than one genus
b
Keller and Petrini (2005) described the subfamily Massosporoideae for this genus; Humber
(2012) found insufficient bases to accept such a subfamily
c
Zoophthora remains phylogenetically supported; other genera in this subfamily may or may not
be retained after more complete phylogenetic analyses, but how many or which genera will be
sustained remains unclear
d
Humber (1989) included Ballocephala and Zygnemomyces in this family, but subsequent evi-
dence supports their reassignments from Entomophthorales to the unclassified subphylum
Kickxellomycotina (see Humber 2012)
confirmed, although the families were redistributed among three new classes—
Basidiobolomycetes, Neozygitomycetes, and Entomophthoromycetes—each of
which includes a single order (Basidiobolales, Neozygitales, and Entomophthorales).
Basidiobolus has phylogenetic and biological properties that have led it to be
treated variously as removed from Entomophthorales but nebulously related to
flagellate fungi (Nagahama et al. 1995) to be placed (correctly) among the ento-
mophthoroid fungi (James et al. 2006). The genome-based retention of
Basidiobolus in the Entomophthoromycota may remain controversial for some
time to come since Basidiobolus (and its close relatives) represent a conundrum
for the understanding of the correct phylogenetic relationships of the most basal
taxa of terrestrial (nonflagellate) fungi. Despite any uncertainties about interpret-
ing the affinities of Basidiobolus from data for a tiny handful of genes, this genus
and its close relatives appear to be indisputably correctly placed in the
Entomophthoromycota based on their more traditional developmental, cytologi-
cal, and general biological characters (Humber 2012). The similarities between
basidiobolomycete and all other entomophthoroid fungi seem to be far greater
than can be accounted for by convergent evolution with phylogenetically unre-
lated (flagellate) fungi.
6 Entomophthoromycota: A New Overview of Some of the Oldest Terrestrial Fungi 129
Major Characters Recognized to Have Traditional

Taxonomic Value
Fungi in the Entomophthoromycota are easily differentiated from all other fungi
by their broad hyphae that are coenocytic to infrequently septate (except in
Basidiobolomycetes, whose broad hyphae have distinctly uninucleate cells) and
production of forcibly discharged conidia with a strong tendency to produce sec-
ondary conidia that are also forcibly discharged. These fungi are best known for
their entomopathogenic habits, but these fungi show a range of habits from saprobic
to obligately pathogenic for insects, other arthropods, or even nematodes, as well
as two uncommon phytoparasitic genera, Ancylistes and Completoria, known from
desmid algae and the gametophytes of ferns, respectively. The most phylogeneti-
cally basal class, Basidiobolomycetes, is primarily saprobic, although Basidiobolus
species can cause mycoses of humans and other mammals, and a related (still unde-
scribed) genus is known only from mycoses of snakes in Europe and the USA.
The long-accepted taxonomy for entomophthoromycotan genera has been based
on a range of traditional characters (Humber 1981; Gryganskyi et al. 2012) such as
the morphologies of primary and secondary conidia, the modes of conidial dis-
charge and dispersal, nuclear cytological characters (nuclear appearance in inter-
phase, mode of mitosis, and the number of nuclei in primary conidia), conidiophore
branching, and on the presence and morphologies of rhizoids and cystidia. Resting
spores (RS), whether formed as zygospores or azygospores, have comparatively
limited taxonomic value (Humber 1981).
Mitotic/Nuclear Characters
Variations in mitotic patterns were once hoped to be used to discover phylogenetic

relationships (e.g., Heath 1978), but those hopes withered in by the end of the last
millennium. The rise of DNA-based phylogenetics, however, brought a new appre-
ciation of nuclear cytology and mitotic patterns as newer DNA-based revisions of
many fungi became available. The ancient fungi in Entomophthoromycota do, how-
ever, underscore the value of nuclear characters that remained unrealized for the
younger fungi in Ascomycota and Basidiomycota.
Among entomophthoroid fungi, the diverse mitotic patterns and types of mitotic
nuclear-associated organelles (NAO) observed in Basidiobolus (Robinow 1963;
Tanaka 1970; Sun and Bowen 1972; McKerracher and Heath 1985), Ancylistes
(Moorman 1976), Entomophaga (Murrin et al. 1984), Strongwellsea (Humber
1975), Pandora (Butt and Beckett 1984), and Neozygites (Butt and Humber 1989)
are now seen to represent distinctive cytologies that help to differentiate the classes
in this phylum. The Basidiobolomycetes have large nuclei with huge central nucle-
oli, numerous tiny chromosomes that align on a central metaphase plate in nuclei
whose nuclear envelopes dissociate during mitosis but whose fragments and
130 R.A. Humber
cytoplasmic membranes isolate the nucleoplasm from the cytoplasm; these fungi
have an enigmatic NAO that is unique among all nonflagellate organisms in having
embedded microtubules (McKerracher and Heath 1985). The Neozygitomycetes
have closed mitoses (inside intact nuclear envelopes) with no known NAO and with
vermiform chromosomes aligning on a central metaphase plate (Butt and Humber
1989). Entomophthoromycete nuclei are more variable, but all pre-anaphase spin-
dles seen so far are so small that they are necessarily laterally located until elonga-
tion during anaphase; chromosomes in this class vary from not obviously condensing
during mitoses (in Ancylistaceae) to exceptionally large chromosomes in
Entomophthoraceae (see Gryganskyi et al. 2013a) that remain partially condensed
and present a granular appearance during interphase as seen by light microscopy
with or even without using such nuclear stains as aceto-orcein.
Entomophthoromycotan Taxa Needing Further

Revisionary Study
The latest multigenic phylogenetic studies of these fungi (Gryganskyi et al. 2012,
2013a) included a much greater range of taxa than earlier such studies, but the
extraction of cleanly amplifying DNA from some entomophthoroids appears to be
more difficult than for most other fungi. The problem of generating good PCR data
is intensified because so many of these fungi have never been cultured, and many
critically important taxa of this phylum are very rare and poorly known. It may also
be germane that entomophthoraceous nuclei are so large (larger than many yeast
cells), contain large amounts of condensed chromatin during interphase, and have
total quantities of DNA much larger than in many other fungi (Murrin et al. 1986).
The total genome sizes for Basidiobolus meristosporus and Conidiobolus incon-
gruus (listed at http://www.ncbi.nlm.nih.gov/genome/browse) are large in compari-
son to other fungi. Polyploidization may have occurred often among fungi in this
phylum (Humber 2012) and might account for some of the large total genome sizes.
However, until the whole genomic sequences of several entomophthoroids can be
compared with the more intensively studied ascomycetes and basidiomycetes, we
may never understand just how unique or anomalous the genomes of entomophtho-
romycotan fungi may actually be.
Basidiobolomycetes The unprecedented nature (and meaning) of the mitotic NAO
with embedded singlet microtubules (McKerracher and Heath 1985) and the dem-
onstrated similarities between some Basidiobolus genes and those of flagellate
fungi need further evaluation. Basidiobolus urgently needs more phylogenetic study
to determine whether the isolates that cause vertebrate mycoses are conspecific with
its saprobic species. At least two undescribed genera—one from plant detritus and
another from mycoses of snakes—whose overall biology, morphology, develop-
ment, and nuclear cytology place them indisputably in this class need more evalua-
tion and documentation before their formal descriptions can be published.
Neozygitomycetes That this group has long been difficult to place among ento-
mophthoroid fungi is not aided by the extreme challenge of obtaining clean DNA
from them. Neozygites species are not common and are among the most difficult to
culture of all entomophthoroid fungi. To date, the only in vitro cultures from this
genus have been of mites pathogens (mostly N. floridana and N. tanajoae); neither
N. fresenii (the generic type species) nor other aphid or hemipteran pathogens are
available in vitro. These technical difficulties with Neozygites species are more
troubling since traditional taxonomic approaches suggest a probable need to split
Neozygites into two genera for the hemipteran and acarine species, respectively.
The genera Thaxterosporium Ben-Ze’ev and Kenneth (Ben-Ze’ev et al. 1987) and
Apterivorax Keller (Keller and Petrini 2005) are also poorly known and remain
unavailable in culture; without any genomic information, the taxonomies of all of
these genera (and their species) remain unverified.
Entomophthoromycetes: Ancylistaceae: Conidiobolus This genus may be the most

widely distributed and commonly occurring in the phylum but has needed taxo-
nomic revision for a very long time. Its first two described species—C. utriculosus,
the type species, and C. minor—have not been re-collected since their description
(Brefeld 1884) and must either be neotypified or the genus must be conserved with
some other extant species. Beyond these nomenclatural issues, the last taxonomic
treatment of this genus (King 1976a, b, 1977) and the descriptions of three sub-
genera separated by secondary conidial characters (Ben-Ze’ev and Kenneth 1982a)
have resolved none of the enigmas plaguing this genus. Recent molecular evidence
(Gryganskyi et al. 2012, 2013a) does not support the subgenera of this genus but
do confirm that this genus includes several phylogenetically distinct lineages.
The systematics of Conidiobolus cannot be resolved until its typification is set-
tled and a determination made about which lineage will continue to bear the name
Conidiobolus.
Entomophthoromycetes: Entomophthoraceae: Zoophthoroid genera (subfamily

Erynioideae) The segregation of genera in this group whose species form uninu-
cleate bitunicate conidia on digitately branched conidiophores (except for simple
conidiophores in Strongwellsea) began with recognition of four subgenera in
Zoophthora (Batko 1966) that were later treated as genera (Humber 1989) and aug-
mented with the genera Strongwellsea (Batko and Weiser 1965) and Orthomyces
(Steinkraus et al. 1998). Whether all of the Batkoan subgenera should be recognized
as separate genera (compare treatments of these taxa by Remaudière and Hennebert
1980; Remaudière and Keller 1980; Ben-Ze’ev and Kenneth 1982b; Humber 1982,
1989; Bałazy 1993; Keller and Petrini 2005) remains controversial: The available
phylogenetic data (Gryganskyi et al. 2012, 2013a) confirm the recognition
Zoophthora for species producing elongate secondary capilliconidia. Too few
genomic data are available for Strongwellsea species, marginally too few for the
diversity of Pandora species, and distinctly too few for Furia or Erynia species, and
absolutely no gene-based data are available for Orthomyces aleyrodis to support
any phylogenetically sound conclusions about genera within this subfamily. It
132 R.A. Humber
seems that still more genes have to be analyzed before relationships among these
fungi can be fully determined.
Miscellaneous Other Poorly Known Taxa No fully comprehensive and stabi-

lized phylogenetic revision of the Entomophthoromycota will be possible without
more pertinent biological and gene-based information about some of the uncultured
and extremely rare taxa in this phylum including the following:
• Two new genera of Basidiobolaceae from plant detritus or soil and from snakes
await formal description. Publications about the snake pathogen include Ippen
(1980), Jessup and Seely (1981), Kaplan et al. (1983), and Dwyer et al. (2006).
• The monotypic family Completoriaceae, Completoria complens (Fig. 6.1; also
see Atkinson 1895), parasitizes fern gametophytes and strongly resembles some
entomophthoraceous entomopathogens in having apparently protoplastic vege-
tative growth and physically large nuclei that appear to have granular contents
during interphase (presumably due to condensed chromosomal material, as in
the Entomophthoraceae).
• The taxa in the Meristacraceae (Humber 1989) form multiple conidia on an
unbranched erect conidiophore but remain little understood and rarely collected.
Meristacrum asterospermum is a rarely seen nematode pathogen (Drechsler
1940), and Tabanomyces milkoi (Couch et al. 1979) was based on a single collec-
tion of tabanid fly larvae. Ballocephala Drechsler and Zygnemomyces Miura
Fig. 6.1 Completoria complens in cells of fern gametophyte. These first published micrographs of
this fungus are of the CUP herbarium collection illustrated and discussed by Atkinson (1895). (a,
b) Apparently wall-less extrusions (arrows) of hyphae from one infected cell filled with vegetative
hyphal bodies through narrow holes in cell walls into adjacent cells. (c, d) Vegetative hyphal bod-
ies in gametophyte cells showing nuclei; cells on right clearly showing large nuclei (at higher
magnification in 1d) with granular contents reminiscent of granular heterochromatin in nuclei of
Entomophthoraceae. (e) Mature, thick-walled resting spores are binucleate (asterisk)
affect tardigrades and strongly resemble Meristacrum, but these genera have
been reassigned to the Zoopagomycotina because of the ultrastructure of their
septa. Both Meristacrum and Tabanomyces (see Humber 1989) need reevalua-
tion and confirmation of their affinities whenever suitable collections become
available.
Nutritional Habits and Host Range
The entomophthoroid fungi exemplify the three main nutritional habits of fungi:
Saprobes depend mostly of nonliving sources of carbon and other nutrients,
although some taxa that are primarily saprobic may act as facultative parasites and
cause mycoses of medical or veterinary importance. Parasites such as species in
the plant-associated genera Ancylistes (from desmid algae) and Completoria (from
fern gametophytes) infect living hosts but do not necessarily cause the host’s death
(Completoria) or do not seriously affect the host population (Ancylistes). The ver-
tebrate mycoses caused by Basidiobolus and Conidiobolus species are also exam-
ples of parasitism. Pathogens infect, develop internally, and later kill their hosts.
Entomopathogenicity—broadly defined as causing fatal disease for insects, mites,
spiders, and, indeed, such other invertebrates as nematodes—is the most impor-
tant (and best known) nutritional habit for the Entomophthoromycota. This habit
appears to have arisen independently and repeatedly among water molds (espe-
cially in Blastocladiomycota), Entomophthoromycota (with obligate entomopatho-
gens occurring in two of the three classes and four of the six families), and among
several phylogenetically diverse classes and orders of Ascomycota, as well as in the
zoophilic rust-like basidiomycetes in the order Septobasidiales (see Humber 2008).
A fascinating and important more general study on fungal evolution (Moore 2013)
also suggests that parasitism is a nutritional habit that has arisen easily and often
among fungi.
The phylogenetic data (Gryganskyi et al. 2012, 2013a) suggest that the
Basidiobolomycetes are basal in this phylum, followed by the Neozygitomycetes
and Entomophthoromycetes, successively. With the exception of the few basidiobo-
lomycete fungi that cause vertebrate mycoses, this class appears to be primarily
saprobic in soil and plant detritus. Even Basidiobolus ranarum—the type species
for its class, whose classic “source” is amphibian dung—is a saprobe in soil that is
easily acquired (probably carried on the cuticles of insects that are also not diseased
by this fungus), is ingested, and survives in the guts of amphibians or reptiles with-
out causing disease.
The mainly saprobic species of Basidiobolus and Conidiobolus from soil or plant
detritus have simple nutritional needs, enabling them to grow on almost any culture
medium. Nonetheless, isolations of these fungi, despite their generalized nutritional
needs, are unlikely to succeed unless using a “canopy plate” technique (Drechsler
1952) in which small amounts of detritus or soil are minced into a small amount of
nutrient agar on a petri dish lid to allow the entomophthoroid fungus to develop and
to discharge its conidia forcibly either upward (or, with a slightly higher chance of
134 R.A. Humber
contamination, downward) onto the main agar surface. Some taxa of both of these
genera are known to cause mycoses of humans or other vertebrates (see Humber
et al. 1989). A small number of Conidiobolus species (especially C. thromboides
and C. obscurus) are best known as entomopathogens, although C. thromboides was
first described as a soil saprobe (Drechsler 1953). Macrobiotophthora affects nema-
todes, and all species of Ancylistes parasitize desmid algae.
All taxa of the Neozygitomycetes and Entomophthoraceae are obligate patho-
gens of arthropods. Within the genus Neozygites, however, there may be a taxo-
nomically significant disjunction between species with smooth, ovoid zygospores
that affect aphids and related hemipterans versus those with globose, rough-walled
zygospores affecting mainly mites. There are too few cultures (all of which are from
acarine hosts) or fresh collections or in vivo colonies (especially from hemipteran
hosts) to allow a phylogenetic evaluation of whether these differences support sepa-
rate genera.
The specificities of pathogenic entomophthoroid genera for their hosts are often
generalized to the order or family of their hosts; individual fungal species may show
higher levels of specificity to their host genera or species. A very common ento-
mophthoroid entomopathogen, Zoophthora radicans, may represent an unresolved
species complex that is known from hosts in an extremely broad range of insect
orders (see Bałazy 1993). Pandora neoaphidis, on the other hand, is another cosmo-
politan and common species on a very large diversity of aphids, but P. neoaphidis
does not appear to be a species complex (Nielsen et al. 2001). The species of
Massospora are completely specialized to their unique gregarious cicada hosts
(Soper 1974), and Strongwellsea species are also apparently specific to their host fly
genera (Humber 1976; Eilenberg and Michelsen 1999).
Infection
Entomophthoroid entomopathogens work against their hosts in a manner very much

like almost all other fungal entomopathogens (Humber 2009, 2011): The infection
process begins with the adherence of infective spores, a primary or secondary
conidium, onto the host cuticle; no entomophthoroid fungi infects any invertebrate
by means of an ingested spore germinating and having the germ tube penetrate the
gut wall. After landing on a potential host or any other substrate, the complex physi-
ological interactions of the infection process begin with the spore’s reaction to its
substrate by germinating, forming a secondary conidium, or doing nothing. The
germ tubes of these fungi may grow across the cuticle (with the cytoplasm always
in the apex of a growing but empty, frequently septate germ tube), possibly growing
toward higher nutrient levels that might leach through thinner areas of the cuticle or
from spiracles, or it may almost immediately turn down to begin to penetrate that
cuticle. Few entomopathogens from this phylum form any differentiated appresso-
rium before growing into the cuticle. Some melanotic host reactions may be induced
in the cuticle by fungal germ tubes (Humber 2009), but the most decisive
fungus–host interactions occur when the invading fungus reaches the hemocoel and
confronts the full force of the host’s humoral and/or cellular immune reactions
(Boucias and Pendland 1998; Humber 2000, 2009). No infection is truly established
until the pathogenic fungus and all host responses are exhausted or evaded and fun-
gal vegetative development has begun. The usual outcome of that growth in the host
will be the death of the host and production of primary conidia or of thick-walled
resting spores. The variations on this quick scenario are too numerous to be dis-
cussed in detail here.
All germ tube growth depends on nutrients and other components packaged into
spores at the time of their formation. A dramatic morphological and physiological
transition occurs at the physical point when an entomophthoroid cell converts from
the exclusive use of internal nutrient resources (in germ tubes) to initiate vegetative
growth as marked by the complete switch to using external nutrient sources (see
Manners 1966).
No fungal infection has been successfully established until the fungus has over-
come or evaded all of the host’s immune responses. One great advantage for many
entomophthoroid entomopathogens to grow at least initially (if not throughout all of
vegetative development) as wall-free protoplasts appears to be the evasion of host
immune responses (Butt et al. 1996; Humber 2009).
The infective unit for most entomophthoroid fungi is either the primary or sec-
ondary conidia. However, there seem to be separate roles for the primary and sec-
ondary conidia as dispersive and infective forms, respectively, in some taxa; a
major clue to this distinction is that some primary conidia routinely begin to form
secondary conidia almost immediately after discharge. Neozygites species almost
always immediately form secondary capilliconidia with a distinctive apical mucoid
droplet whose sole purpose is to attach the capilliconidium to a passing host; the
primary conidia in this genus are not discharged to any great distance from the host
and only very rarely form forcibly discharged secondary conidia. Species of
Zoophthora may form forcibly discharged secondary conidia but show a very strong
tendency to form secondary capilliconidia soon after the discharge of the primary
conidia. Entomophthora species show not only an immediate production of second-
ary conidia from the primaries, but their conidial morphologies and modes of dis-
charge are also distinct. The secondary conidia of Entomophthora are discharged by
papillar eversion, but the primary conidia are affixed to the substrate by cytoplasmic
contents discharged from the conidiogenous cell as part of a cannon-like discharge
mechanism. However, it is necessary to acknowledge that there are conflicting
interpretations of the mucoid material in which Entomophthora primary conidia are
embedded (Humber 1981; Eilenberg et al. 1986).
Mycoses of humans or other vertebrates can be caused by some entomophtho-
roid fungi. These mycoses are comparatively rare, occur mostly in tropical and
subtropical locations, and are generally caused by only a handful of Conidiobolus
and Basidiobolus species. Humber et al. (1989) offer the most biologically sensible
hypothesis about the ontogenies of the primarily nasopharyngeal conidiobolomyco-
ses of humans, equines, and other mammals. Basidiobolomycoses are less common
but have a greater tendency to become either systemic in humans or occur as
136 R.A. Humber
disseminated subcutaneous infections not affecting the head as is the case with most
conidiobolomycoses. An undescribed genus in Basidiobolaceae sometimes referred
to (but still not formally described) as Schizangiella is reported only from captive
snakes in Europe (Ippen 1980) and North America (Jessup and Seely 1981; Kaplan
et al. 1983; Dwyer et al. 2006) and grows vegetatively with a unique pattern of
repeated internal cleavage of globose cells into clusters of up to 16 cells by mutu-
ally perpendicular series of cell divisions that recall the yeastlike (but microaero-
philic or anaerobic) darmform growth pattern of Basidiobolus cells. It seems highly
likely that this snake pathogen is a soil- or detritus-borne saprobe that can become
a facultative subcutaneous to systemic pathogen of snakes after inoculation through
skin abrasions or cuts.
Development
Established infections by entomophthoroid fungi typically grow in the hemocoel of

the host as hyphal bodies (short hyphal segments) that multiply rapidly and circu-
late freely throughout the body cavity. Hyphal bodies are short structures whose
morphologies and general behaviors will depend, among other factors, on whether
they are walled or protoplastic rather than growing as long, continuous walled (or
protoplastic) hyphae. Even wall-less hyphal bodies, however, can vary dramatically
in their appearance from being rodlike and more or less cylindrical throughout their
length to highly amoeboid shape-shifting cells typical of Entomophaga species
affecting lepidopterans that were first reported by Tyrrell and Macleod (1972) to
much more rodlike cells in many genera (Humber 1975; Latgé et al. 1988; Butt
et al. 1990). Protoplastic development by entomophthoroid fungi is not universal—
and is not known for any taxa in Basidiobolomycetes or Ancylistaceae—but occurs
in a wide range of taxa. Some Neozygites species may develop in their hosts (and in
culture) as protoplasts. In the Entomophthoraceae, protoplasts are known from
Entomophthora, Entomophaga, and Massospora but not from Batkoa species within
the subfamily Entomophthoroideae; among the zoophthoroid genera (Erynioideae),
protoplasts are known from at least the early developmental stages (vegetative pro-
liferation up to the point where the host is moribund or dead, and the fungus is
preparing itself for reproduction) in the genera Strongwellsea and Pandora but may
be absent (or uncommon) in Zoophthora and remain unconfirmed from the other
genera of this subfamily. Curiously, vegetative development by the fern-parasitic
Completoria complens clearly appears to be protoplastic; the fungus spreads from
cell to cell injecting highly irregularly shaped cells through tiny holes in the adja-
cent cell walls (Fig. 6.1; also see Atkinson 1895).
Entomophthoroid entomopathogens mostly follow a common developmental
pattern in their hosts involving proliferation and dispersal of cells throughout the
body in the hemocoel. Few entomophthoroid entomopathogens grow primarily as
longer hyphae (rather than hyphal bodies) throughout their development. Significant
spatial restriction of development, usually to the abdomen, is limited mainly to
Massospora and Strongwellsea species. In the case of Strongwellsea, whose species
affect muscoid fly adults, vegetative development is as protoplastic hyphae that

eventually form a hollow ball in the abdomen, become walled toward the interior of
that ball so that (walled) conidiophores line the interior of the hollow ball, and dis-
charge conidia through an expanding hole in the abdominal pleuron of living flies
(Humber 1976; Eilenberg and Michelsen 1999). Despite the major development of
Strongwellsea in the abdomen, the hyphae can interpolate themselves into the host’s
nervous system to proliferate in the thoracic ganglion and even throughout the brain
while few if any hyphae are found in the thoracic or cephalic hemocoel (Humber
1975, 1976). Massospora species, all of which are known only from gregarious
cicadas, produce protoplastic hyphal bodies but are spatially restricted entirely to
the terminal (gonadal) segments of the abdomen that are separated from the rest of
the host’s body by a membrane (Speare 1921).
Entomophthoroid development in invertebrate hosts usually remains in the
hemocoel until the fungal growth occludes that space and blood circulation is
impeded. The host is moribund by this time, and the fungus then begins excreting
enzymes that digest the host’s organs; all of these events—nutrient starvation, loss
of excretory clearance of now rapidly accumulating cellular breakdown products,
and loss of circulation and of oxygenation of the host’s body—cause the host’s
death. By this time, any fungal protoplasts will have acquired walls, and walled
hyphae begin penetrating through the cuticle immediately prior to external conidio-
genesis or, mostly inside the host body, to produce the thick-walled resting spores.
Arthropods killed by entomophthoroid fungi may produce rhizoids and/or cys-
tidia shortly before or soon after death. These two types of specialized cells emerge
before the conidiophores; their morphologies and taxonomic significance were dis-
cussed by Humber (1981). Rhizoids descend to the substrate from the ventral surfaces
of the host and frequently form some type of terminal holdfast to anchor the host.
Cystidia emerge ventrally and facilitate the passage of developing conidiophores by
their multiple perforations of the host cuticle. Except for extending far beyond the
level of the conidiogenous hymenium that will form on the surface of infected host
cadavers, cystidia (much like rhizoids) may be little differentiated from vegetative
hyphae in their thickness or may (e.g., in Erynia species) be markedly thicker and
may have characteristic apical branchings (Thaxter 1888; Humber 1981).
The only entomophthoroid entomopathogens that sporulate from living hosts are
the species of Strongwellsea and Massospora, as well as Entomophthora erupta
(Hall 1959).
Reproduction
Conidiogenesis and Dispersal
Zygomycete propagules were formerly always considered to be sporangiospores—

spores formed inside a cell (sporangium) whose cytoplasm is repackaged into one or
more sporangiospores, each of which acquires a newly generated cell wall. However,
138 R.A. Humber
the thin-walled (usually) forcibly discharged spores of Entomophthoromycota and

also the asexual spores of some taxa of Zoopagomycotina (Saikawa 2011) are not
sporangiospores: The wall layers covering these spores are identical to those of
the cells producing them (Humber 1975, 1981; Benny et al. 2001); no new wall is
generated inside a parental cell wall, and these spores must be treated as conidia
formed in the same manner as the mitospores of innumerable ascomycete taxa. In
slide preparations of uninucleate conidia from taxa in the subfamily Erynioideae
(Entomophthoraceae), a wall is often seen to separate and to lift away from the spore
surface; although this appearance suggests these spores might be monosporic sporan-
gioles, transmission electron microscopy of such “bitunicate” conidia (Remaudière
and Hennebert 1980) confirms that these are conidia with the same wall layers of all
other entomophthoroid cells except for having an outer (electron dense) wall layer
that is extensible and easily detachable from the inner (electron lucent) layer.
On arthropod hosts with comparatively thick cuticles (e.g., flies, grasshoppers,
beetles, etc.), the conidiophores of entomopathogens emerge through the thinner
cuticle of intersegmental membranes of the abdomen, around the eyes and mouth-
parts, from leg joints, or in other thin sections of cuticle to form densely packed
hymenial bands of conidiogenous cells. On hosts with comparatively thin cuticles
(e.g., aphids and some lepidopterans), the conidiophores often form hymenia com-
pletely covering the host cadaver. The conidiophores are unbranched (or, rarely,
sparingly branched well below the conidiogenous apices) in all taxa except those in
the subfamily Erynioideae (Entomophthoraceae), in which conidiophores are digi-
tately branched just below the conidiogenous cells (although the conidiophores of
Strongwellsea remain simple). The primary and also secondary conidiophores of
many of these fungi are highly phototropic and orient both the production and dis-
charge of conidia toward a light source (Page and Humber 1973). Positive photot-
ropism may be very important for the dispersal of saprobes growing in soil or plant
detritus (to get the spores discharged freely into the air) but may also be observed in
the cultures of many entomopathogenic taxa.
Several different mechanisms for active discharge of conidia are found within
the phylum, but the most common and important is that of papillar eversion (see
Humber (1981) for a characterization of all of the conidial discharge mechanisms in
this phylum), in which the septum between the conidiogenous cell and conidium
forms by the growth of the inner layer of the cell wall and projects into the develop-
ing conidia (possibly because cytoplasm is being transferred hydraulically from the
conidiogenous cell into the developing conidium while this septum is developing).
Once the septum is closed, very nearly all cytoplasm has been transferred from the
conidiogenous cell into the conidium. The hydrostatic pressure builds inside the
conidium (possibly driven by the hydrolysis of some storage products) and is sub-
sequently relieved by the sudden eversion of this papilla in a manner that breaks the
outer cell wall layer so that the spore flies away to a considerable distance. Two
other modes of primary conidial discharge are found in the genera Basidiobolus and
Entomophthora; differing hypotheses about how primary conidial discharge is
accomplished in Entomophthora species are discussed by Humber (1981) and
Eilenberg et al. (1986).
Secondary Conidiogenesis
Secondary conidiogenesis is a key character of Entomophthoromycota. Primary

conidia formed by the developing vegetative fungus are forcibly discharged (except
in Massospora), and, if they land on substrates unsuited to forming germ tubes (or
are preprogrammed not to germinate), the primary conidium produces one (or more)
of several possible types of secondary conidium that were classified by Ben-Ze’ev
and Kenneth (1982a) according to their morphologies and modes of formation.
Forcibly discharged secondary conidia formed singly on short conidiophores are the
most typical for these fungi; the formation and phototropically oriented discharge of
these conidia was illustrated and discussed in detail by Page and Humber (1973).
The production and forcible discharge of multiple secondary “microconidia” from
single primary conidia are seen in several species of Conidiobolus and one of
Basidiobolus. The production of a single, passively dispersed capilliconidium on a
long, slender capillary conidiophore is a key character of species of Zoophthora
(Ben-Ze’ev and Kenneth 1982a, b), but a somewhat variant morphology in the com-
panion zoophthoroid genus Orthomyces (Steinkraus et al. 1998) suggests that the
slightly papillate secondary conidium in Orthomyces may, in fact, be forcibly dis-
lodged to fall from the capillary apex, whereas all other capilliconidia produced
within the phylum are passively dispersed (from much narrower capillaries on
which the secondary capilliconidia have no visible basal papilla). Other forms of
secondary capilliconidia with various orientations on the conidiophores and with or
without terminal mucoid droplets to aid attachment to a passing host are seen in
species of Neozygites, Basidiobolus, some Conidiobolus species, and also in the
nematode-pathogenic genus Macrobiotophthora—in which short, flexible capillary
conidiophores are caught and rapidly bent by surface tension in the fluid layer on a
contacting nematode and are literally slapped onto the cuticle of the unfortunate
nematode (Humber, unpublished).
A bit more about the production of passively dispersed secondary capilliconidia
on long, capillary conidiophores is justified. This mode of sporogenesis does not
appear to be an ancestral state for the Entomophthoromycota. While capilliconidio-
genesis may be uncommon elsewhere, it is known for the ascomycete entomopatho-
gen Hirsutella aphidis (Bałazy 1985). There is a good reason to believe that the
formation of such secondary conidia in four families (Basidiobolaceae,
Neozygitaceae, Ancylistaceae, and Entomophthoraceae) and all classes of the phy-
lum suggests that this mode of sporogenesis has arisen multiple times. It probably
arose easily as a modification of forcibly discharged secondary conidiogenesis with
the secondary conidium forming at a greater height (to aid dispersal to possible
hosts) with the consequence that elongating conidiophores necessarily became
increasingly narrower, eventually too narrow to allow forcible discharge by papillar
eversion. The hypothesized forcible dislodgement of minutely papillate secondary
capilliconidia of Orthomyces aleyrodis from unusually broad capillaries (whose
apices also show a correspondingly tiny eversion like that seen with discharge by
papillar eversion) seems to support this supposition (Steinkraus et al. 1998).
140 R.A. Humber
Specialized Adaptations of Conidiogenesis in Entomophthorales
Species of diverse entomophthoraceous fungi infecting aquatic larvae of nematoc-

eran dipterans (especially Simuliidae) or their female adults (that may become
waterlogged during oviposition on moss-covered rocks in splash zones and die soon
thereafter while drying off on the backsides of these rocks) may produce conidia
with several stout arms projecting forward from the apex of primary conidia or
backward from around the papillae of secondary conidia; these spores are referred
to as “coronate” or “stellate” conidia, respectively (Descals et al. 1981; Descals and
Webster 1984). These branched conidia so not appear to promote spore dispersal (as
for the “tetraradiate” conidia of many waterborne ascomycetes) but rather to retard
or to prevent their dispersal from the site where susceptible, healthy hosts can be
infected by the small, globose, forcibly discharged secondary (or tertiary) conidia
discharged from these modified conidial forms.
Cryptoconidiogenesis (Humber and Ramoska 1986) is an unexpected variation
on entomophthoroid fungal sporulation that confirms many basic principles about
reproduction by these fungi. Populations of spur-throated grasshoppers, Melanoplus
spp. (Acrididae: Melanoplinae), may support large epizootics of Entomophaga
calopteni, but all infected grasshoppers appear to produce thick-walled resting
spores without any customary conidial state. How such a fungus could spread dis-
ease in its host populations without producing a forcibly discharged conidia (since
the resting spores are not involved in horizontal transmissions) remained unan-
swered until it was noted that hosts infected by this fungus often showed marked
stretching of the abdominal intersegmental membranes. When these thinned and
very fragile membranes rupture by the movements of other grasshoppers or by can-
nibalism of cadavers, the masses of hyphal bodies that had not (yet) produced rest-
ing spores are exposed to full atmospheric oxygen levels, and these hyphal bodies
can produce forcibly discharged primary conidia directly without ever forming any
“normal” conidiophores. These unexpected spores formed by cell types not known
for such an ability once supplied with sufficient oxygen were referred to as crypto-
conidia. The critical role of oxygen was proven because exposed populations of
hyphal bodies from infected hosts produced no cryptoconidia when enclosed in jars
with a piece of burning paper (resulting in depleted oxygen and augmented carbon
dioxide) or when incubated in pure nitrogen gas (Humber and Ramoska 1986).
Resting Spores and Sexuality in Entomophthoromycota
Entomophthoroid fungi challenge many mycological stereotypes, and the durable

resting spore states of these ancient fungi pose some especially difficult challenges
(Humber 1981, 2012). The locations and importance of sexuality in the life his-
tories of ascomycete and basidiomycete fungi are clear. Meiosis occurs in asci or
basidia; ascospores and basidiospores are haploid meiospores; and the overall life
histories of these fungi are understood. The thick-walled durable spores produced
by zygomycete fungi are, however, more problematic. Classic studies by Cutter
(1942a, b) demonstrated several distinct patterns of sexuality among mucoroid
fungi that produce thick-walled zygospores and azygospores. The thick-walled
spores of zygomycetous taxa, of chytrid and blastocladian fungi, and of oomyce-
tous straminopiles usually fit in the expected pattern of life histories, with these
durable spores being sexually derived and with meiosis occurring either during the
formation or the germination of these spores. Among the (formerly) zygomycete
fungi, the thick-walled durable spores are regarded to be zygospores (if formed
after a gametangial conjugation) or azygospores (if formed without any gametan-
gial conjugation), and it is almost always assumed that zygospores are sexual
spores while azygospores are not.
These morphological definitions of sexuality, unfortunately, ignore the issue of a
diametrically opposed definition of sexuality based on genetic events in a life his-
tory: The genetic definition of sexuality requires the alternation of meiosis and kary-
ogamy in a life history. In almost every well-known and widely studied group of
organisms, there is no separation of the morphological and genetic definitions of
sexuality, and most biologists have fallen into the conceptual trap of expecting the
distinction between these two definitions to be trivial or nonexistent. The fungi in
the Entomophthoromycota—especially the numerous and highly diverse fungi in
the Entomophthoraceae (see Table 6.1)—both set and spring this trap on the unwary.
These fungi shine a strong light onto the need to be aware of these disparate defini-
tions of sexuality and on the role of sexual reproduction in phylogenetic radiation:
Unlike nearly all other fungi, homothallism is the observed rule in
Entomophthoromycota. Heterothallism, with its obligatorily outcrossed pair-
ings of disparate mating types, has never been definitively demonstrated in
Entomophthoromycota. Only very indirect, poorly supported suggestions (e.g.,
Gryganskyi et al. 2013b) exist that outcrossing might occur among these fungi.
McCabe et al. (1984) showed that there is a more or less free mixture of the two
important morphological and developmental hallmarks of sexuality within
Entomophthorales—of zygosporogenesis versus azygosporogenesis and of whether
nuclear numbers reduce to two or not resting spore maturation—with all four pos-
sible combinations of these two processes being demonstrable. The assumed pattern
that when mature resting spores are binucleate that the nuclei do fuse is reinforced
by the demonstration (McCabe et al. 1984) of a single, notably larger nucleus in the
germinating resting spore of Conidiobolus thromboides that was interpreted as a
possible zygotic nucleus that would undergo meiosis as germination proceeded; the
nuclei of all other cells of all entomophthoroid fungi are haploid.
If (a)zygosporogenesis in the Entomophthoromycota is entirely homothallic and
the involvement of gametangial conjugations immaterial, then other questions can
be raised: Do these fungi contain genetic suggestions of mating type genes or other
similar markers of outcrossing organisms? Are the fungi in Entomophthoromycota
the only fungi showing evidence for an ancient abandonment of (traditional) sexual-
ity like that demonstrated for bdelloid rotifers (Normark et al. 2003) in the animal
kingdom?
142 R.A. Humber
An alternative route to allow gene flow among organisms with known impor-
tance for many ascomycetes, for example, is parasexuality, in which fusions of
genetically different cells result in heterokaryons in which genetically dissimilar
nuclei may undergo fusions and some type of genetic exchange (whether by “mitotic
crossing-over” or meiosis in “unusual” places at “unusual” times). Parasexuality is,
however, a mechanism that may be wholly unavailable in the Entomophthoromycota:
No cell-to-cell fusions have been observed at any time among these fungi except
when forming zygospores, but even these fusions may be between genetically iden-
tical adjacent cells (e.g., as in Conidiobolus or adjacent uninucleate cells in
Basidiobolus) or, in an insect, between hyphal bodies that might be derived from a
single nucleus (e.g., in infections by zoophthoroid genera whose conidia are uni-
nucleate). Curiously, even in the protoplastic vegetative cells of entomophthoroid
taxa, there is no verified record of cellular fusions despite intimate or prolonged
cell-to-cell contacts. Dunphy and Nolan (1977) reported fusions in vitro of spindle-
shaped protoplasts of Entomophaga aulicae (as Entomophthora egressa), but this
report is now known to be a misinterpretation of hypertrophic swelling of single
protoplasts in nutrient-deficient liquid medium; cells that progressively become
large, highly multinucleate “spheroplasts” with gigantic central vacuoles will, when
transferred to fresh medium, spin off new fusoid, mobile protoplasts and disappear
from the culture.
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Chapter 7
Latest Developments in the Research of Rust
Fungi and Their Allies (Pucciniomycotina)
Merje Toome-Heller
Introduction
Pucciniomycotina is one of the three subphyla of Basidiomycota, known members of

which have disc-like spindle pole bodies and simple septal pores that lack pore caps and
dolipores (Bauer et al. 2006; Swann et al. 2001). Pucciniomycotina species are divided
into nine classes: Agaricostilbomycetes, Atractiellomycetes, Classiculomycetes,
Cryptomycocolacomycetes, Cystobasidiomycetes, Microbotryomycetes, Mixiomycetes,
Pucciniomycetes, and Tritirachiomycetes (Aime et al. 2014). Nearly all these fungi
are microfungi, ranging from psychrophilic or animal pathogenic yeasts to moulds,
various plant pathogens, and saprobes (Aime et al. 2006, 2014). There are also a few
sporocarp-forming species in Pucciniomycotina, which form either minute—up to
a few millimetres high—(some species of Atractiellomycetes, Agaricostilbomycetes,
and Microbotryomycetes) or resupinate fruiting bodies (members of Septobasidiales
and Helicobasidiales in Pucciniomycetes; Swann et al. 2001).
The vast majority of Pucciniomycotina species (ca. 7500 of nearly 8500) belong
to a single order Pucciniales (formerly Uredinales) exclusively containing the obli-
gate parasitic fungi that cause rust diseases of plants. While all rust fungi share rela-
tively similar life cycles, morphology, and biology, their allies, i.e. the remaining
members of Pucciniomycotina, are very diverse. The next largest orders, in terms of
species, are the entomophilic Septobasidiales (ca. 280 species) and the phytopatho-
genic anther smut fungi from Microbotryales (ca. 125 species). All other described
orders of Pucciniomycotina contain few known species, ranging from 1 to 70 (Aime
et al. 2014; Kirk et al. 2011) and the only other larger groups in Pucciniomycotina
are the polyphyletic anamorphic yeast genera Rhodotorula and Sporobolomyces,
which contain 162 and 93 known species, respectively.
M. Toome-Heller, Ph.D. (*)

Plant Health and Environment Laboratory, Ministry for Primary Industries,
Auckland 1140, New Zealand
e-mail: merje.toome@mpi.govt.nz

DOI 10.1007/978-3-319-29137-6_7
148 M. Toome-Heller
Plant pathological and systematic research of rust fungi has a long history, mostly
due to their economic significance in agriculture and forestry, and their easily
noticeable symptoms (Cummins and Hiratsuka 2003). In contrast, the remaining
members of Pucciniomycotina have been placed together relatively recently and
comparatively less is known about them (Aime et al. 2006, 2014; Bauer et al. 2006;
Swann et al. 2001). The availability of molecular research and identification tools
has greatly facilitated the research of Pucciniomycotina, especially the systematics
and species discovery of the anamorphic yeasts and rust fungi. Genome sequencing
has opened new opportunities to study the phylogenetic relationships within
Pucciniomycotina and to use rusts and their allies for better understanding the biol-
ogy of fungi in general (e.g. Ianiri et al. 2011; Raffaele and Kaumon 2012; Tavares
et al. 2014). Albeit, as is true for all groups of fungi (Blackwell 2011), a great deal
of research is still to be completed in both the areas of biology and biodiversity.
This chapter focuses on the latest developments in Pucciniomycotina research—
which are mainly in the area of biodiversity research, systematics, taxonomy, and
genomics—and on highlighting some of the challenges related to working with this
group. For further general information on this subphylum, please refer to previously
published overviews, which provide detailed information about the major groups of
Pucciniomycotina and their general characteristics (Swann et al. 2001; Aime et al.
2006, 2014).
Biodiversity
Species Discovery
While the majority of the species now classified in Pucciniomycotina were described
during the 1900s, new species discovery continues to be important for this group,
with 375 (or ca. 5 % of the total) new species described since year 2000. When
comparing the average new species members between the 2000s and the first half of
2010s, a trend towards a rise was detected as on average 21 new species were
detected for the first period and 27 for the latter (Fig. 7.1a). Although increased spe-
cies discoveries could be expected due to improved accessibility to molecular data
analysis and therefore greater ability to detect new to science and cryptic species,
the following years and further analyses should reveal whether the species discov-
ery is truly a rising trend in Pucciniomycotina or whether this has been an artefact
of the timing of publications.
Taxonomic distribution of the new species between Pucciniomycotina classes
followed greatly the distribution of already known species. More than 70 % of new
species belonged to Pucciniomycetes, the most species-rich class of Pucciniomycotina,
and around 17 % belonged to Microbotryomycetes, the second-most species-rich
class. Over half of the remaining species were determined to be members of
Cystobasidiomycetes, and a quarter belonged to Agaricostilbomycetes. The remain-
ing species (less than 2 % of total new species) belonged to Atractiellomycetes,
7 Latest Developments in the Research of Rust Fungi and Their Allies… 149
Fig. 7.1 Information about the new Pucciniomycotina species described between 2000 and 2015.
(a) The yearly total number of new species (black squares) and the 5-year average values (blue
bars). (b) Distribution of the number of new species between the nine Pucciniomycotina classes.
(c) The number of species by the type of organism. (d) Geographic distribution of the newly
described species
Tritirachiomycetes, and Cryptomycocolacomycetes, and no new taxa were added to

Classiculomycetes and Mixiomycetes (Fig. 7.1b). It remains to be seen if these
trends are in accordance with the actual number of Pucciniomycotina species in
nature or the smaller groups are just greatly under-sampled due to limited number of
researchers working on these groups and/or their preference for under-studied envi-
ronments. However, examining the rate at which new species are added to different
classes, a further increase in microfungal species discovery could be expected, espe-
cially those of the saprobic yeasts. Proportionally, the greatest contribution to the
total species numbers was made to Cystobasidiomycetes, of which over 50 % of
known species were discovered over the last 15 years. Nearly all those new additions
represent anamorphic yeast species, indicating that the discovery of the unicellular
Pucciniomycotina species could be on a rise.
Although rust fungi comprise the most thoroughly studied group of
Pucciniomycotina, a considerable number of new species are still described for this
group every year. In fact, more than half (i.e. 234 species) of all the new
Pucciniomycotina species discoveries made since 2000 were for rust fungi. These
novelties were from 41 different genera, ten of which represent new genera, and
therefore entirely new genetic lineages. Most of the new rust fungi species were
described in the genera Puccinia (66 new species; Abbasi et al. 2002; Abbasi and
Darvishnia 2015; Afshan and Khalid 2008, Afshan et al. 2009, 2010a, b; Aliabadi
and Abbasi 2012; Bahcecioglu and Gjaerum 2003, 2004; Bahcecioglu et al. 2005,
2009; Berndt 2007, 2009, 2010, 2013a; Berndt and Freire 2004; Berndt and
150 M. Toome-Heller
Uhlmann 2006; de Carvalho and Hennen 2012; Hüseyin and Kirbag 2003; Iqbal
et al. 2009; Kabaktepe 2015; Khalid and Afshan 2009; Kirbag et al. 2001, 2011;
Liu and Hambleton 2012; McKenzie 2008; McKenzie and Johnston 2004;
Mennicken and Oberwinkler 2004; Okane et al. 2014; Perdomo-Sanchez and
Piepenbring 2008; Sotao et al. 2007; Thaung 2011; Zhuang and Wei 2001, 2011),
Uromyces (28 new species; Agarwal 2003; Bahcecioglu 2014; Bahcecıoglu and
Gjaerum 2004; Berndt 2002a, 2004, 2009, 2013b; Berndt and Baiswar 2009;
Berndt and Uhlmann 2006; Berndt et al. 2007; Doungsaard et al. 2014; Hernandez
et al. 2005; Mennicken and Oberwinkler 2004; Perdomo-Sanchez and Piepenbring
2014; Rezende and Dianese 2003; Thaung 2009; Walker and van der Merwe 2009;
Wood and Scholler 2005; Zhuang and Wei 2003), Uredo (16 new species; Berndt
2002b, 2004, 2009; Berndt and Freire 2004; Berndt and Uhlmann 2006; Berndt and
Wood 2012; Berndt et al. 2007; Cao et al. 2000; Hernandez et al. 2005; Mennicken
and Oberwinkler 2004; Zhuang and Wei 2011, 2012), Prospodium (12 new spe-
cies; Berndt 2002b; Berndt et al. 2007; de Carvalho and Hennen 2010), and
Phakopsora (11 new species; Bagyanarayana et al. 2001; Beenken 2014; Berndt
and Wood 2012; Berndt et al. 2008; Ferreiea et al. 2001; Maier et al. 2015; Ono
2000; Ono et al. 2012; Ritschel et al. 2007). Interestingly, species descriptions for
rust genera follow the same trend as was seen for classes, i.e. the most species-rich
genera (Puccinia, Uredo, and Uromyces) had the highest number of new species
discovered.
Among the remaining genera of Pucciniomycotina, most of the new species were
revealed for the insect associated hyphal fungi in Septobasidium (32 new species;
Chen and Guo 2011a, b, c, d, 2012a, b, c; Li and Guo 2013; Li et al. 2013; Lu and
Guo 2009a, b, c, 2010a, b, c, 2011; Lu et al. 2010), for the anamorphic red and white
yeast genera Rhodotorula (30 new species; Bai et al. 2004; Belloch et al. 2007; Fell
et al. 2011; Golubev and Scorzetti 2010; Huang et al. 2011; Laich et al. 2013;
Libkind et al. 2010; Margesin et al. 2007; Nagahama et al. 2001, 2003, 2006; Pohl
et al. 2011; Satoh et al. 2013; Shivaji et al. 2008; Singh et al. 2014; Thanh et al.
2004; Vishniac and Takashima 2009; Zhao et al. 2002) and Sporobolomyces (23
new species; Bai et al. 2002; Fell et al. 2002; Hamamoto et al. 2002; Libkind et al.
2005; Nakase et al. 2003, 2005a, b; Satoh and Makimura 2008; Takashima and
Nakase 2000; Valerio et al. 2002; Wang and Bai 2004; Zhao et al. 2003), and for
anther smuts in Microbotryum (17 new species; Chlebicki and Sukova 2005;
Denchev 2007; Denchev et al. 2009; He and Guo 2008; Lutz et al. 2005, 2008;
Piatek et al. 2012, 2013; Vanky 2004; Vanky and Berner 2003). These four are the
remaining largest genera in Pucciniomycotina, confirming the trend that more new
species are found for already known species-rich groups.
Diverse Habitats and Geographic Distribution
When analysing the new species data by their organism type, 61 % of all new spe-
cies were rust fungi, 22 % yeasts, 10 % sporocarp forming fungi, 5 % anther smuts,
and the remaining 2 % hyphal microfungi (Fig. 7.1c). Most of the Pucciniomycotina
species have been found to have an association with plants. While rust and anther
smut fungi strictly inhabit only the above-ground parts of living plants, other groups
have been isolated from plant roots, stems, as well as dead plant materials. Non-
plant habitats include soil, fresh and marine water (including ice), air, animals
(including insects), and other fungi. Most of the 83 new yeast species described
since 2000 have been recovered from plant surfaces (47 new species; e.g. Golubev
and Scorzetti 2010; Wang et al. 2003) where they are believed to be epiphytic sap-
robes. Other habitats of the remaining yeasts are marine environments, often associ-
ated with the sea floor animals (10 new species; e.g. Laich et al. 2013, 2014;
Nagahama et al. 2003) and soil (9 new species; Vishniac and Takashima 2009).
Nearly all new sporocarp-forming species were of the scale insect-associated
Septobasidium species, which grow on various tree branches (34 new species; e.g.
Chen and Guo 2012a; Li et al. 2013), three were isolated from beetle galleries
(Hausner et al. 2008; Oberwinkler et al. 2006), and one from palm litter (Toome and
Aime 2014). The few new hyphal microfungi species have been recovered from soil
(Bauer et al. 2009; Nguyen et al. 2014), aquatic environments (Manohar et al. 2014),
or in association with bark beetles (Kirschner et al. 2001).
Some Pucciniomycotina species can grow in extreme or remote environments,
which are more difficult to access and study. For instance, some of the most recently
described Pucciniomycotina species have been isolated from flare pit soils in
Canada (Nguyen et al. 2014), anoxic costal sediments of Arabian Sea (Manohar
et al. 2014), Antarctic marine sponge (Laich et al. 2014), cryoconite holes in Arctic
(Singh et al. 2014), tropical rain forests in South America (Toome et al. 2013), and
desert soil crusts in China (Zhang et al. 2013).
The general geographic distribution of new rusts and their allies is dominated by
previously less documented areas (Fig. 7.1d). Increased mycological activity in
Asia has resulted in a concomitant increase in new species discovery from these
regions, totalling nearly 40 % of all new records, including all new Septobasidium
species, 73 new rusts, and 44 new yeasts (e.g. Crane 2005a; Pohl et al. 2011; Yang
et al. 2014) described since 2000. Nearly 20 % of the new species are from South
America, the majority of which are rust fungi described from Brazil (e.g. Beenken
et al. 2012; Rezende and Dianese 2001; Berndt and Freire 2000). The studies in
Africa have also been greatly biased towards the rust fungi with 43 new rusts
described mostly from South Africa (e.g. Berndt and Wood 2012; Wood and Crous
2005). In Europe, a more even distribution was seen between the new species of
Pucciniomycotina with 21 yeasts, 20 rusts, six anther smuts, and four hyphal micro-
fungi (e.g. Lutz et al. 2005; Margesin et al. 2007; Valerio et al. 2008). All species
found from Arctic and Antarctic environments (2 and 3, respectively) were yeasts
(e.g. Laich et al. 2013; Singh et al. 2014; Turchetti et al. 2011).
The role of many non-rust Pucciniomycotina species in nature is not yet well
understood. For example, some species belonging to Atractiellomycetes were iso-
lated from the roots of orchids and poplar trees (Bonito et al. 2010; Kottke et al.
2010). Although a mycorrhizal or a similar symbiotic relationship between those
fungi and plants is suspected, additional studies are needed to clarify the exact
nature of this association. The majority of the yeasts isolated from plant surfaces are
believed to be saprobes, although some research also show that they could be pro-
152 M. Toome-Heller
tecting the plants from pathogens (e.g. Robiglio et al. 2011; Vero et al. 2013), and
there is evidence that some may be pathogenic on cultivated mushrooms (e.g. Xu
et al. 2014) or animals, including humans (Chitko-McKown et al. 2014; Tsiodras
et al. 2014). In fact, more human infections by Pucciniomycotina species have been
reported over the past years (Wirth and Goldani 2012). In the majority of these
cases, infections are associated with a weakened immune system and central venous
catheter, indicating that these fungi are opportunistic pathogens and do not affect
healthy humans. One such example is the red yeast Rhodotorula mucilaginosa that
has become a serious human pathogen over the last few years. This species appears
to be present in the normal microflora of several animals (Park et al. 2012; Raggi
et al. 2014) and is present in various foods, but it can also cause serious infections
in hospitals because it readily adheres to plastic surfaces. The yeast can cause super-
ficial skin or eye infections or more serious fungemia, which have led to the death
of the patients in nearly half of the cases (Wirth and Goldani 2012). Pathogenicity
towards humans has also been reported for a hyphal species Tritirachium oryzae,
which can cause eye, nail, and scalp infections (Moraes et al. 2010; Naseri et al.
2013; Schell et al. 2011).
Challenges in the Biodiversity Research
The availability of verified reference sequence data is critical for efficient identifica-
tion of hidden biodiversity. While most new species descriptions today include the
publication of sequence data for at least one or two gene regions, obtaining good
sequence coverage for members of Pucciniomycotina can still be challenging.
Thanks to the sequencing completed by culture collections (e.g. CBS—
Centraalbureau voor Shimmelcultures, ATCC—American Type Culture Collection)
and the Assembling the Fungal Tree of Life (AFTOL) project, the majority of cul-
turable Pucciniomycotina species now have publicly available sequence data.
Moreover, as a result of a yeast sequencing initiative, the LSU region (and in most
cases also the ITS region) of all the known yeast species has been sequenced and
made publicly available (Kurtzman et al. 2011), allowing the identification and dis-
covery of new Pucciniomycotina yeasts based on sequence data. On the other hand,
for the fungal groups that are not culturable (e.g. rust fungi) and for rarely collected
sporocarp-forming and hyphal taxa that are known only from herbarium material,
sequence data may not be available. Today, less than 10 % of all known rust fungal
species have published sequence data (Toome and Aime 2015). Therefore, taxo-
nomic studies and species identification in rust fungi still rely greatly on non-
molecular methods. Since it can be challenging to obtain sequence data from old
herbarium specimens, new collections and thorough molecular studies of all the
collected material in the future (especially of the ones with few morphological char-
acters) will allow revealing some unknown biodiversity of Pucciniomycotina with
greater confidence.
There are also a few challenges related to next generation environmental sequenc-
ing projects. Several pipelines and programs for data analysis have been developed
to manage the high volumes of generated data and to annotate and identify the spe-
cies that are recovered (e.g. Abarenkov et al. 2010; Dannemiller et al. 2014).
However, these identifications are, of course, only as good as the reference sequence
databases. Therefore, the identification of inadequately sequenced lineages (e.g.
rust fungi) cannot be reliable until the taxa are thoroughly sequenced. Additionally,
since the anamorphic yeast genera Sporobolomyces and Rhodotorula contain spe-
cies that span several orders but the types only belong to Microbotryomycetes,
under- or overestimation of the representativeness of some of the taxa can occur and
may not illustrate the true biodiversity of the studied communities. These issues can
be resolved only via verified and improved databases.
Although new species cannot be described solely based on sequence data and
without a voucher specimen, environmental community sequencing projects can
provide very valuable information about the hidden biodiversity and hint, where the
future studies should focus to find the hidden species of Pucciniomycotina. For
example, the species Mixia osmundae of the monotypic Mixiomycetes was known
only from fern leaves in North America and Asia, but published environmental
sequencing data analysis indicates that conspecific or congeneric species are also
present in Europe and could be associated with other hosts (Toome et al. 2014).
Similarly, unknown members of Atractiellomycetes have been isolated from tree
roots in a number of locations, indicating that in addition to the known habitats in
North and South America (Bonito et al. 2010; Kottke et al. 2010), they are also pres-
ent in the Seychelles (Suvi et al. 2010). Some other examples of the community
studies where various Pucciniomycotina taxa have been recovered include the study
of Arctic soils (Timling et al. 2014), deep-sea sediments in the Central Indian Basin
(Singh et al. 2011), Quercus macrocarpa phyllosphere in Kansas, USA (Jumpponen
and Jones 2010), acid mine drainage biofilm in California, USA (Baker et al. 2009),
and ice of the Baltic Sea (Majaneva et al. 2012).
Phylogenetic Classification
Higher-Level Phylogenetic Relationships
The placement of Pucciniomycotina within Basidiomycota is not yet fully resolved.

While several multigene studies have reported this subphylum to be a sister clade to
Agaricomycotina and Ustilaginomycotina, i.e. the most early diverging basidiomy-
cetes (James et al. 2006; Matheny et al. 2006; Ebersberger et al. 2012; Floudas et al.
2012), others have found that Ustilaginomycotina is the basal group and
Pucciniomycotina is sister to Agaricomycotina (Medina et al. 2011). Similarly, the
deeper nodes within Pucciniomycotina have not been resolved. The best resolution
to date was provided in Aime et al. (2006) and in Schell et al. (2011), which both
strongly support all class level lineages but do not allow clarifying the evolutionary
relationships between the classes. Although the availability of fungal genome data
has greatly improved our ability to perform phylogenomic studies of fungi to reach
154 M. Toome-Heller
better resolution for deeper nodes (e.g. Floudas et al. 2012), this approach has not
yet been applied to gain knowledge of evolutionary relationships within
Pucciniomycotina. This is because the complete genomic coverage of all the
Pucciniomycotina classes has not been available. Albeit, this situation is most likely
to change over the next few years as new Pucciniomycotina genomes are becoming
available through the 1000 Fungal Genomes Project (Grigoriev et al. 2013).
Taxonomic Improvements as a Result of Improved

Understanding of Phylogenetic Relationships
Over the last 16 years, 23 new genera have been described in Pucciniomycotina.
Eight of those genera have been described as a result of a thorough analysis of exist-
ing species and revision of existing genera. These are the rust fungi genera
Desmosorus (Ritschel et al. 2007), Esalque (Hennen et al. 2000), Pelastoma (Yepes
and de Carvalho 2012), Puccorchidium (Beenken and Wood 2015), Racospermyces
(Walker 2001), and Sphenorchidium (Beenken and Wood 2015); a yeast genus
Leucosporidiella (Sampaio et al. 2003); and a mould genus Paratritirachium
(Beguin et al. 2012). The remaining 15 new genera were described based on new
species discovery. These are the sporocarp-forming genera Basidiopycnis,
Proceropycnis (Oberwinkler et al. 2006), Basidiopycnoides (Hausner et al. 2008),
and Pycnopulvinus (Toome and Aime 2014); the hyphal microfungi genera
Cystobasidiopsis (Bauer et al. 2009) and Colacosiphon (Kirschner et al. 2001); rust
fungi genera Bibulocystis (Walker et al. 2006), Canasta (de Carvalho and
Hennen 2010), Cratea (Yepes and de Carvalho 2012), and Diaphanopellis (Crane
2005b); and yeast genera Bannoa (Hamamoto et al. 2002), Curvibasidium (Sampaio
et al. 2004), Glaciozyma (Turchetti et al. 2011), Meredithblackwellia (Toome et al.
2013), and Microbotryozyma (Suh et al. 2012). These new genera have greatly
improved the taxonomy of Pucciniomycotina species as several of them have
enabled to re-classify members of polyphyletic and anamorphic genera. As a result,
numerous new combinations have been proposed to already existing species and
several species complexes have been identified to species level, greatly improving
our understanding of Pucciniomycotina phylogeny (e.g. Berndt 2011, 2013b;
Turchetti et al. 2011; Yurkov et al. 2015).
Above genus level classification has also seen some improvements. Since the
major revision of Pucciniomycotina published by Bauer et al. (2006), a few new
families and orders have been introduced to provide better higher level classifica-
tion. The most significant change since the last major revision has been the descrip-
tion of a new class Tritirachiomycetes and lower level groups Tritirachiales and
Tritirachiaceae (Schell et al. 2011). Tritirachium species were previously classified
in Ascomycota, but a thorough molecular study revealed that these fungi are mem-
bers of Pucciniomycotina, forming a separate lineage in it. The other higher level
change has been within Microbotryomycetes, where Kriegeriaceae and Kriegeriales
were described to provide higher level classification for a group of anamorphic
yeasts and a sedge pathogen (Toome et al. 2013). More of such studies are needed
as there are still numerous incertae sedis taxa in nearly all Pucciniomycotina classes
(Aime et al. 2014).
Challenges in Phylogenetic Studies
Due to their complicated life cycles including two different host plants and up to
five or more different spore stages, many species of rust fungi have been given
numerous names. As a result, we can find nearly 10,000 different names registered
for rust fungi in MycoBank (www.mycobank.org). While new names are created, it
is important to evaluate the suitability of the existing names. This is one of the chal-
lenges researchers who work with rust fungi need to face as often there are only few
morphological characters on the type specimens. Moreover, since the DNA extrac-
tions from old rust herbarium specimens have a low success rate it might be nearly
impossible to obtain DNA sequences for old specimens. Most of the 133 recognised
genera of rust fungi have only one or less than ten known species (Cummins and
Hiratsuka 2003) and examining sequence data might sometimes be the only way to
determine, whether they really represent separate genera/species or they could be
part of some other groups. This is likely going to be one of the greatest challenges
in rust research due to the poor sequence coverage and the need for new
collections.
A few studies have been already completed to address this issue. In one of them,
sequence data from the two species of the rust genus Frommeëlla were analysed,
which determined that these species do not represent a separate genus, but are actu-
ally part of a larger genus Phragmidium (Yun et al. 2011). A contrary situation was
revealed in a recent study on mayapple rust, a fungus that was first described as
Aecidium podophylli, then placed in genus Allodus and thereafter transferred to
Puccinia, based on morphological characters. After completing phylogenetic analy-
ses, it was determined that the species actually is different from Puccinia and the
genus Allodus was resurrected (Minnis et al. 2012). In addition to genus level
improvements, thorough molecular revisions are needed at the species level as well
since some studies of rust fungi have uncovered a great number of cryptic species.
For example, analyses of the Melampsora epitea complex in the North American
pacific northwest determined the existence of 14 different phylotypes within this
one morphospecies (Bennett et al. 2011) and studies within the Endoraecium digi-
tatum (Berndt 2011) and Dasyspora (Beenken et al. 2012) species complexes are
revealing similar patterns of cryptic speciation.
Great taxonomic changes are also needed for the yeasts that have been placed in
anamorphic catch-all genera, especially Sporobolomyces and Rhodotorula. Several
research groups have already started this progress by proposing new genera for
anamorphic Pucciniomycotina yeasts (e.g. Bauer et al. 2009; Toome et al. 2013;
Turchetti et al. 2011; Yurkov et al. 2015), while other researchers still choose to
place new discovered species into the anamorphic genera until further data is avail-
able (e.g. Laich et al. 2013; Singh et al. 2014). Thanks to the complete availability
156 M. Toome-Heller
of yeast sequence data, more changes are expected to be made to create meaningful
taxonomy for the yeasts during the coming years.
Genomics
As is true for mycological studies in general, the availability of draft genomes has
greatly advanced the research of the biology of the members of Pucciniomycotina.
For example, access to genomic data enables us to study the gene functions and the
evolution of gene families, discover effector proteins, and perform detailed popula-
tion studies (e.g. Duplessis et al. 2014; Hacquard et al. 2012; Persoons et al. 2014).
Further availability of genomes over a range of different Pucciniomycotina lineages
will also enable us to better understand the phylogeny of rust fungi and their allies
and the evolutionary pathways that have led to such a diverse group of fungi. The
number of sequenced genomes for Pucciniomycotina is still relatively small com-
pared to other fungal subphyla, with published genomes being available for only 12
species to date—one of these was published in 2015, six in 2014, one in 2012, and
three in 2011 (Table 7.1). Several other genomes have been completed recently as
part of the 1000 Fungal Genomes project (Grigoriev et al. 2013) and other sequenc-
ing projects (see Duplessis et al. 2014) and they should become available in the next
few years.
Genomes of Rust Fungi
The first annotated rust genomes were published for the poplar leaf rust fungus
Melampsora laricis-populina and the wheat stem rust fungus Puccinia graminis f.
sp. tritici (Duplessis et al. 2011), followed by two genomes for the stripe rust fungus
P. striiformis f. sp. tritici (Cantu et al. 2011; Zheng et al. 2013). These genomes
have facilitated the research of several new aspects of rust fungi, especially the
genome-based analysis of effector genes and transcriptomic studies that are eluci-
dating interactions between rusts and their host plants (e.g. Cantu et al. 2013;
Hacquard et al. 2012; Persoons et al. 2014). For a detailed overview of the genome
research on these fungi, please refer to Duplessis et al. (2014). More recently, the
draft genomes of four other rust species have been published. These are for the
myrtle rust fungus P. psidii (Tan et al. 2014), the flax rust fungus M. lini (Nemri
et al. 2014), the coffee rust fungus Hemileia vastatrix (Cristancho et al. 2014), and
the broad bean rust fungus Uromyces fabae (Link et al. 2014; Table 7.1).
Several years before the rust fungi genome sequencing was completed, genome
size studies by flow cytometry estimated that these fungi have large genomes,
reaching hundreds of millions of base pairs (Eilam et al. 1994). The first completed
rust genomes of M. laricis-populina and P. graminis f. sp. tritici showed that the
genomes of those obligate parasites are indeed large, with over 101 and 88 Mb,
7
Table 7.1 Genomic characters of the Pucciniomycotina species with published genomes
Species Strain Order Genome size (Mb) Number of gene models Reference
Hemileia vastatrix 8 different isolates Pucciniales 333 14,445 Cristancho et al. (2014)
Melampsora laricis-populina 98AG31 Pucciniales 101 16,399 Duplessis et al. (2011)
Melampsora lini CH5 Pucciniales 189 16,271 Nemri et al. (2014)
Mixia osmundae IAM14324 Mixiales 13 6903 Toome et al. (2014)
Puccinia graminis f. sp. tritici CDL7S-36-700-3 Pucciniales 89 17,773 Duplessis et al. (2011)
Puccinia psidii PBI 115012-Mr Pucciniales 103–145 19,000 Tan et al. (2014)
Puccinia striiformis f. sp. tritici PST-130/CY32 Pucciniales 79/110 22,815/25,288 Cantu et al. (2011)/Zheng
et al. (2013)
Puccinia triticina 1-1 (BBBD) Pucciniales 135 14,880 Duplessis et al. (2014)
Rhodosporidium toruloides MTCC457 Sporidiobolales 20 5993 Kumar et al. (2012)
Rhodotorula glutinis ATCC204091 Sporidiobolales 20 3359 Paul et al. (2014)
Sporidiobolus salmonicolor CBS6832 Sporidiobolales 20 5147 Coelho et al. (2015)
Uromyces fabae I2 Pucciniales 329 23,153 Link et al. (2014)
Latest Developments in the Research of Rust Fungi and Their Allies…
157
158 M. Toome-Heller
respectively (Duplessis et al. 2011). Moreover, the genome sizes of the other com-
pleted rust fungi genomes range from 80 to 333 Mb (Table 7.1). A recently pub-
lished thorough flow cytometry study which examined the genome sizes of ten
different genera of rust fungi provided further support that some rusts have even
larger genomes (Tavares et al. 2014). Based on their data, the largest known
genomes belong to Gymnosporangium confusum (893 Mb), Puccinia chrysanthemi
(806 Mb), Phakopsora pachyrhizi (720 Mb), and Uromyces vignae (712 Mb). These
are clearly the largest fungal genomes ever measured (Tavares et al. 2014). The
availability of the genome size estimates is important for successful completion of
new genome projects because this enables the researchers to calculate the sampling
depth needed to cover the genome with the minimum number of gaps. Since Tavares
et al. (2014) revealed the genome sizes for several rust genera that had no previous
information this now facilitates further sequencing of the representatives of other
rust families. Interestingly, possible connection between the rust host plant and the
fungal genome size has been observed, most likely due to the close co-evolution
between these plant pathogens and their hosts (Tavares et al. 2014). Future research
will determine if the species with similar host range share similar genomic traits
specific to the hosts, or each lineage has evolved to obtain a unique set of genes to
overcome host resistance.
Although it might be assumed that these large genomes might be needed due to
the macrocyclic life cycles of rust fungi, where gene sets for infecting two different
host plants and producing up to five different spore stages are required, comparative
analysis of protein coding genes does not seem to support this (Duplessis et al.
2014). The reasons for such large genomes of rusts are still not fully understood, but
it is clear that the vast majority of their genomic content is comprised of repetitive
and transposable elements (Duplessis et al. 2011, 2014). In fact, based on currently
sequenced genomes, rust fungi have just between 14,000 and 25,000 protein coding
genes (Table 7.1), despite their great genome sizes. Initial analyses show that many
of those genes belong to unique gene families which are not shared with other
basidiomycetes, including the other Pucciniomycotina species (Duplessis et al.
2014; Toome et al. 2014; Zheng et al. 2013).
Other Pucciniomycotina Genomes
Only a few genomes are publicly available for non-rust Pucciniomycotina species,
four of these from Microbotryomycetes. The first two, the red yeasts Sporobolomyces
roseus and Rhodotorula graminis, were sequenced and assembled in 2006 and
2010, respectively (http://genome.jgi.doe.gov), but have not been officially pub-
lished to date although genomic data of these species have been included in various
comparative studies (e.g. Coelho et al. 2010; Horns et al. 2012). Three additional
Microbotryomycetes, the yeasts Sporidiobolus salmonicolor, Rhodosporidium tor-
uloides, and Rhodotorula glutinis, have been sequenced and published (Coelho
et al. 2015; Kumar et al. 2012; Paul et al. 2014). Outside of Microbotryomycetes
and Pucciniomycetes, genome data have been published only for the fern pathogen
Mixia osmundae, the monotypic representative of Mixiomycetes (Toome et al.
2014).
The genome sizes of rust relatives are considerably smaller than those of the rust
fungi. Both S. roseus and R. graminis have small genomes at around 21 Mb (http://
genome.jgi.doe.gov) and the genomes of S. salmonicolor, Rh. toruloides, and
R. glutinis are sized at 20 Mb (Table 7.1). While these are already fairly small
genome sizes compared to other sequenced fungi (average fungal genome size is
37.7 Mb, Tavares et al. 2014), the genome of M. osmundae is even smaller, rep-
resenting the smallest known genome among all Basidiomycota and one of the
smallest genomes in fungi at only 13 Mb (Toome et al. 2014). Genomes for the
representatives of five additional classes have been completed and are being anal-
ysed at the moment (Aime et al., unpublished), which should give us a better under-
standing of general genomic traits of other Pucciniomycotina representatives and
enable to reveal unique traits of this subphylum.
Acknowledgements I am grateful to Dr. M. Catherine Aime for her continuous support and
contribution to this chapter and for sharing her invaluable knowledge of Pucciniomycotina with
me during the past years. Drs. Wellcome Ho and Gregory Heller are also acknowledged for their
comments on an earlier version of this manuscript.
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Chapter 8
Conidiogenesis: Its Evolutionary Aspects
in the Context of a Philosophy of Opportunity
(Lectics)
Richard C. Summerbell and James A. Scott
In 1682, botanist John Ray published his Methodus Plantarum Nova (Ray 1682),
revealing a new method of classifying plants. While still holding to the traditional
division of plants into trees, shrubs, and herbs, Ray devised a refined system that
was based on details of plant anatomy, including the numbers and arrangement of
flower parts, stem parts, and seed leaves. This method revealed the hidden, funda-
mental distinction between monocotyledonous and dicotyledonous angiosperms for
the first time. Ray was sufficiently awed by the hidden correspondences in anatomi-
cal plant symmetries that the finding became a cornerstone of his theology. He
propounded the idea that God expressed hidden wisdom throughout nature and out-
lined this credo in a later book The Wisdom of God Manifested in the Works of the
Creation, 1691 (Ray 1691).
The factor that made the contrast of monocots and dicots appealing was that
many different sorts of character distinctions, none of which betokened obvious
functional differences, correlated to distinguish the groups. The number of cotyle-
dons emerging from the seed differed, even though there was no obvious reason to
imagine the two seed leaves of the dicot would perform significantly differently
from the single seed leaf of the monocots. The numbers of parts in the flower—
petals, stamens—differed, again, without clear functional significance: multiples of
three for the monocots and multiples of four or five for the dicots. The leaf venation
R.C. Summerbell, Ph.D. (*)

Sporometrics Inc., 219 Dufferin Street, Suite 20-C, Toronto, ON, Canada M6K 1Y9
Dalla Lana School of Public Health, Gage Occupational and Environmental Health Unit,
University of Toronto, 223 College Street, Toronto, ON, Canada M5T 1R4
e-mail: rsummerbell@sporometrics.com
J.A. Scott, Ph.D.
Division of Occupational and Environmental Health, Dalla Lana School of Public Health,
e-mail: james.scott@utoronto.ca

DOI 10.1007/978-3-319-29137-6_8
170 R.C. Summerbell and J.A. Scott
tended to differ, parallel in the monocots and reticulate in the dicots, and even
though this character showed a degree of correlation with elongate leaves in the
monocots and more radially symmetric leaves in the dicots, the members of each
angiosperm subgroup possessed a wide variety of leaf shapes. Such divergent char-
acter appositions, then, when they seemed not to indicate counterposed functional
differences, could be envisioned as clues to the thought process of intelligent design.
Later in biological history, after evolution was generally accepted, the same charac-
ter differences could be seen as unusual factors that were so fundamental that they
could be maintained through all the stresses of natural selection. They appeared to
be arbitrary formats of construction that could be viable in essentially any niche
inhabitable by the type of organism in question. Occasionally, one of these basic
developmental formats showed variation—for example, in the yam, Dioscorea,
reticulate leaf venation occurred in a monocot. In general, however, there was curi-
ously little indication that natural selection was directly involved in shaping the
form of the defining monocot/dicot differences.
Fungi, being arguably of lower morphological complexity than plants, showed
few such characters. In the relatively complex agarics, however, a number of seem-
ingly arbitrary construction formats were found to correlate with each other and to
indicate apparently co-derived lineages. The macroscopic characters used at first
included hymenial form, lamellar attachment, veil types, and basidiospore mass
coloration. Emphasis on the last was one of the great contributions of Elias Fries
(1825), who believed that major differences in agaric spore coloration followed a
spiritual trend in nature to divide and subdivide taxa into clusters of four, with each
member of a tetrad marked by fundamental distinctions in design. Later in history,
as the protective functions of melanin began to be appreciated, there might have
been stimulus for scientists to speculate that brown and black basidiospores would
be more robust in sunlight than pale ones. To our knowledge, however, basidio-
spore colour, to this day, has not been shown to be a salient selective factor in any
natural situation. Although most lineages once defined on basidiospore colour are
now known, through DNA analysis, to include some exceptions, there remain large
blocks of related species that, no matter how diverse the habitats they grow in,
retain the characteristic spore colour of their lineage.
When microanatomy came to mushroom work, other quasi-arbitrary develop-
mental formats such as lamellar tramal structures and pileipellis constructions were
revealed (Fayod 1889; Singer 1986). They joined the list of characters that appeared
to be lineage-revealing, selectively near-immutable evolutionary whims. Since
mycologists have mostly tended to Neodarwinism, the idea that these character dif-
ferences were perhaps not directly selected for by environmental exigencies has
tended to receive little attention, even though biologists have generally accepted that
some developmental characters appear to be constrained towards self-perpetuation.
In the microscopic conidial fungi, taxonomy for many decades remained at the
level analogous to the trees, bushes, and herbs of primitive plant taxonomy. In the
definitive work of Saccardo (1886), the conidial fungi were divided into form taxa
called Hyphomycetes (essentially, branches), Sphaeropsideae (flasks, i.e., pyc-
nidia), and Melanconiae (erumpent tufts, i.e., acervuli). The Hyphomycetes were
then subdivided into Mucedineae (pale branches), Dematiae (dark branches),
8 Conidiogenesis… 171
Stilbeae (erect fascicles of branches, i.e., synnemata), and Tuberculariae (superfi-

cial tufts of short branches, i.e., sporodochia).
In 1953, Hughes (1953) revolutionized hyphomycete taxonomy by crystallizing
and extending our knowledge of the microanatomy of conidiogenesis. Specifically,
he discerned that conidiogenous structures provided a degree of the correlative tax-
onomic insight provided by developmental design characters in other organisms,
such as mushrooms and plants. Additional studies in subsequent years only under-
scored the importance of his scheme of organization. Aspergillus conidiogenous
states, for example, strongly featured phialides, and their associated sexual states
tended to be cleistothecia producing equatorially ornamented ascospores.
Scopulariopsis conidiogenous states featured annellides (a term originally coined
by Hughes as ‘annellophores’) where the phialide-like conidiogenous cells extended
their necks via percurrent proliferation, and any sexual states found were clearly
related species of Microascus. Entire orders of fungi were characterized by distinct
patterns of conidiogenous development, e.g. the Onygenales, with thallic conidio-
genesis producing conidia dehiscing via lytic disruption of small, empty separating
cells. Conidiogenesis, for the first time, gave hyphomycete experts a somewhat reli-
able means of predicting the phylogenetic affinity of most of the asexual fungi they
studied.
Up until a few years ago, review articles dealing with conidiogenesis would have
been primarily focused on reviewing the patterns of conidiogenous development
and morphogenesis [an excellent example is that of Cole (1986), in tandem with a
discussion of the taxonomic information these structures revealed. Now, however,
conidiogenesis has been superseded as a biosystematic indicator by DNA sequences.
Many misplaced or hard-to-place asexual forms have thereby been correctly
assigned to higher taxa and the clades they are based on. The study of conidiogen-
esis, we can now see, was by no means a taxonomic panacaea (as Cole 1986) had
already pointed out), but it was truly the best of the available microanatomical char-
acters that could have been used to order the Hyphomycetes.
The eclipsing of the study of conidiogenesis by DNA-based biosystematics
raises the danger that treasured information will be downgraded in importance and
ultimately ignored. If our primary concerns are now divided into systematics, the
discipline allowing identification; ecology, the discipline characterizing the interac-
tions of organisms with each other and ourselves; and the -omics (genomics, pro-
teomics, metabolomics), gauging the functioning and potential functioning of
organisms as biotechnological machinery, how much attention is left to observe the
development of seemingly arbitrary, industrially unexploitable characters like
annellations and sympodial proliferations? In an effort, then, to show that conidio-
genesis transcends being a superseded taxonomic character (still useful to micros-
copists in their ever-cloudier identifications of DNA-defined taxa) and a minor
developmental curiosity, we would like to elaborate a succinct evolutionary classi-
fication of the characters possessed by organisms and show where the characters of
conidiogenesis fit in. That is to say, we wish to accord these characters a place in the
story of fungal evolution, not just in taxonomy, ecology, or development.
The classification of taxonomic characters used henceforth arises from a philosophy
of the background conditions such characters address. In the broadest conception,
these conditions are called ‘evolutionary opportunities’, and when organisms are
conceived of as having settled into exploitation of a subset of these opportunities, the
limited locus of recurring energy interchange is referred to as a ‘niche’. In order that
an ‘opportunity’, in the evolutionist’s sense of the term, not be seen as vague hand-
waving toward ‘something out there’, we are going to define it exactly: it is a pos-
sibility for ‘benefit’, or increase, that may or may not be undertaken by a
self-reconstituting entity in a modifiable system. This definition has the following
dimensions:
1. Energy transfer and its constructive (including reproductive) use. Biology is
based upon solar energy, and the phrase ‘benefit or increase’ refers, ultimately,
to gaining a portion of that energy and putting it to use in life and in perpetuation
of the lineage.
2. Random or chosen optionality. Fundamental to the notion of an opportunity is
the idea that the entity confronted by the opportunity is not completely con-
strained to undertake it. If it were to be so constrained, one would have a com-
plex deterministic system, like air flow or water flow, not an opportunistic system
like biology. The finch reaching the Galapagos Islands may evolve to specialize
in eating seeds or insects, and, regardless of its level of prefigurement (e.g. arriv-
ing as a seed-eater), it is not wholly constrained a priori in its recruitment towards
one or the other of these opportunities or niches. The optionality intrinsic to
evolution as an opportunistic system is indicated by the ‘may or may not’ phrase
in the definition of opportunity. Evolutionary optionality is a matter addressed
by the lineage, and the arrival of the lineage at one side of the option (e.g. seed-
eating) or the other (insect-eating) is a matter of chance. Terminology for the
arbitration among these options is awkward and encumbered by teleology. We
have, thus, borrowed a common Greek root used (sometimes via Latin) in selec-
tion, election, lectotypy, and so on and coined the word ‘paralection’, meaning
chance-derived pseudo-choice, for this retrospectively seen process. Its ant-
onym, ‘lection’ applies to any deliberate choice made by a conscious entity, if
the concept of ‘free will’ is accepted, a matter we need not get into here. The
consequences of a theory of opportunity (‘lectics’) for conscious beings have
been adumbrated elsewhere (Rogerson 2013).
3. Self-reconstitution. The fundamental difference between opportunistic and
deterministic systems is that the acting (discrete, causally connected) elements in
the former can paralectically (or lectically) modify and regenerate the funda-
mental basis of their interactions. Tungsten always acts as tungsten, and water as
water, but a finch lineage (in contrast to the individual finch) may change over
time in any or all of its evolutionarily interacting characters and be transformed
from the production of dull-coloured seed-eaters with the digestive and meta-
bolic characters fitting that niche to the production of brightly coloured nectar-
eaters with radically altered digestion, metabolism, flight, breeding habits, and
so on. Insofar as entities address opportunities, they are ipso facto not static, but
rather are changeably regenerative.
4. The modifiable system. The non-static nature of the ‘paralectont’ (the non-
deliberating entity addressing the opportunity; in biology, the lineage) is only of
possible significance in the context of a larger, ambient system that also allows
modification. In evolution, an evolving species tends to change all the species it
interacts with, e.g. a nectar-feeding insect may induce a flowering species,
through cyclical reinforcement of benefit, to alter its floral coloration to better
attract the insect as a successful pollinator. Moreover, through shading, bio-
chemical secretion, and so on, species also alter the conceptually abiotic ele-
ments of their environments, such as soil and water. These modifications to the
abiotic substrata then redound to influence the progressive evolution of all the
species interacting with those substrata. The defining ‘logical’ feature of these
interactions is progressively altering cyclicality. If the interactions are pro-
grammed into a computerized model, they become elaborately interlocking
recursive functions that are ultimately unable (extremely unlikely) to re-attain
exactly any previously seen configuration. No moment in evolutionary history
can ever be duplicated later. Neither can any of the interacting causal agents and
causal momentum-carriers, except at the level of simplest chemistry, be dupli-
cated. Convergence occurs but is never absolute.
In order to assess where conidiogenesis fits into the systematics of evolutionary
opportunity, we need to consider the question of how the characters seen in biologi-
cal organisms address or interact with the opportunities/niches from which the spe-
cies obtain increase.
A consideration of the features of organisms shows that these features may be
approximately divided into two conceptual groups, which we trust will soon become
apparent as operationally different even though the distinction may seem abstract at
first. Again, these criteria apply to opportunistic situations outside biology as well
as inside it, but we will restrict the scope of our comments to biology.
1. Characters for which the form is obligate to the performance. Any organism
evolved to propulsively fly must have wings that can be used to overcome grav-
ity by directing air flow. Whether these wings are feathered or membranous, the
character ‘has functional wings’ must pertain. Any fungus producing dissemi-
nating conidia must have a dehiscence mechanism, whereby disseminable cells
or cell groups break away in a genetically determined manner from the fixed
structures that formed them. ‘Has functional wings’ and ‘has a conidial dehis-
cence mechanism’ are examples of this type of character obligate to a function
that is an essential element addressing the current niche. The ‘character’ consid-
ered here may only be an aspect of a discrete structure, e.g. the lift functionality
of a wing, but it is nonetheless conceptually individuated as an adaptively neces-
sary aspect in relation to the niche. Drawing on two etymologically related Latin
verbs, both with present active form appellō and their Romance-language deriva-
tives such as the French ‘rappeler’, ‘to recall’, we have designated this category
of character ‘rappellative’—the character, in its form, directly ‘recalls’ the form
of the niche exigency to which it responds (e.g. ‘has functional wings’ responds
to ‘needs to locomote by air to survive in its niche’). The niche exigency can
metaphorically be said to ‘pull back’ (etymology: ‘re’—again + ’ad’—
towards + ’pello’ push) the lineage if any of its members deviate from the needed
form (offspring born wingless in a species that needs to fly will not survive).
(This assumes that the deviation is not of the rare serendipitous type that opens
up a novel path to survival.) The metaphor of a mountain climber ‘rappelling’
down a cliff face and being recurrently pulled back to the rigid limit of the moun-
tain is also apt when extended to this type of evolutionary situation. The produc-
tion by any type of organism living predominantly on lactose of at least one
functional lactase enzyme can be seen as an archetypical rappellative character.
2. Characters that represent one of a number of alternate forms that can fulfil the
performance. Wings may rely on feathers, stretched skin, or chitinous membrane
to support flight. Thallic conidia may dehisce by weakening of a cell wall middle
lamella or by rupture of an entire dead, empty separating cell. It is not obvious
how adaptive forces could selectively replace one of these characters with one of
its (approximately) equally functional alternative forms, since the already func-
tioning character will be likely to hold the advantage over any as yet undevel-
oped possible replacement in any case of selective pressure. As an antonym to
‘rappellative’, we refer to the characters juxtaposed in these contrasts as ‘arbitra-
tive’, based on the Latin arbitro, ‘I judge’ or ‘I consider’. This root word in
English has long been used to allude to the concept of impartial justice and has
given us the adjective ‘arbitrary’ to describe indifferent conscious decision pro-
cesses. The indifference due to evolutionary chance is likewise alluded to in the
concept of an ‘arbitrative’ character. Such characters could also be referred to by
the tantalizing adjective ‘fungible’, but that word has the slight disadvantage that
some might take it to imply the existence of a force or agent that could effect a
switch among the different types.
Arbitrative characters come in approximately four subtypes, listed below.
2a. Novel neutral alleles or mutations. As Kimura (1983) pointed out, many
evolutionary divergences from parental stock may begin as mutations in
protein-coding genes that are initially synonymous or, if non-synonymous,
have no marked effect on the function of the proteins involved. In some
cases, non-synonymous neutral mutations may become consequential when
new adaptive stresses emerge. For example, a mutation that confers fortu-
itously more heat tolerance than its predecessor may begin to be selected for
when a habitat begins to trend as warmer over the years. Alternatively, ini-
tially neutral mutations may, through the complexities of protein folding and
through reconfiguring of active domains, fortuitously lend functionality to
later-arising novel mutations that occur in more evolutionarily constrained,
enzymatically functional regions.
2b. Coordinating characters. These are characters that are interchangeable in
principle, interacting with other biological characters that are also inter-
changeable in principle. Such characters tend to be held in relative stasis by
coordination, whether coordination within cells, or within structures, or
within interacting members of the population, or within interacting species
in the ecosystem. Enzymes, hormones, etc., that interact with other cellular
components must in some way chemically interlock with them. The bio-
chemical and physical forms of mating type A must be compatible with
those of mating type B if mating is to occur. Where sensory perception

exists, the members of species must be able to interact with one another
through recognition of shape, colour, olfactory impact, and so on.
Woodpecker species A has a red cap, used in visual recognition of conspe-
cifics; its sibling-species B has a yellow cap, used for the same function.
Mushroom species A, based on interactions within its own inner develop-
mental schema, develops complex, layered lamellae widened out with inter-
woven hyphal growth, while species B develops equally complex lamellae
widened with parallel tramal tissue. Hormone A only stimulates a signal
when it meets compatible receptor A’. Hormone B in a closely related spe-
cies may be co-derived genetically from a common ancestor with hormone
A, and it may be identical in function and nearly so in structure; nonetheless,
it only stimulates receptor B’. Such characters appear arbitrative when they
are juxtaposed against one another by an observer, but in practice, they are
locally near-obligatory to newly generated members of the species, unlike
characters of type 2a. They can be conceived of as rappellative characters
within an arbitrative framework. Based on their property of near-obligatory
coordination within adaptive subsystems, they are designated ‘interrappella-
tive’ characters. Interrappellative characters intergrade with rappellative
characters in situations where coordination occurs as a standoff among
antagonistic species, e.g. where a predator uses odour to recognize its prey,
and the prey species takes steps to conceal its odour against the predator’s
specific methods of detection. In such cases, discoordination, such as a
change of odour by the prey, may be adaptively favoured, if it occurs.
Cooperative interrappellatives have evolutionary advantage in remaining
synchronized, while antagonistic interrappellatives may entail advantage in
developing asynchrony, especially for species being preyed upon, parasit-
ized, or grazed. Much like inclement physical conditions, then, predators,
grazers, allelopaths, and other ‘hostile’ species tend to promote adaptive
change.
2c. Vestigial characters. The human vermiform appendix is a well-known
example of a character that is considered to exist as a developmental relict
form, arguably lacking any functional significance that would outweigh the
death risk it poses. Evolution is efficient, but in complex organisms, essen-
tially functionless developmental leftovers may remain. These, then, are
purely arbitrative characters, in that closely related species may either pos-
sess them or not. We are not aware of any clear mycological examples, but
see further comment below sub Trichophyton rubrum.
2d. Characters without strictly specified role, elaborated by adapted processes
that intrinsically generate variation. Cells and organisms operate various
processes through which the rates of adaptive change can be varied. DNA
repair, for example, can adapt to be more or less rigorously accurate, lower-
ing and raising the rate of stabilized mutation. Genes can become arranged to
undergo recombination or positional reshuffling with greater or lesser facil-
ity, depending on diverse factors such as methylation, regulatory proteins,
histones, repetitive regions between genes, and genomic expansion processes

featuring retention and eventual redeployment of originally homologous
gene copies on multiple chromosomes. Each human receives a unique face,
set of fingerprints, and set of ‘genetic fingerprints’ that are determined by
genetic characters, or by genetically enabled epigenetic processes, or by
early developmental generators of quasi-random variation. The extent to
which such processes operate in other species is often unclear, but can hardly
be thought to be absent in most or all cases.
Some clearly arbitrative character types are remarkably evolutionarily
labile, sometimes perhaps ‘locking in’ interrappellative relations with fea-
tures of interacting organisms, and sometimes appearing functionally mys-
terious. A good example is the secondary metabolite spectra elaborated by
all mycelial organisms that densely occupy volumes of substrate, including
Hyphomycetes and Streptomycetes, among others. Each species, even in
recently evolved phylogenetic clusters, generally produces a distinct array of
several characteristic ‘extrolites’, sometimes consisting of a shuffling from a
short list of metabolites found in multiple related species, and sometimes
including one or more metabolites unique to the species in question. A given
unusual metabolite may pop up in isolation in very distantly related species,
e.g, penicillin in Penicillium chrysogenum and Trichophyton rubrum, a hap-
penstance implying either a common exogenic origin of the character
(Peñalva et al. 1990) or the existence of a randomization process in which
the spate of permutations is constrained in chemical possibility and thus has
some tendency towards producing coincidental results.
Though extrolites sometimes become typical examples of features locked
into interrappellative relationships with competing species—for example,
the case of mycotoxins adapted to deter grazing of the mycelial domain by
poisoning the major grazers—many have no known interrappellative func-
tion, either within the producing species or in the ecosystem. A previous
paper (Summerbell 2000) has proposed that selection for anomaly or non-
recognizability, as an ecologically interactive feature in its own right, can
occur, for example when an array of unusual metabolites distinguishes a
mycelial colonization zone as ‘unrecognizable and/or indigestible as food’
to a non-specific array of grazers, even though the metabolites are neither
toxic nor notably repugnant.
Heath (2000) developed and tested a sophisticated theory of host vs. non-
host defence mechanisms in plants. The former (‘host resistance’) mecha-
nisms become involved in a near-rappellation, based on progressive
adaptation of the plant to recognize established pathogens by acquiring new
means of detecting them as they, in turn, altered their chemistries to evade
recognition. The latter (‘non-host resistance’) mechanisms consist of the
generation of features generally adapted to deter a wide and possibly ever-
changing group of potential, unspecialized antagonists. Some of these non-
host defence features may consist of the production of chemically distinctive
‘flavour’ compounds arguably analogous to the extrolites of fungi—again, a

group of chemicals including some strongly deterring (‘antifeedent’) or
toxic members, and also members arguably merely anomalous: distinctive,
odd chemicals, ‘un-food-like’ to the perceptions of many grazers, and per-
haps somewhat inaccessible to many species’ digestive enzymes.
Axiomatically, evolution demands the ability to adapt, that is, to change.
Genomic information systems may have various mechanisms for second-
order logic, controlling their own adaptability, functioning to set the rate of
change. Adapted constitutive changeability has to be seen as to some degree
prospective: a lineage has no way of predicting when change will become
obligatory. Thus, an exigency is exerted upon evolving species to produce
initially merely anomalous changes responding, in effect, to a general need
for change. Species that anomalize themselves to grazers and predators may
be responding to an enhanced need to generate an ever-progressing unrecog-
nizability, like a cryptographic device using fresh encryption settings. Such
changes can be viewed in models of evolution as a search of the evolutionary
landscape for advantage in semi-randomized novelties. Some of these anom-
alies, as mentioned, may become stabilized as interrappellatives, but in
anomalizing selection, some may be stabilized for a time as anomalies. We
will make a case elsewhere that the bright colours of many mushroom spe-
cies are a quintessential example of this pattern in evolution. Because these
characters evolve in processes ‘recalling’ (responding to) the general evolu-
tionary conditions favouring the constant elaboration of new diversity, they
are designated ‘general-rappellative’ characters. This conceptually difficult
character type can be conceived of as the output of adapted innovation
mechanisms prefiguring the generation of other, more specific adaptations.
Character type 2a is very closely related to 2d and may be seen as a subset
of it. It is not clear how much genetic mutation is truly unavoidable—no
doubt some errors fail to be repaired by chance alone, but the efficiency of
such repairs is itself an adaptable feature. Rapidly evolving animals such as
humans, canines, and rats are notoriously cancer prone, arguably as a conse-
quence of inadequately repaired DNA lesions. Some lineages such as sharks
(Lee and Langer 1983; Luer and Luer 1982) and decapod crustacea (Vogt
2008) have at least a preliminary reputation as being less prone to carcino-
genesis (In sharks, it must be noted, discussion of this idea has been clouded
by tendentious argument about the extreme ‘straw man’ position that ‘sharks
never get cancer’, something allegedly propounded by people touting shark
cartilage as an alternative medicine remedy; see Ostrander et al. (2004).
Regardless of what the true story is with the sharks, it remains very much an
evolutionary possibility that our intrinsic human carcinogenic disease con-
trol, based on oncogenic resistance mechanisms involving accurate repair of
damaged DNA, prompt apoptosis of affected cells, and other processes may
have been partly sacrificed in the achievement of our recent evolutionary
success.
An analysis of the characters seen by early botanists as showing hidden spiritual

symmetries in nature, together with later-described taxonomically revelatory ana-
logues such as mechanisms of conidiogenesis, shows that these characters are, to a
large extent, developmental interrappellatives. The few purely rappellative features
that are seen in the different conidiogenesis mechanisms mainly consist of features
spacing newly forming conidia apart from one another in conidiogenesis adapted
for airborne dispersal. Clearly, sympodulae, producing conidia well distanced from
each other, are less likely to be involved in producing mucoid conidial masses
favouring arthropod transmission than are phialides, which can readily be adapted
to produce many conidia amassed in a small volume of space. Other features, such
as the percurrent extensions seen in annellides, the cell wall splitting and separating
cells seen in different subclasses of thallic conidiogenesis, and the retrogressive
progress of conidiation seen in species like Trichothecium roseum, are relatively
pure developmental patterns held in stasis by the need for conidia, as more-or-less
standardized propagules, to be sequentially formed and released in an orderly man-
ner. That these conidiogenesis mechanisms are perhaps closely ontogenetically
related and, in principle, truly fungible can be seen in a few species like Trichothecium
indicum (Summerbell et al. 2011), which produces retrogressive conidia, sympodial
conidia, and phialoconidia. Another example lies in the members of the main
Exophiala-Cladophialophora-Fonsecaea-Phialophora-Rhinocladiella-Capronia
‘black yeast’ clade, arguably all one genus, in which multiple species can produce
two or more elements of a list including acropetally extending, branching blastoco-
nidial chains, annelloconidia, sympodial conidia, meristematic fission cells, and
phialoconidia. In the case of ‘black yeasts’, there may be some rappellative parti-
tioning among the conidial types into airborne and subaqueous or arthropod-
dispersed forms, with a complex ecology favouring polymorphism, but clearly, even
in the relatively few organisms featuring multiple types of conidiogenesis, each type
is stably reproduced with its spate of distinctive features, such as the relatively large,
vase-shaped, darkened collarettes seen in many of the phialides produced by the
‘black yeast’ clade.
No evolution towards efficiency in the relatively stripped-down Hyphomycetes
can eliminate the details of conidiogenous development, and thus most species are
distinctly fixed in one of the major variants of this system. Even in yeasts and other
sequentially reproducing single-celled fungal states, where conidiogenesis (when-
ever non-ballistic) tends to produce progeny cells immersed in highly physically
comparable aqueous or oily habitats, arbitrative differences in conidiogenesis are
prominent. There are splitting cells, as seen in Schizosaccharomyces, blastoconidia,
as seen in most yeasts, phialoconidia, as seen in most Malassezia species and the
yeasty states of Lecythophora species, annelloconidia as seen in the ‘black yeast’
clade as well as Hortaea, and even sympodially produced conidia, as seen in
Malassezia sympodialis, Bullera, Kurtzmannomyces, and, most extravagantly,
Sympodiomycopsis.
An interesting study in the fungibilities of fungal conidiogenesis is seen in the
organisms in the scattered Eurotiomycete lineages that have adapted to aerially
infect mammalian bodies and use them as perennating structures (Plate 8.1).
Plate 8.1 Conserved and apomorphic conidiation in medically important Eurotiomycetes. (a–c)
Blastoconidial particulate morph of Blastomyces dermatitidis. (a) Nanic form, in sputum. (b)
Normal form, in tissue, Gomori methenamine silver (GMS) stain. (c) Conversion from filaments
to particulate phase, in vitro, Nomarski. (d) Arthroconidial host phase of Talaromyces marneffei.
(e–f) Coccidioides posadasii. (e) Spherule particulate morph in tissue, Periodic acid-Schiff stain.
(f) Disjunctive arthoconidia. (g) Blastomyces dermatitidis. Aleurioconidium (lateral disjunctive
arthroconidium) formation in filamentous form. (h–j) Histoplasma capsulatum. (h) Aleurioconidia.
(i) Blastoconidial particulate morph in vitro. (j) Blastoconidial particulate morph in human macro-
phage cells, GMS stain
Although experimental confirmation of this scenario has proved elusive for these
species, their inoculum appears to persist in the infected host until the animal dies,
whereupon the fungus is released into nitrogenously enriched soil. The species
involved make up most of the small group of human and animal invaders referred
to as ‘dimorphic systemic pathogens’. They are Blastomyces spp. (Ajellomyces pro
parte), Emmonsia spp., Paracoccidioides spp., Histoplasma capsulatum, and
Coccidioides spp. in the Onygenales, and Talaromyces marneffei in the Eurotiales.
They have all developed forms of conidiogenesis within the host that are radically
different from the conidiogenesis normally seen in vitro and, presumably, in the
non-host environment.
The non-host, ‘environmental form’ conidia of the Onygenalean species are all
variations on a theme of aleurioconidia (Plate 8.1g, h) and alternate arthroconidia
(Plate 8.1f), which are the lateral and intercalary manifestations of the same conid-
ial release mechanism (Sigler 1989). These conidia separate from subtending cells
via the lysis and breakage of an empty disjunctor cell. Single-celled conidia released
in this way can readily become airborne and, in species possessing the necessary
virulence characters, can infect the respiratory systems of hosts. Likewise, the typi-
cal penicillium-type catenulate phialoconidia of T. marneffei are specialized for air-
borne dissemination.
Within the host, however, the spread of the dimorphic-systemic organisms through
the bloodstream and lymph system requires a planktonic assimilative form able to
pass through vessels. In many species, particulate fungal cells are incorporated into
host macrophages (Plate 8.1j), where any linear growth would tend to destroy these
sheltering host cells. In addition, the host form, free of the need to resist desiccation
and of the need to exert and integumentally restrain the turgor force of hyphal pene-
tration, is evolutionarily free to develop a biochemically modified cellular integu-
ment that will minimize the diversity of potential antigens presented to the immune
system. The yeast phase of Histoplasma capsulatum (Plate 8.1i, j), for example,
produces proportionately less of the highly immunoreactive ß-1,3 glucan than the
hyphal form and, in virulent chemotypes, tends to produce more α-1,3-glucan
(Guimarães et al. 2011). Conidial reproduction in the host is thus modified to accom-
modate the exigencies and opportunities particular to that habitat.
The particulate assimilative phases that have evolved in rapprochement with
this mammalian niche are unexpectedly diverse. Blastomyces, Histoplasma,
Paracoccidioides, and Emmonsia pasteuriana have revived or re-evolved path-
ways for producing budding yeast structures, although the ‘budding on a broad
base’ yeasts of Blastomyces (Plate 8.1a–c) and the multipolar budding yeasts of
Paracoccidioides are highly distinctive in morphology. Only Histoplasma pro-
duces a yeast phase that can be confused morphologically with those seen in dis-
tant basal Ascomycete lineages in the Saccharomycetes and Taphrinomycotina.
Blastoconidiation in all these cases occurs without any formation of the disjunctor
cell seen in all conidiation occurring off the host.
Coccidioides, as seen in the host, has evolved completely differently from other
Onygenales, producing structures of a type rarely seen in the fungi. Endoconidia are
produced by the internal fragmentation of multicelled, spherical mother cells (Plate
8.1d). Just a very few Ascomycetous genera are known to produce endoconidia in
sacs or cysts, namely Phaeotheca, Phaeothecoidea, Hyphospora (Seifert et al.
2011), Endoconidioma (Tsuneda et al. 2004), and, arguably, the obligate pulmonary
pathogen Pneumocystis. Environmental conidia of Coccidioides species are alter-
nate arthroconidia separated by disjunctors (Plate 8.1f).
Meanwhile, T. marneffei, in its host conidiogenesis, has evolved to recapitulate
the fission-yeast form (Plate 8.1d) best known from extremely remotely related
Schizosaccharomyces, placed by Schoch et al. (2009) in the Taphrinomycotina.
Schizosaccharomyces is the genus most closely related to Pneumocystis.
Pneumocystis itself can be mentioned as a possible remote analogue of the host
states of Eurotiomycetous dimorphics. It produces two kinds of propagules, ‘intra-
cystic bodies’ and ‘trophozoites’. These names hearken back to the time when this
genus was thought to be protozoan. The eight intracystic bodies that form in each
reproducing cyst appear to be diploid ascospores forming within an ascus (De Souza
and Benchimol 2005). These ascospores, upon release from the ascus, germinate to
give rise to the irregularly shaped trophozoites, which were sometimes termed
‘amoeboid’ in the past. These forms, however, are non-motile, mitotically dividing
fission cells, with a thin integument of typical fungal cell wall polysaccharides con-
taining mannose and N-acetyl-glucosamine residues. They produce static, filiform
projections that may aid in attachment to cells, but otherwise, they are typical par-
ticulate assimilative cells reproducing by schizolytic conidiogenesis. Only shape
and habitat distinguish them from the fission cells of the related Schizosaccharomyces.
Pneumocystis is simply another fungus that has utilized an unusual non-filamentous
form of conidiation in its particulate growth in mammalian tissue.
The ‘conservatism’ of the non-host conidiogenesis in the Eurotiomycetous
dimorphic systemic pathogens, contrasted with the ‘liberality’ of the divergent
conidiogenous adaptations developed in the host forms, illustrates the stabilizing
effect of interrappellative developmental systems. Only when opportunities change
significantly, and highly altered, quasi-rappellative host–pathogen exigencies begin
to pertain, do the very stable processes of thallic-disjunctive or phialidic conidio-
genesis become replaced or supplemented with completely different mechanisms.
The ability to evolve radically different conidiogenous mechanisms can be said to
lie latent in any of these organisms, but usually only to manifest when an environ-
ment renders the established interrappellatives non-functional or developmentally
disengaged. (As an example illustrating what we mean by the latter descriptive,
typical alternate arthroconidia would fail to be produced in Coccidioides in its
growth in the host in part because formation of the filaments from which they derive
is not induced.)
A relict of the hypothetical, protean transitional form through which most of
these Eurotiomycete dimorphic host phases may have arisen is perhaps seen in
Emmonsia parva and E. crescens. These species infect the lungs of small mammals
as their relatives do, but undergo no conidiogenesis or other cellular reproduction
there. (Their close relative, Emmonsia pasteuriana, produces budding yeast).
Instead, the airborne conidia, caught in the respiratory passages, simply swell up
and develop a progressively thickened cell wall. The resulting highly enlarged,
tough ‘adiaspores’ may cause death of the host by occlusion of the lower airways.
One can easily imagine that the more complex forms produced by related
Onygenalean species originally arose from conidia that persisted and, while possi-
bly shedding some outer cell wall layers in incipient germination, swelled in the
lungs rather than germinating as filaments. Eventually, after this simple form of
mammalian colonization became reinforced by favouring survival of the fungus in
stressful habitats, new, particle-producing methods of cellular proliferation devel-
oped, increasing the success of each affected species by allowing it to disseminate
through the circulatory system and colonize tissues beyond the lungs.
The fission cells produced by T. marneffei distinctly resemble fragmenting
hyphae in culture at 37 C (Plate 8.1d) and may have arisen through a genetically
relatively simple development producing cell wall schizolysis along the septal
plane. Their ontogenetic similarity to Pneumocystis trophozoites may be purely

coincidental or may carry some degree of symplesiomorphy in the retention of
enzymatic programmes for lysing middle lamellae of walls in adjacent cells.
Outside the Eurotiales and Onygenales, there are other pathogen host states
that illustrate the potential for conidiogenous plasticity in Hyphomycetes.
Chaetothyrialean fungi of the ‘black yeast’ clade, e.g. Cladophialophora carrionii
and Phialophora verrucosa, causing subcutaneous ‘chromoblastomycosis’ infec-
tions, produce schizolytic fission cells in infected tissue. Similar cells may also be
produced in stems of infected plant hosts (de Hoog et al. 2007). That this is an evo-
lutionarily highly localized form of conidiogenesis is perhaps illustrated by the fail-
ure of the nominally congeneric Cladophialophora bantiana to produce a particulate
vegetative state in its typical cerebral infections, and by the prevalence of blastic or
minutely annellidic budding yeast cells in closely related Exophiala species. Some
typical ‘black yeast’ fungi, like Exophiala phaeomuriformis, can produce thecate
‘meristematic’ cell clumps under certain metabolic conditions, and these appear to
bear some ontogenetic relationship to the developing fission cells of the chromo-
blastomycosis agents, though they also resemble pseudothecial initials. In the chro-
moblastomycosis agents, however, the process of schizolytic conidiogenesis
producing individual cells from meristematic clumps has been regularized.
Dermatophyte fungi in the Onygenales (Arthrodermataceae), infecting the super-
ficial layers of human and animal skin, may have life cycles where they spend part
of the life history growing on shed keratinous materials such as rodent hairs in the
environment, or they may be essentially entirely restricted to growth on their hosts,
as far as is known. In the latter case, as seen in the most common species on humans,
Trichophyton rubrum, their conidiogenesis is altered to a fission process involving
adjacent swollen cells within the skin. This ‘endoarthric’ process, based on produc-
tion and schizolysis of a septum derived from the inner cell wall within a friable
outer cell wall (Hashimoto et al. 1984), produces conidia referred to as ‘substrate
arthroconidia’. In culture, however, the species produces completely distinct, dac-
ryoid to clavate microconidia and subcylindrical macroconidia, as well as disjunctor-
separated arthroconidia, typical of Onygenales. This production of classic
thallic-disjunctive, aerial conidia in T. rubrum and other species known only from
growth on the host may be a truly vestigial feature, a latent programme only eluci-
dated by growth, after many thousands of years of diverging evolution, in a labora-
tory. Some human-associated dermatophytes like Trichophyton schoenleinii and T.
concentricum have been completely divested of this programme and never produce
aerial conidia, but can still produce the separable swollen cells in host material that
serve as the ecologically functional conidia. These substrate-arthroconidia appear to
produce lectin-like materials enabling them to bind efficiently to host skin
(Esquenazi et al. 2004), and as small separable particles, thus have an advantage
over any larger structures, such as filaments in shed skin fomites, which might also
be physiologically capable of infecting a new host. Here again, then, though the
typical Onygenalean conidiogenesis usually remains, it has been largely superseded
in the natural ecology by a de novo form, in this case one visually suggestive of the
sessile chlamydospores formed by other fungi in substrate materials.
It can take surprisingly little time for a fungal lineage to be permanently trans-
formed by host conditions. To give an illustration: as part of studies on Acremonium
species, we dealt early on with an unusually fast-growing species called A. falci-
forme that was known only from a slowly progressing, chronic human subcutane-
ous infection called mycetoma. (It grew more rapidly than other Acremonium
species, where a slow growth rate was a genus-defining character, though it grew
more slowly than any common Fusarium species.) It was unusual in producing
some curved conidia that were occasionally one-septate. Upon reading that an inky
blue to violet-purple pigment was sometimes seen in fresh isolates of this species
(e.g. Gams 1971), we were seized by the idea that it was probably a host-adapted
member of the Fusarium solani complex, members of which sometimes produce
such pigments. In part, the idea that a Fusarium could be so morphologically trans-
formed was influenced by years of work with aberrant host-adapted Aspergillus
fumigatus isolates, not to mention dealing with the polymorphous skin pathogen
T. rubrum (Summerbell 2000). It struck us also that the completely morphologically
different Cylindrocarpon lichenicola, also occasionally making bluish pigments
and involved in subcutaneous infections (Summerbell and Schroers 2002), could be
an F. solani sensu lato. After sequencing by Hans-Josef Schroers showed both
hunches to be correct, and the species were recombined into Fusarium, we realized
that there was a likely third example on the list of medically important fungi. The
species in question was named for the same blue pigment that had provided a clue
in the other cases. The ex type isolate of this fungus, originally isolated from a sub-
cutaneous infection (de Vries et al. 1984; De Bruyn et al. 1985), was revived from
the CBS collection as CBS 518.82 and secondary isolation CBS 637.82. Study
revealed a dense, slow-growing, off-white clump of thin-walled, colourless chla-
mydospores connected by short hyphal segments. Hyphae occasionally produced a
relatively long, tapering phialide that yielded some unicellular, ellipsoidal conidia.
No blue pigment matching that noted in the description was formed. To the essen-
tialist eye of the medical mycologist, if not to the formalist eye of the morphotax-
onomist, this enigmatic form was strikingly suggestive of Fusarium solani.
Sequencing by Hans-Josef Schroers disclosed that indeed, this species, originally
described as Phialophora cyanescens (de Vries et al. 1984) and later cannily trans-
ferred to Cylindrocarpon by Zoutman and Sigler (1991), represented a distinct hap-
lotype in the F. solani complex. The F. solani complex has recently been segregated
as the uninominate genus Neocosmospora by Lombard et al. (2014), and F. falci-
forme and F. lichenicola have become N. falciforme and N. lichenicola. Phialophora
cyanescens is recombined here as
Neocosmospora cyanescens (G.A. de Vries, de Hoog & Bruyn) Summerbell,
Schroers and Scott, comb. nov. (MycoBank MB 813864)
Basionym: Phialophora cyanescens G.A. de Vries, de Hoog & Bruyn, Antonie van
Leeuwenhoek 50 (2): 150 (1984) [MB 107121]
≡ Cylindrocarpon cyanescens G.A. de Vries, de Hoog & Bruyn) Sigler, Journal of
Clinical Microbiology 29 (9): 1858 (1991) [MB 499349]
The point of describing this essentialist detective work here is to illustrate how
extremely malleable Hyphomycetes may be under any selective pressure. After 33
years in human tissue (De Bruyn et al. 1985), CBS 518.82, which at the time of
initial infection was probably a completely orthodox, rapidly growing F. solani
capable of producing normal macroconidia and microconidia, had stably adapted to
become a lump of aberrant chlamydospores with simplified, sparse conidiogenesis.
(Evaluation of this hypothesis of rapid morphological adaptation awaits isolation of
another representative of the exact same haplotype from nature.) Under immediate
host selection, also, it showed hyperproduction of a pigment that may fortuitously
have had some immunomodulatory effect. Fungal anthro- and naphthoquinone pig-
ments, including some incompletely characterized metabolites from F. solani (You
et al. 2013) as well as dermatophyte xanthomegnin (Alvi et al. 2000), may suppress
the inducible nitric oxide synthase pathway of mammalian immune response. This
protective pigment was soon lost or switched off in the culture collection. That
hyphomycetes can be so extremely plastic under emergent selection concomitantly
shows how remarkable the stability of their normal characters is.
Stability in fungal morphological characters can be achieved either through con-
stant ecologically mediated stabilizing selection (rappellative) or through some type
of developmental momentum, whether it be the functionally necessary coordination
needed for morphogenesis to produce well-adapted structures, or the functionally
superfluous vestigial holdovers from the production of previously functional struc-
tures (fungal equivalents of the vermiform appendix, if any). Conidiogenesis
appears to be unusually rich in such arbitratives because (1) coordinated, stably
reproducible development is required to produce ecologically optimized, morpho-
logically stereotypical conidia and (2) there are relatively few rappellative exigen-
cies that potentially come to bear on this process. Rappellative exigencies exerted
on conidiogenous cells per se mainly constrain:
1. Structures capable of producing airborne conidia
2. Structures capable of producing mucoid conidia or water-borne conidia (many
of these structures overlapping with items in the previous category, e.g. phialides)
3. Structures capable of producing intra-matrical and specialized host conidia
4. Structures capable of supporting very heavy conidia, unusually shaped conidia,
or other conidial types exerting or subject to unusual physical stress or requiring
unusual volumes of space (a theoretical possibility yielding few obvious exam-
ples; one that comes to hand, perhaps, is the production of large, rounded
Pleospora [previously Stemphylium] conidia apically on well-separated annel-
lides in a clade where most related genera, like Bipolaris, produce narrower
conidia closely spaced together on tretic sympodulae)
This short list is in contrast to the considerably longer preliminary list of rappel-
lative factors that can be compiled for the form of conidia themselves, including
(with short mnemonic words for each category in parentheses),
1. Structures facilitating airborne dispersal, e.g. small size or small aerodynami-
cally equivalent size, fragile attachment, surface roughening (‘flying’)
2. Structures and external chemistry physically facilitating arthropod dispersal

(‘sticking’, ‘hooking’)
3. Structures and dimensions facilitating penetration of the physical boundary
layer about a surface subject to moving air or water (‘impaction’, ‘hooking’,
‘entangling’)
4. Structures mediating buoyancy in water or affinity for the interface, where
applicable (‘floating’)
5. Structures physically conferring resistance to grazing (‘deterring’)
6. Structures and chemistry (e.g. melanin) conferring resistance to physical fac-
tors such as drought and light (‘resisting’)
7. Structures facilitating germination of resistant forms (‘splitting’)
8. Structures facilitating detachment of mucously attached conidia from arthro-
pod vectors (‘detaching’, examples being the pointed, crescent-shaped ends of
many Fusarium macroconidia and, in general, the curvature of Fusarium and
many Neonectria macroconidia)
9. Structures physically stabilizing the conidium on the conidiophore during for-
mation (‘stabilizing’, e.g. the extended portion of the ‘foot cells’ of Fusarium
macroconidia)
10. Structures and dimensions allowing storage of sufficient food reserves to ger-
minate on recalcitrant substrata (‘storing’)
Thus, when one observes the forms of conidia, one mainly sees characters that
directly reflect the physical ecology of the organism, sometimes intermixed with
basic physical modifications influencing vector dispersal and anti-predation interac-
tions. The influence of physical or ecological stressors is only minimally seen when
one examines the forms of conidiogenous cells, particularly the aspects focused on
in twentieth century taxonomy. This is the most likely explanation of why conidi-
ogenous cells tend to remain true to form within lineages. The structures are prob-
ably not highly developmentally constrained: it is perhaps not a major genetic leap
to interconvert sympodial and annellidic conidiogenesis, for example, and there are
genera like Pseudeurotium where ambiguous structures resembling both of these
categories have long puzzled investigators. Likewise, to diminish percurrent prolif-
eration so that an annellide becomes a phialide may not be genetically ‘difficult’, if
there is any selection pressure tending to impose such a transition. Because the
conidiogenous cell structures, however, are probably so strongly equally functional
in most habitats and niches, there are likely to be stronger interrappellative
(coordination-based) genetic factors tending to maintain them than environmental
(rappellative) or genetic (drift/mutational or general-rappellatively programmed-
explorational) factors tending to alter them.
At this point in biosystematic history, we can look through the conidial micro-
fungi and begin to determine how closely the taxa partly based on consistent conid-
iogenesis reflect the underlying phylogenetic structure. Ultimately, perhaps, a
phylogenetic overview of conidiogenesis types in all ascomycetes and basidiomy-
cetes, including lichenized forms, would be interesting, following the prototype set
for ascomatal characters such as ascus dehiscence mechanisms in Schoch et al.’s
(2009) supplemental Fig. 6. Conidiogenesis structures are somewhat more variable

than the ascomatal structures annotated there, and the result of such an effort would
be compendious. A beautifully illustrated preliminary survey of the Hyphomycetes
and their conidiogenesis has been published by Seifert et al. (2011) in the introduc-
tory overview to the Genera of Hyphomycetes. It excludes many items of interest in
the context of this chapter, including yeasts, pycnidial fungi, and specialized host
infection conidia.
Fungi of human medical interest make up a limited survey of species, mostly
ascomycetous, that have by now been well covered by molecular biosystematics, at
least at the preliminary level. Table 8.1 shows the patterns of conidiogenesis seen in
a selection of these species, as well as a few of their close relatives and other fungi
well known from the clinical laboratory and its impinging indoor air. Only ascomy-
cetous fungi are included. The order followed is that seen in Schoch et al.’s
Supplemental Fig. 6, and follows the biosystematic spine of the dendrogram
(Schoch et al. 2009), not the cut-and-paste individual figures (6A, 6B, etc.) used to
show details.
Chlamydospores that are not known to have a regular dehiscence mechanism are
not included in Table 8.1.
As can be seen, the division of conidiogenesis into its airborne, moisture-borne,
and intramatrical rappellatives considerably clarifies the significance of the diver-
sity of forms. For example, Pleosporales producing the typical heavy, dark, leaf-
impacting airborne conidia of the group mainly produce tretic-sympodial, cylindrical
phragmoconidia or blastic or annellidic dictyoconidia. Alternaria (inclusive of
Ulocladium) has an intermediate form, a tretically formed dictyoconidial type, with
sympodial proliferation inconspicuous in some species. The variably disarticulating
blastic sympodial chains of Cladosporium, producing single conidia, short chains,
and longer chains, branched and unbranched, are an elegant adaptation to simulta-
neously make an extensive range of aerodynamically different configurations using
a single mechanism. On the other hand, Pleosporales producing potentially arthro-
pod- or splash-dispersed mucoid conidia tend to feature pycnidia with small
phialides or annellides.
Phialides, an item found in members of most groups other than Onygenales and
Saccharomycetes, are equally used for airborne and moisture-borne conidiation, but
very rarely for intramatrical forms, the exception being found in a few organisms like
Lecythophora with yeast-like repetitive conidiation carried out by phialidic cells.
The Hypocreales are almost completely dedicated to phialidic conidiogenesis, except
in a few cases where budding yeast forms are produced in infected insects (Beauveria,
Metarhizium) or where warty, resistant aleurioconidia are produced (Sepedonium,
Mycogone). We have classed the latter as substrate conidia even though they are
produced on aerial hyphae in culture, in that their heavy form and relatively sturdy
attachment suggest they may persist in the substrate (if soil) or near its location (if it
was, for example, an infected agaric) rather than dispersing. Such structures may be
adapted for heat resistance in fire ecosystems (Hambleton et al. 2005).
The overall impression given by Table 8.1 is that multiple class-level groups of
conidial ascomycetes have reproduced, whether as symplesiomorphies or as a series
Table 8.1 A survey of conidiogenesis types seen in a phylogenetic arrangement of medically important ascomycetous fungi, common clinical contaminants,
and selected related genera
Mucoid/aquatic conidia
(including pycnidia with
Clade Genus Aerial conidia desiccating mucoid cirrhi) Substrate or host conidia
Sordariomycetes Beauveria Blastic, sympodial, Blastic (budding yeast)
8 Conidiogenesis…
Hypocreales sometimes synnematal

Purpureocillium Phialidic, chains with
connectives
Stachybotrys Phialidic, chains Phialidic, heads
Metarhizium Phialidic, chains, Blastic (budding yeast)
sporodochial
Fusarium Phialidic, chains Phialidic, heads, single, and
sporodochial
Acremonium Phialidic, chains Phialidic, heads
Sarocladium Phialidic, chains Phialidic, heads
Trichothecium Blastic, retrogressive; Phialidic, heads
Blastic, sympodial
Sepedonium Phialidic, heads Blastic, aleurioconidia
Sordariomycetes Scopulariopsis Annellidic, chains
Microascales
Scedosporium Annellidic, mucoid
Ceratocystis Phialidic, chains Blastic, aleurioconidia
Thielaviopsis Phialidic, chains Blastic, aleurioconidia
Sordariomycetes Togninia (Phaeoacremonium), Phialidic, heads
Calosphaeriales Pleurostoma
(Pleurostomophora)
(continued)
187
188
Sordariomycetes, Sporothrix Blastic, sympodial Blastic, aleurioconidia in
Ophiostomatales substrate; Blastic budding
yeast in host
Leptographium Annellidic, mucoid
Sordariomycetes, Phialemoniopsis Phialidic, heads, single, and
unnamed order sporodochial
Sordariomycetes, Chaetomium (Taifanglania) Phialidic, chains Blastic, aleurioconidia
Sordariales
Sordariomycetes, Lecythophora Phialidic, heads Phialoconidiation, repetitive
Coniochaetales
Sordariomycetes Phialemonium Phialidic, chains Phialidic, heads
Cephalothecaceae
Sordariomycetes, Arthrinium Blastic, basauxic
Xylariales
Ascotricha (Dicyma) Blastic, sympodial
Leotiomycetes, Botrytis Blastic, simultaneous
Sclerotiniaceae
Leotiomycetes, Cadophora, Phialocephala Phialidic
Helotiales
Leotiomycetes, Scytalidium Arthric-schizolytic
unnamed order
Leotiomycetes, Oidiodendron, Geomyces Arthric-disjunctive
Myxotrichaceae
Amorphotheca Blastic, branching chains
Leotiomycetes, Pseudeurotium Blastic, obscurely sympodial
Pseudeurotiaceae
R.C. Summerbell and J.A. Scott
Eurotiomycetes, Exophiala/Rhinocladiella (often Blastic, sympodial Annellidic, blastic (budding
Chaetothyriales synanamorphs) yeast)
Cladophialophora, Blastic, sympodial Phialidic Fission cells schizolytic
Fonsecaea branching chains
Phialophora, Phialidic Fission cells schizolytic
Cyphellophora, (Phial. verrucosa), Blastic
8 Conidiogenesis…
Phaeomoniella budding cells (Phaeom.

zymoides, etc.)
Eurotiomycetes, Aspergillus Phialidic, chains with Blastic, aleurioconidia (A.
Eurotiales connectives terreus)
Talaromyces Phialidic, chains with Fission cells schizolytic (T.
connectives marneffei)
Penicillium Phialidic, chains with
connectives
Paecilomyces Phialidic, chains with
connectives
Phialosimplex Phialidic, chains Phialidic, mucoid heads
Monascus Blastic, retrogressive Arthric –schizolytic
Eurotiomycetes, Trichophyton, Arthric-disjunctive (empty Arthric –schizolytic
Onygenales Microsporum, separating cell)
Epidermophyton
Histoplasma, Arthric-disjunctive Blastic (budding yeast)
Blastomyces,
Paracoccidioides
Coccidioides Arthric-disjunctive Thecate, endoconidial fission
Emmonsia Arthric-disjunctive Blastic (budding yeast) (E.
pasteuriana)
(continued)
189
190
Dothidiomycetes, Cochliobolus (Bipolaris, Tretic (blastic-cicatrized)
Pleosporales Curvularia) with sympodial
proliferation
Alternaria Tretic (blastic-cicatrized)
with sympodial
proliferation
Pleospora (Stemphylium) Tretic (blastic-cicatrized)
with percurrent
proliferation
Phoma Phialidic, in pycnidia
Coniothyrium Annellidic, in pycnidia
Nigrograna Phialidic, in pycnidia
Paraconiothyrium Phialidic, in pycnidia
Epicoccum Blastic or polyblastic Phialidic, in pycnidia
aleurioconidia in
sporodochia
Dothidiomycetes, Lasiodiplodia Blastic, in pycnidia
Botryosphaeriales
Neoscytalidium Arthric–schizolytic Blastic, in pycnidia
Dothideomycetes, Aureobasidium Blastic, synchronous
Dothideales
Dothidiomycetes, Cladosporium Blastic, cicatrized, in
Capnodiales, sympodial branching chains
Davidiellaceae
R.C. Summerbell and J.A. Scott
Dothidiomycetes, Hortaea Annellidic Annellidic yeast-like
Capnodiales, particulate assimilative phase
Teratosphaer
iaceae
Stenella Blastic, sympodial
Dothidiomycetes, Septoria Blastic, sympodial, with few
8 Conidiogenesis…
Capnodiales, annellations, in pycnidia

Mycosphaerellaceae
Saccharomycetes, Candida, Saccharomyces Blastic; mainly budding yeast Blastic; mainly budding yeast
Saccharomycetales
Taphrinomycota, Pneumocystis Fission cells, schizolytic
Pneumocystidiales
191
of apomorphies, most of the basic possibilities of conidiogenous construction, viz.,

phialide, blastic, annellidic, sympodial, and the two major forms of arthric conidio-
genesis, schizolytic and disjunctive [of which ‘rhexolytic’, in the strict sense, is a
subcategory that lacks obvious autolytic processes, fide (Sigler 1989). At the same
time, there is extensive stability seen, with orders, families, and large generic com-
plexes sharing consistent patterns in conidiogenesis, sometimes partitioned to dif-
ferent mechanisms for airborne, liquid-borne, and intramatrical dispersal. In the
future, genome sequencing and subsequent data mining studies, combined with
genetic knockout and targeted mutation, may clarify how many times features such
as the percurrent proliferation seen in annellides have evolved independently. This
type of study will yield insight not only into the degree of convergent evolution that
has occurred historically in conidiogenous morphogenesis, but also into the degree
of intrinsic informational complexity involved in engendering interrappellative sta-
bility in the continuing production of such structures. If features like annellidic and
sympodial proliferations turn out to be long-lasting, highly conserved symplesio-
morphies that are, in effect, switched on and off within major lineages, that suggests
that these processes entail complex coordination that is not easily convergently
evolved. If, on the other hand, such features emerge repeatedly as apomorphies on
the genetic level, then the degree of coordination involved in the morphogenetic
processes involved should be found to be relatively low.
As a final note, to anyone speculating that this information is all less interesting
than a dendrogram precisely locating conidial fungi into their evolutionary histori-
cal context, we would only point out that lectical considerations apply just as much
to DNA sequences as to morphological forms. Pure lineal history is indicated by
arbitrations, minimally constrained DNA mutations, assumed to reflect, via an
assumption of near-randomness, a time clock. When aligning regions like ribo-
somal DNA sequences, one sees regions that are ‘conserved’, highly functionally
constrained, either via interrappellation (selection for correct ribosomal folding and
coordination with other biomolecules) or rappellation (assuming a configuration
with adequate tolerance of heat, osmotic stress, and efficient speed of protein pro-
duction). In coding genes, the third bases of codons may, of course, be a fraction
less constrained. These areas are subject to rappellative and interrappellative stabi-
lizing selection and, when they do evolve significantly, may do so in a punctuated
way, where a change in a small number of items may entail an acceleration of
coordination-optimizing changes in many others. One evolutionary question that
we know next to nothing about is whether rappellative exigencies or opportunities
can entail significant convergent evolution even in DNA sequences. For example, if
two organisms evolve a lactase enzyme completely independently of one another, is
there sufficient fungibility in the potential form of the actively catalytic protein
regions involved that the sequence of enzyme A can be completely different from
that of enzyme B? What about cases where the organisms involved are related at the
level of the taxonomic class and share some common genetic background—will
their independently evolved lactases be highly likely to share sequence motifs?
Nothing, of course, necessarily constrains such a predisposed convergence as the
organism’s only solution to the opportunity presented by lactose, but the actual
responses of organisms to such opportunities are subject to statistics and probability.

We know relatively little, so far, about the statistics of convergent and semi-
convergent (convergence from shared symplesiomorphic basis) evolution in DNA
sequences coding for functional proteins. We do not know the extent to which
including coding regions in phylogenies will, in effect, lead us back towards a sys-
tematics based on trees, shrubs, and herbs.
In marked contrast to the constrained sequence regions, some areas within spacer
regions are so labile that they may be poorly alignable except within tightly related
clades. They are subject to coincidental convergence at the level of the individual
base pair. Microsatellite repeat regions likewise are predictably vulnerable to statis-
tical coincidence, e.g. when investigators contrast lineages whose sequences show
six repeat motifs to those with sequences showing seven repeats. Pure neutrality,
arbitrative condition 2a, above, may be inscrutable when it is found in DNA
sequences.
Fortunately, there are regions of possible low-specificity rappellative and/or
interrappellative constraint, as seen in the more highly alignable regions of the ribo-
somal internal transcribed spacer. Mutation about these regions may be constrained
by inter- and intramolecular interactions arising from functionally necessary nucleic
acid folding or twisting, or from coordination (whether amino-acid-specific or at the
general van der Waals level) with regulatory proteins or nucleic acids, or from a
need for precise physical qualities (e.g. pH). The somewhat constrained DNA-base
fungibility applying to these regions arguably provides the best available molecular
clock for dating the divergence of lineages. Ultimately, understanding of the subcat-
egories of constraint on DNA sequences may provide a mathematical means of
selecting regions that truly give the best independent phylogenetic signal, optimally
free (to use metaphors) of both the flywheel noise arising from excessive mutation
and the brake noise arising from excessive rappellative constraint.
The bottom line, however, is that DNA is a highly complex, self-interacting
construction template and, as such, is a vast repository of developmental, interrap-
pellatively modulated taxonomic characters. It, thus, provides the same fundamen-
tal type of information that the study of conidiogenous mechanisms provided, but
does so in much higher quantity. Awareness of this typological correspondence
should allow us to maintain our respect for the insights Hughes and contemporaries
gained from studying conidiogenesis. Even if such revelatory structures are not
seen as the mycelial needlework of God, their discovery should still stimulate due
awe at the ability of good scientists to unlock the deep secrets of reality.
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Chapter 9
Fungal Diversity of Central and South
America
Rafael F. Castañeda-Ruiz, Gabriela Heredia, Luis F.P. Gusmão,

and De-Wei Li
Introduction
Since the last two decades of the twentieth century, numerous novelties of hypho-
mycetes have been published from tropical Central and South American forests and
semiarid areas. These fungi were collected from tropical forests, jungles, and semi-
arid vegetation as well as from decaying plant debris accumulated on the leaf litter
surface or submerged in streams and rivers (Castañeda-Ruiz et al. 1998; da Cruz
et al. 2008; Gusmão et al. 2008; Heredia et al. 2007; Monteiro et al. 2014, 2015;
Pereira-Carvalho et al. 2009). The quantity and diversity of fungi found suggests the
R.F. Castañeda-Ruiz, Ph.D. (*)

Instituto de Investigaciones Fundamentales en Agricultura, Tropical “Alejandro de
Humboldt” (INIFAT), Académico Titular de la Academia de Ciencias de Cuba,
Calle 1 Esq. 2, Santiago de Las Vegas, C. Habana C. P. 17200, Cuba
e-mail: rfcastaneda@inifat.co.cu; rfcastanedaruiz@gmail.com
G. Heredia, Ph.D.
Red de Biodiversidad y Sistemática, Instituto de Ecología,
A. C. Km. 2.5 Antigua Carretera a Coatepec No. 351 Congregación El Haya, C.P. 91070
Xalapa, Veracruz, Mexico
e-mail: gabriela.heredia@inecol.mx
L.F.P. Gusmão, Ph.D.
Laboratorio de Micologia, Departamento de Ciências Biológicas, Universidade Estadual,
de Feira de Santana, BR116 KM03, 44031-460 Feira de Santana, BA, Brazil
e-mail: lgusmao@uefs.com
D.-W. Li, Ph.D.
e-mail: Dewei.Li@ct.gov

DOI 10.1007/978-3-319-29137-6_9
198 R.F. Castañeda-Ruiz et al.
existence of an indefinite number of undiscovered fungi still remaining in the tropi-

cal and subtropical forests in these geographical regions (Mueller and Schmit 2007)
according to the estimation that 1–1.5 million of fungi are present on Earth
(Hawksworth 1990, 2001).
In this chapter, the authors discuss the novelties of fungal diversity of Central and
South America described during 2000–2015, with a focus on hyphomycetes, and
provide the detailed methodology proposed by Castañeda-Ruiz (2005) and used by
the authors in the collection and incubation of samples colonized by hyphomycetes.
Collection of Samples
The choice of location to collect study materials depends on the collection area and
its surroundings. Small streams in the forests of mountainous regions sometimes
produce cracks in the soil where plant residues accumulate. The plant materials are
often in the areas near the course of the stream subject to constant washing.
Generally, the degree of colonization in these substrates is lower than those on the
sides or near the shore, farther from the current course, and receiving a soft wash,
sometimes almost imperceptible. The fragments of bark, branches, pods, flowers,
and other debris submerged in relatively quiet areas close to the rapids, cascades,
and waterfalls of the stream where bubbles or foam accumulates are ideal for col-
lecting hyphomycete-colonized materials (Figs. 9.1, 9.2, 9.3, 9.4 and 9.5).
The samples may consist of leaves, twigs, bark, stems, flowers, pods, or other
structures of plant origins and are collected in plastic bags or plastic flasks for trans-
portation to the laboratory.
The terrestrial samples of the hyphomycete-colonized plant materials are usually
found on steep hillsides, which are clad in dense, almost impenetrable vegetation.
The trees are covered in epiphytic bromeliads and orchids, and the dead leaves that
yield many of the new hyphomycetes are found lying in small crevices in the rocks,
or hidden underneath the branches of shrubs or creepers. Cryptic, undisturbed
microhabitats appear to maintain the leaves, branches, bark, and other plant debris
at a relatively high humidity during much of the year (Castañeda-Ruiz and Kendrick
1991). Numerous uncommon or new hyphomycetes can be collected under the big
trunks of the fallen trees in the rainforest, and sometimes several hyphomycetes are
growing on a piece of debris about 2–3 cm2 (Castañeda-Ruiz et al. 2011).
Sample Washing
Undesirable particles and sediments are washed off with tap water. It is important
that the jet of tap water does not directly hit the plant materials because in some
cases the material is weakly colonized by the assimilative hyphae. It should be
enough to let a flow of water passing through the whole sample for 20–45 min to
9 Fungal Diversity of Central and South America 199
Fig 9.1 Collection site of tropical hyphomycete diversity
Fig 9.2 Collection sites (a different site) of tropical hyphomycete dicersity

Fig. 9.3 A boat view showing plant debris accumulating along river banks in a tropical area
Fig. 9.4 Sampling materials on the soil in the riparian zone in a rainforest
Fig. 9.5 Sampling aquatic hyphomycetes
wash off surface soil particles, protozoa, nematodes, insect, and other organisms in
the samples. A soil sieve and a vegetable washing basket are used to wash different
diameters and sizes of the samples for the study of anamorphic fungi, since tiny col-
onies in fragments or reduced size can be recovered and will not be lost (Fig. 9.6).
Sample Drying
Plant debris is placed on a paper towel or a filter paper for 1–2 h to remove excess
water before putting them in moist chambers or sterile water according to the origin of
the sample and the ecological group of hyphomycetes studied. The tap water film on
the surface should completely disappear before the incubation (Figs. 9.7, 9.8 and 9.9).
Incubation
The washed plant material is divided or cut into two parts. A part of the washed
material is dipped into plates or containers filled with sterile distilled water in a Petri
dish and placed in an incubator at a temperature of 20–25 °C or a cooler (Fig. 9.10)
at room temperature.
Fig. 9.6 Samples washed with tap water
Fig 9.7 Samples placed on paper towels to remove excessive water

Fig 9.8 A close view of the samples placed on paper towels to remove excessive water
Fig 9.9 Another close view of the samples placed on paper towels to remove excessive water
Fig. 9.10 A cooler used for sample incubation
The other part of the sample is placed in a Petri dish or a flask with a perforated
lid prepared with a filter paper and absorbent paper or cotton at the bottom. Several
drops of water are added to provide the necessary moisture within the container. The
surface of litter incubated should be free of water to facilitate sporulation and pre-
vent other undesirable organisms from developing. The plates or containers with the
selected materials are placed within a 50 L cooler in which the inner walls were pre-
viously covered with wet absorbent paper towel and added 100–400 ml of distilled
water and 2 drops of glycerol to maintain a moist environment. The lid of the cooler
is positioned such that there is a slit of 2–3 mm and is situated opposite to an oscillat-
ing fan at its lowest speed throughout the incubation period to facilitate aeration and
air exchange inside the cooler. This is of great importance to ensure the production of
conidia in anamorphic fungi from aquatic resources (Figs. 9.10, 9.11, 9.12 and 9.13).
The cover of the cooler should be open for 30 min after 24 h of incubation or
ventilated with a fan at a low speed for 30 min. The water lost by evaporation is
replaced periodically. The relative humidity in the cooler is maintained between 99
and 100 %, the observation under the stereoscopic microscope starts after 48 or 72
h of incubation, and observations are repeated for up to 30 days.
Fungal Diversity in Central and South American Neotropic
The Neotropic is a terrestrial ecozone that includes South and Central America, the
Mexican lowlands, the Caribbean islands, and Southern Florida. It includes the
largest area of tropical and subtropical forests on the planet (Olson and Dinerstein
2002), which are considered the most important reserves of biodiversity on Earth.
Fig. 9.11 Petri dishes with plant materials on the filter papers in the incubating cooler
Fig. 9.12 The partially opened incubating cooler with a fan for ventilation
Among the 17 countries considered as megadiverse, six are located in the

Neotropic including Brazil, Colombia, Ecuador, Mexico, Peru, the United States,
and Venezuela. The diversity of microfungi inhabiting the rainforest and the corre-
lations with host plants and their debris have been estimated in several studies
(Hawksworth 2001; Blackwell 2011), and the authors have remarked on the impor-
tance of collecting and documenting the fungal diversity of tropical regions because
such regions have the greatest diversity of plants and we can therefore also expect a
Fig. 9.13 A hygrometer

used to monitor
temperature and relative
humidity in the incubating
cooler
large number of fungi. During the last 30 years, 3757 taxa of microfungi were
described from the Central and South American Neotropic, mainly from the rainfor-
est, cloud forest, Cerrado, and semiarid areas of Brazil, Costa Rica, Cuba, Ecuador,
Mexico, Panama, Peru, and Venezuela. These novelties were mostly collected from
soil, plant debris, and living plants, sometimes as plant pathogenic fungi and sub-
merged plant materials.
Microfungi from Living Plants
Most of the microfungi novelties were described from living plants as plant
pathogenic fungi, sooty molds, and endophytic fungi or associated with trichomes;
445 new taxa of the genera Cercospora, Cercostigmina, Distocercospora,
Mycovellosiella, Passalora, and Pseudocercospora and other cercosporoid fungi
were described by numerous authors such as Braun and Castañeda-Ruiz (1991),
Braun and Urtiaga (2012, 2013a, 2013b), Hernández-Gutiérrez and Dianese (2008),
Castañeda-Ruiz and Braun (1989), Crous et al. (1997, 1999), and Pons and Sutton
(1988). Other nectrioid fungi associated with living plants were discovered mainly
from Brazil by Alfenas et al. (2013) and Crous et al. (1993, 1998). Sooty molds and
other epiphyllous fungi were treated by Pinho et al. (2012a, 2012b, 2013, 2014),
Rodríguez-Hernández (1985), Rodríguez-Hernández and Camino (1987), Rodríguez
and Piepenbring (2007), and Rodríguez et al. (2015). Several interesting novel gen-
era as Alveariospora, Echinoconidiophorum, Globoconidiopsis, Globoconidium,
Helminthosporiomyces, Microtrichosphaera, Phaeoidiomyces, Phragmoconidium,

Trichomatoclava, Trichomatomyces, Trichosporodochium, and Vesiculohyphomyces
occurring on trichomes of plants in diverse localities of Brazil were added by
da Silva et al. (2012), Dornelo-Silva and Dianese (2004), and Pereira-Carvalho
et al. (2009), and some of these genera showed a curious combination of sympodial
and percurrent extensions of conidial development.
Microfungi from Soil, Plant Debris, and Submerged

Plant Materials
Along with its enormous diversity and endemism, Neotropical forests are subject to
many kinds of threats, which occur in other tropical regions of the world, consisting
of conversion of natural habitats to farmland and grasslands and forest degradation
as a result of overexploitation of hunting and logging (Zunino and Zullini 2003).
The documentation of fungal diversity-associated plant debris, soil, and sub-
merged plant materials in the forests of the Central and South American Neotropic
has increased due to the effort of mycologists of several institutions in Brazil, Cuba,
Mexico, Venezuela, and other countries. They described more than 2800 taxa which
included 104 new genera (Table 9.1, Figs 9.14, 9.15, 9.16, 9.17, 9.18, 9.19, 9.20,
Table 9.1 Genera of Genera

hyphomycetes and
Acarocybiopsis J. Mena et al. 1999
coelomycetes described from
soil, plant debris, and Amoenomyces R.F. Castañeda et al. 1996
submerged plant materials Amphophialis R.F. Castañeda et al. 1998
from Central and South Ampullicephala R.F. Castañeda et al. 1998
America (1985–2015) Anabahusakala L.T. Carmo et al. 2014
Anacraspedodidymum C.R. Silva et al. 2014
Anaexserticlava T.S. Santa Izabel et al. 2015
Anaselenosporella Heredia et al. 2009
Anaseptoidium Heredia et al. 2012
Ancorasporella J. Mena et al. 1998
Anungitopsis R.F. Castañeda & W.B. Kendr. 1990
Arachnospora R.F. Castañeda et al. 2003
Arthrodochium R.F. Castañeda & W.B. Kendr. 1990
Arthrowallemia R.F. Castañeda et al. 1998
Atrogeniculata J.S. Monteiro et al. 2014
Barretomyces Klaubauf et al. 2014
Bibanasiella R.F. Castañeda & W.B. Kendr. 1991
Bisseomyces R.F. Castañeda 1985
Brachycephala J.S. Monteiro et al. 2015
Brachyconidiella R.F. Castañeda & W.B. . Kendr. 1990
(continued)
Table 9.1 (continued) Genera

Brachyconidiellopsis Decock et al. 2004
Brevicatenospora R.F. Castañeda et al. 2006
Briansuttonia R.F. Castañeda et al. 2004
Brunneodinemasporium Crous & R.F. Castañeda 2012
Bulbocatenospora R.F. Castañeda & Iturr. 2000
Carrismyces R.F. Castañeda & Heredia 2000
Castanedaea W.A. Baker & Partr. 2001
Cheiromyceopsis Mercado & J. Mena 1988
Chlamydopsis Hol.-Jech. & R.F. Castañeda 1986
Chrysofolia Crous & M.J. Wingf. 2015
Circinoconiopsis A. Hern.-Gut.2014
Cubasina R.F. Castañeda 1986
Curvicladiella Decock & Crous 2006
Cylindrosympodium W.B. Kendr. & R.F. Castañeda 1990
Dentocircinomyces R.F. Castañeda & W.B. Kendr.1990
Digicatenosporium S. M. Leão et al. 2015
Digitoramispora R.F. Castañeda & W.B. Kendr. 1990
Distophragmia R.F. Castañeda et al. 2015
Ellisembiopsis T.S. Santa Izabel & Gusmão 2013
Elotespora R.F. Castañeda & Heredia 2010
Endogenospora R.F. Castañeda et al. 2010
Enridescalsia R.F. Castañeda & Guarro 1998
Falcocladium S.F. Silveira et al. 1994
Helensiella Minter et al. 2015
Helicodochium J.S. Monteiro et al. 2014
Herreromyces R.F. Castañeda & W.B. Kendr. 1991
Hyalocladosporiella Crous & Alfenas 2014
Hyalopleiochaeta R.F. Castañeda et al. 1996
Idriellopsis Hern.-Restr. & Crous 2015
Inesiosporium R.F. Castañeda & W. Gams 1997
Inifatiella R.F. Castañeda 1985
Kostermansindiopsis R.F. Castañeda 1985
Lauriomyces R.F. Castañeda 1990
Luxuriomyces R.F. Castañeda 1988
Luzfridiella R.F. Castañeda & W.B. Kendr. 1991
Menidochium R.F. Castañeda & W.B. Kendr. 1990
Mercadomyces J. Mena 1988
Micropustulomyces R. W. Barreto 1995
Minimelanolocus R.F. Castañeda & Heredia 2001
Minteriella Heredia et al. 2012
Mirimyces Nag Raj 1993
Mycelephas R. F. Castañeda 2009
(continued)
Table 9.1 (continued) Genera

Nagrajia R.F. Castañeda & W.B. Kendr. 1991
Neopenidiella Quaedvlieg & Crous 2013
Neosporidesmium Mercado & J. Mena 1988
Nigrolentilocus R.F. Castañeda & Heredia 2001
Paraceratocladium R.F. Castañeda 1987
Paraidriella Hern.-Restr. & Crous 2015
Paraulocladium R.F. Castañeda 1986
Perelegamyces R.F. Castañeda & W.B. Kendr. 1990
Phaeocandelabrum R.F. Castañeda et al. 2009
Phaeomoniella R.F. Castañeda et al. 2007
Phaeoschizotrichum Silva et al. 2015
Phellinocrescentia Crous & Decock 2014
Phialosporostilbe Mercado & J. Mena 1985
Phragmospathulella J. Mena & Mercado 1986
Physalidiopsis R.F. Castañeda & W.B. Kendr. 1990
Piricaudiopsis J. Mena & Mercado 1987
Polyphialoseptoria Quaedvlieg et al. 2013
Porobeltraniella Gusmão 2004
Pseudocanalisporium R.F. Castañeda & W.B. Kendr. 1991
Pycnovellomyces R.F. Castañeda 1987
Readerielliopsis Crous & Decock 2015
Repetoblastiella R.F. Castañeda et al. 2010
Rogergoosiella A. Hern.-Gut. & J. Mena 1996
Sanjuanomyces R.F. Castañeda & W.B. Kendr. 1991
Scolecobeltrania Iturr. et al. 2013
Selenodriella R. F. Castañeda & W.B. Kendr. 1990
Selenosporopsis R. F. Castañeda & W.B. Kendr. 1991
Solicorynespora R. F. Castañeda & W.B. Kendr. 1990
Soloacrospora W.B. Kendr. & R.F. Castañeda 1991
Stephembruneria R. F. Castañeda 1988
Ticosynnema R. F. Castañeda et al. 2013
Trinosporium Crous & Decock 2012
Venustosynnema R. F. Castañeda & W.B. Kendr. 1990
Veracruzomyces Mercado et al. 2002
Wojnowiciella Crous et al. 2015
Xenocylindrocladium Decock et al. 1997
Xenopenidiella Quaedvlieg & Crous 2014
Ypsilomyces D.A.C. Almeida & Gusmão 2014
Zeloasperisporium R. F. Castañeda 1996
Zelodactylaria A.C. Cruz et al. 2012
Zelosatchmopsis Nag Raj 1991
Zelotriadelphia R. F. Castañeda et al. 2005
Fig. 9.14 Ancorasporella mexicana. (a) Conidiophores and conidia. (b) Conidium. (c) Conidium
and conidiogenous cell
Fig. 9.15 Carrismyces proliferatus. (a) Conidiophore and conidium. (b) Conidium. (c)
Conidiophore and conidium
Fig. 9.16 Elotespora mexicana. (a) Conidioma and conidium and conidium. (b) Conidioma “cup”
and conidium
Fig. 9.17 Lauriomyces bellulus. (a) Conidiophores, conidiogenous cells, and conidia
Fig. 9.18 Minimelanolocus limpidus. (a) Conidiophore and conidia. (c) Conidiogenous cells and
conidium. M. curvisporus. (b) Conidiogenous cells and conidium. (d, e) Conidium
Fig. 9.19 Minimelanolocus navicularis, conidiophore, and conidia

Fig. 9.20 Minteriella cenotigena. (a) Conidioma and conidia. (b) Conidiophore, conidiogenous
cells, and conidia. (c, d) Conidium
9.21, 9.22 and 9.23) in the past 30 years (Seifert et al. 2011; Index Fungorum 2015;
MycoBank 2015), and some mycological resources on the Internet provide useful
information about the regional inventories of Central and South American Neotropic
(Cybertruffle 2015).
Acknowledgments The authors are very grateful to Drs. Josep Guarro, Marcos Fabio Marques,
Margarita Hernández-Restrepo, and Xiuguo Zhang for providing photos.
Fig. 9.21 Stephembruneria

elegans. (a) Conidiophores,
conidiogenous cells, and
conidia. (b) Conidia
Fig. 9.22 Phaeocandelabrum joseiturriagae. (a) Conidiophore, conidiogenous cell, and conid-
ium. (b) Venustosynnema ciliatum. Conidioma
Fig. 9.23 Venustosynnema

ciliatum. Conidioma,
marginal setae,
conidiogenous cells, and
conidia
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Caatinga biome of Brazil. New and interesting Dictyochaeta species. Mycotaxon 106:15–27
da Silva M, Castañeda-Ruiz RF, Pereira OL, Barreto RW (2012) Alveariospora, a new anamorphic
genus from trichomes of Dimorphandra mollis in Brazil. Mycotaxon 119:109–116
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Brazilian cerrado. Mycologia 96(4):879–884
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servation. Mycol Res 95:641–655
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Heredia Abarca G, Castañeda-Ruiz RF, Arias RM, Saikawa M, Stadler M (2007) Anamorphic
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Chapter 10
Mesofungi
Bryce Kendrick
The title of the book is Microfungi. This is a short ‘bridging’ chapter, in which I
suggest that readers might usefully extend that term in the course of their collecting
activities to include numerous but largely overlooked taxa that do not quite fit that
description. Not because I do not find microfungi fascinating, indeed, after I first
discovered them, I spent 60 years looking at, thinking about, investigating and writ-
ing about them, and I look forward to reading this book.
Fruiting macrofungi draw visual attention to themselves, unless they are buried
in the earth (hypogeous fungi) when olfactory clues, at least for some mammals,
take over. Microfungi, despite their virtual invisibility, also began to be studied cen-
turies ago, Persoon making 1801 a banner year. Later, they were realized to be the
causes of food spoilage and of many plant and animal diseases, the source of myco-
toxins, important agents of food processing, and the producers of antibiotics and
other pharmaceuticals that helped to stem the tide of various bacterial plagues that
had previously ravaged the human race and our domestic animals. This led to a
string of increasingly detailed and sophisticated compilations (e.g., Sutton 1980,
Nag Raj 1993), the most recent appearing only 2 years ago (Seifert et al. 2012).
But there are fungi which fall through the cracks—neither micro nor macro—
which are too small to be regularly collected by most people, of no interest to
mycophagists, and in most cases of unknown biochemical potential.
A recently published book has the title Ascomycete Fungi of North America: A
Mushroom Reference Guide (Beug et al. 2014). The first phrase is factually correct,
the second not so much. It is appropriate to apply the term mushroom to such things
as morels, false morels and saddle fungi and even truffles, all of which can be mac-
roscopic and are often considered in handbooks alongside the agarics and boletes,
B. Kendrick, Ph.D., D.Sc., F.R.S.C. (*)

8727 Lochside Drive, Sidney-by-the-Sea, BC, Canada V8L 1M8
Department of Biology, University of Waterloo, Waterloo, ON, Canada
e-mail: bryce@mycolog.com

DOI 10.1007/978-3-319-29137-6_10
220 B. Kendrick
while their ecological roles and edibility and toxicity are widely pondered or experi-
mentally investigated. But when we consider many of the other organisms illus-
trated in the Ascomycete book, the word mushroom would not spring to our lips.
Indeed, most fungivorous humans would fail to see them, or would spurn or ignore
them. They are just too small.
When I wrote the second edition of my mycology textbook The Fifth Kingdom, I
knew I could not exclude the Oomycota. They are not true fungi, since for many valid
reasons they belong to Kingdom Chromista, beside the brown algae and diatoms, but
their hyphae are such good mimics of those produced by what we call the true fungi
(Kingdom Eumycota), having been molded by processes of convergent evolution, that
they had to be included. But the myxomycetes and mycetozoa were clearly beyond the
pale. Not only are they absolutely not fungi at all, since for much of their life cycles
they don’t even have cell walls, but biologists have long since placed them among the
extremely diverse Kingdom Protozoa (or Protoctista). Yet before the appearance of the
third edition of my text, I was asked by many of my teaching colleagues at other uni-
versities to put them back in. Their response to my objections was that if we mycolo-
gists didn’t teach them, then no one would. This argument was enough for me to bring
what I now call the Myxostelida and other kinds of ‘slime mould’ back from the wilder-
ness. This helped to broaden my mind and possibly those of some students. The spec-
trum of organisms awaiting study is still huge and partially unperceived.
During my doctoral studies at the University of Liverpool, I spent thousands of
hours examining forest soil from various horizons, seeking out and exploring the
ecology of truly microscopic fungi—in some cases, exciting new taxa—and the
other organisms with which they shared that environment. In the process, I came
across many small invertebrate animals—beetle mites, nematodes, enchytraeid
worms and springtails—and soon found that although taxonomically disparate, they
interacted in important ways with the microfungi and were placed by soil scientists
in an informal but well-recognized category called the meiofauna or mesofauna.
Wikipedia now tells us that this category contains ‘organisms (animals) that will
pass through a 1 mm mesh, but will be retained by a 45 μm mesh’. This term is usu-
ally applied to benthic (aquatic) fauna, but is easily and conveniently transferred to
a wide range of soil invertebrates.
In mycological terms, when defining mesofungi, I am including fungi that do not
require a microscope to be seen, but which are small enough, or fruiting in such unex-
pected places or such unusual forms as to be passed over by most collectors. If unseen
or ignored, they will fail to be collected and studied at the microscopic—or any other—
level. I have included a number of illustrations to give a sample of such fungi and places.
Mesofungi are most likely to be seen by those looking for microfungi or macro-
fungi in the field, and if we are eventually to get a good hold on the full spectrum of
what is fruiting out there, and injecting spores into the atmosphere, some of us must
raise our sights and widen our horizons a little.
I have gone through the most recent book on Ascomycete Fung of North America
(mentioned above) and have found that a fairly large number of the 600 taxa dealt with in
detail are very small and unlikely to draw much attention. I counted about 50 genera in
which this was the case. For example, Orbilia luteorubella has ascomata that are between
10 Mesofungi 221
0.2 and 1.5 mm in diameter, 200–1500 μm; Nectria cinnabarina, 200–400 μm;
Helminthosphaeria clavariarum, up to 300 μm; and Roseodiscus subcarneus,
400–1500 μm. Whether these organisms are observed or not may depend on their colour.
The first is yellow, the second red, the third black and the fourth white. Mollisia cinerea
ranges from 500 to 3000 μm, but is grey and may not be noticed or thought worth collect-
ing even when its ascomata are present in numbers on the surface of rotting wood.
Helminthosphaeria is often missed altogether because it is just assumed to be a
very dark specimen of its clavariaceous host. On a field trip in Algonquin Park,
Ontario, many years ago, one of my former students made me think that he might have
the makings of a future professional mycologist when he spotted this colour as denot-
ing a parasitic fungus. Fortunately for our discipline, my prediction was correct.
Other mesofungi may be seen or missed because of the degree of aggregation or
clustering of their ascomata, or its absence. A few ascomata of Godronia urceolus
(500–1500 μm) would probably be missed: a large aggregation would probably
draw attention to itself—but in most cases would not be collected. Many senescing
leaves develop dark spots in fall, but unless there is a pattern of multiple similarly
sized spots, as in species of Rhytisma, Coccomyces or Trochila, these may not be
recognized as fungal fructifications. In the case of Rhytisma punctatum, which
attacks Acer macrophyllum on the west coast, each of the spots, which represents an
original single-spore infection, may be quite extensive and encompasses a fairly
large number of tiny ascomata (Fig. 10.1). Less conspicuous, because each spot
incorporates a single ascoma, are Trochila ilicina (Figs. 10.2, 10.3 and 10.4) and
Coccomyces dentatus (Figs. 10.5 and 10.6) which will become recognizable as dis-
comycetes only when incubated in a damp environment.
Fig. 10.1 Colonies of Rhytisma punctatum producing large spots in senescent leaves of Acer
macrophyllum (bigleaf maple). Each spot is developing numerous black apothecia which will not
mature until the following year
222 B. Kendrick
Fig. 10.2 A dead leaf of

Ilex aquifolium (holly)
with very numerous black
apothecia of Trochila
ilicina
Fig. 10.3 Close-up of developing apothecia of Trochila ilicina in dead holly leaf
10 Mesofungi 223
Fig. 10.4 Open apothecia

of Trochila ilicina in dead
holly leaf, showing greyish
hymenium
Fig. 10.5 Black apothecia of Coccomyces dentatus in dead Mahonia (Oregon grape) leaf, triangu-
lar valves of lid just opening
224 B. Kendrick
Fig. 10.6 Open apothecia of Coccomyces dentatus revealing pale grey hymenium
The genus Taphrina produces extensive symptoms on hosts such as peach and
poplar, in which areas of the leaves are stimulated to some hypertrophy and distor-
tion, and turn red in peach and yellow in poplar (Fig. 10.7). These will often remain
unrecognized as fungal fructifications, though there is an extensive ascal hyme-
nium. The manifestations of Taphrina on alder are different and may even be taken
for something non-fungal (Fig. 10.8).
Other mesofungi also fruit above the ground, as does Claviceps purpurea, its
stromata initially replacing grass fruits in fall (Fig. 10.9), but developing its com-
plex stipitate perithecial ascomata only in spring (Fig. 10.10).
Although the lobster fungus, Hypomyces lactifluorum attacking Russula brevipes,
draws attention to itself by its brilliant colouration, Hypomyces luteovirens
(Fig. 10.11) blankets its Russula host less flamboyantly and might be overlooked.
Nectriopsis violacea (Fig. 10.12) attacks the aethalial fructifications of the
myxostelid, Fuligo septica, and is also easy to miss.
I also found a few fungi that had apparently flown under the radar of the authors.
For example, Colpoma, widespread and often dominant on conifer branches at
higher elevations and a little difficult to recognize as a fungus until incubated in a
damp chamber (Figs. 10.13 and 10.14), is not mentioned in the book. Nor is
Didymascella thujina, the cause of Keithia blight of western red cedar (Fig. 10.15).
Onygena species (Fig. 10.16) fruit, often inconspicuously, on hoof or horn, sub-
strates most people would not examine closely. Diatrype (Fig. 10.17) covers wood
surfaces under bark and because of its extent and uniformity may easily be missed
or misinterpreted as scorched wood. Members of the Erysiphales, Thelebolales,
Myxotrichaceae and Pseudeurotiaceae, as noted in Beug et al. (2014), do not pro-
duce fruit bodies large enough to attract attention and so are obvious candidates for
10 Mesofungi 225
Fig. 10.7 Taphrina populina fruiting on leaves of Populus (poplar)
Fig. 10.8 Taphrina alni (alder tongue) fruiting on cones of Alnus rubra (alder)
226 B. Kendrick
Fig. 10.9 Dark sclerotia of Claviceps purpurea on Leymus mollis (Poaceae)
Fig. 10.10 Germinated sclerotia of Claviceps purpurea with stalked, complex perithecial
ascomata
10 Mesofungi 227
Fig. 10.11 Hypomyces luteovirens fruiting all over the gills of a species of Russula
Fig. 10.12 Nectriopsis violacea fruiting on an aethalium of the slime mould Fuligo septica
228 B. Kendrick
Fig. 10.13 Black, lirellate (elongate) apothecia of Colpoma crispum on dead conifer branch
Fig. 10.14 Apothecia of

Colpoma crispum opening
to show whitish hymenium
10 Mesofungi 229
Fig. 10.15 Didymascella

thujina (Keithia blight) on
Thuja plicata (western red
cedar)
Fig. 10.16 Whitish mazaedia of Onygena equina on deer antler

230 B. Kendrick
Fig. 10.17 Extensive fruiting stroma of Diatrype stigma. The black dots are the ostioles of peri-
thecial ascomata. The stroma can be mistaken for scorched wood
inclusion in the mesofungi. One can certainly see the tiny dark ascomata of many
Erysiphales occurring in gradually maturing clusters on host leaves (Figs. 10.18 and
10.19), but unless they are collected and properly examined, the beauty and infor-
mation content of these teleomorphs are apt to be missed.
Another problem with mesofungi is that they may have a long ripening period
and are often immature when collected, especially in northern regions, so
mature asci and ascospores cannot be found. Even in the macrofungal
Geoglossaceae, where the fruit bodies are relatively large, the ascospores are
often found not to have completed their pattern of septation: ‘Are they 3-septate
or 7-septate? Are they mature?’ Molecular techniques present a way around this
problem.
Not so many years ago, we had good excuses for not studying such fungi. Their
ascomata were too small and were probably immature anyway. But now with read-
ily available and progressively less expensive sequencing techniques, we have
largely lost those excuses.
Then there are the Basidiomycetes. One of the best examples I can think of is
Mucronella (Fig. 10.20), a genus represented in the immediate vicinity of my
home by at least three species. The problem with locating this genus during its
extended fruiting period is that the basidiomata tend to develop inside deep cracks
in the rotten wood of large Douglas fir stumps and are unlikely to be seen unless
directly sought for. The elicitation of the seeking behaviour (usually one of the
cognoscenti tells you where to look) suggests a certain level of prior knowledge
that most people lack.
10 Mesofungi 231
Fig. 10.18 A group of dark ascomata of Uncinula (Erysiphales) on a living leaf
Fig. 10.19 Ascomata of Phyllactinia (Erysiphales) on a leaf
I have spent many years looking for microfungi fruiting in their natural habitats,
whether these be rotting logs, dead leaves or wallboard. But rotting wood is not
reserved for microfungi, and I have inevitably come across many tiny ascomycetes
and basidiomycetes, not to mention Myxostelida, which are not microscopic but
may not be large enough or conspicuous enough to be spotted by casual observers.
In many cases, they are small enough to discourage further investigation by some-
one already focused on other kinds of target. How can you get a spore print from an
agaric with a cap 1 mm across? Recently, Oluna and Adolf Ceska (pers. comm.)
232 B. Kendrick
Fig. 10.20 Icicle-like

basidiomata of Mucronella
pulchra on rotting stump
of Pseudotsuga menziesii
(Douglas fir)
found a species of agaric with a basidioma of that size, but on close inspection, they
were able to find mature angular pink basidiospores typical of Leptonia (Entoloma)
cephalotrichum (Fig. 10.21).
One of the best lab exercises I ever adopted as a teacher was for students to fol-
low the succession of fungi colonizing and fruiting on herbivore dung. We usually
looked at the digestive by-products of horses which, if the horses were being fed
naturally in pastures and we were able to discourage the insect larvae, almost
always gave us a diverse and interesting sequence, including zygomycetes, asco-
mycetes and basidiomycetes (and sometimes dictyostelids and myxobacteria).
Although some of the fungi, for example, Pilobolus (Fig. 10.22), were large enough
to see with the unaided eye (with sporangiophores over 1 cm tall), many such as
Saccobolus (Fig. 10.23) and Ascobolus, though their ascomata could be picked out
by the naked eye, required a microscope for any further investigation. And it was
only because students were focusing on the dung that they even noticed these
fungi. The sequence matured with small species of coprinoid agarics, which tended
to be extremely ephemeral.
If people go out in the field and look for fungi, what are the chances that the aver-
age amateur will find Sphaerobolus stellatus and Heyderia abietis (Fig. 10.24), both
of which I spotted during a recent foray by lying down on the ground and poking
around assiduously in the litter.
Here are some others that qualify. Bisporella citrina has ascomata that are
1–3 mm in diameter. However, they are yellow and often present in large
10 Mesofungi 233
Fig. 10.21 Basidioma of Leptonia cephalotrichum with pink basidiospores
Fig. 10.22 Erect sporangiophores of Pilobolus on horse dung
numbers, and so they can be easily detected. To a lesser extent, this is true of
Mollisia cinerea which ranges from 0.5 to 3 mm—and is much less visible
because it is grey.
But Rosellinia subiculata, with perithecia of only 0.5 mm, is less likely to be
spotted, as is Roseodiscus subcarneus, at 0.4–1.5 mm, and Orbilia luteorubella,
0.2–1.5 mm (check Beug et al. 2014 for pictures). Heyderia abietis, already men-
tioned, with a head 3 × 2 mm, is very likely to be missed.
234 B. Kendrick
Fig. 10.23 Hemispherical apothecia of Saccobolus
Fig. 10.24 Stipitate apothecia of Heyderia abietis
I have merely presented a gesture sketch of the mesofungi and must leave it to
those who collect microfungi in the field to consider both widening their search and
their intellectual horizons in the service of their discipline.
10 Mesofungi 235
References
Beug MW, Bessette AE, Bessette AR (2014) Ascomycete fungi of North America. University of
Texas Press, Austin, TX, 488 pp
Nag Raj TR (1993) Coelomycetous anamorphs with appendage-bearing conidia. Mycologue
Publications, Sidney, 1101 pp
Persoon CH (1801) Synopsis methodica Fungorum. Gottingen
Seifert KA, Morgan-Jones G, Gams W, Kendrick B (2012) The Genera of Hyphomycetes CBS,
Utrecht 997 pp
Sutton BC (1980) The coelomycetes. CABI Publishing, Wallingford, 696 pp
Chapter 11
Evolution of Fungi and Update
on Ethnomycology
De-Wei Li, R.F. Castañeda-Ruiz, and James LaMondia
In the past 20 years, research on fungal evolution, archaeology, and ethnomycology

has made significant advances and produced enormous results. The latest develop-
ments allow us to better understand fungi and their origins and usage of fungi by
human beings in ancient times. The new results have made it possible for us to
examine these topics from a new angle with a new vision. This chapter intends to
cover the areas which are often not addressed in the scientific literature.
D.-W. Li, Ph.D. (*)

R.F. Castañeda-Ruiz, Ph.D.
Instituto de Investigaciones Fundamentales en Agricultura Tropical ‘Alejandro de Humboldt’
(INIFAT), Académico Titular de la Academia de Ciencias de Cuba, Calle 1 Esq. 2,
Santiago de Las Vegas, C.P. 17200 La Habana, Cuba
e-mail: icastaneda@ceis.cujae.edu.cu; rfcastanedaruiz@gmail.com; rfcastaneda@inifat.co.cu
J. LaMondia, Ph.D.
e-mail: James.lamondia@ct.gov

DOI 10.1007/978-3-319-29137-6_11
238 D.-W. Li et al.
Evolution of Fungi
Early Evolution of Fungi
It is important to understand the origin and evolution of fungi and the diversification
of major fungal lineages. Fossil records were used as direct evidence of fungal evo-
lution for much of the past (Takamatsu 2013). Fungi were mainly preserved in two
ways: amber and chert. Fungal fossils are scarce and rare due to their fragile nature
and difficulty in preservation (Braun and Cook 2012). Studies on the evolution,
diversity, ecology, and reproductive biology of fossil fungi and fungal-like micro-
organisms have lagged behind those of other fossil organisms for many years due to
a number of reasons (Schwendemann et al. 2009). Indisputably, the lack of fungal
fossil is one of the major reasons. Uncertainty in fossil ages and their phylogenetic
positions are the two other reasons (Hipsley and Müller 2014). Molecular methods
offer new ways to study fungal evolution. Using DNA sequences to date evolution-
ary events of fungi is gradually becoming more popular (Rutschmann 2006; Prieto
and Wedin 2013), and molecular dating has become an important method to tempo-
rally determine diversification of the Tree of Life (Tamura et al. 2012).
However, there are limits in molecular dating techniques and there is no single
best molecular dating method available (Rutschmann 2006). One of the limits is the
lack of reliable clock calibration (Tamura et al. 2012). The limits had led to discrep-
ancies of dating divergences for major fungal lineages in the literature (Prieto and
Wedin 2013; Beimforde et al. 2014). For example, the date of the first divergence
in Ascomycota was estimated from 325 to 1316 million years ago (Ma) in previous
studies (Prieto and Wedin 2013; Beimforde et al. 2014; Berbee and Taylor 1993,
2007; Gueidan et al. 2011; Lücking et al. 2009). Beimforde et al. (2014) indicated
that recent developments in fungal phylogenies and molecular clock models have
allowed mycologists to establish increasingly more realistic models than those in
previous studies for fungal evolution, contributing to the discrepancies in dating
fungal diversification. It is obvious that the dates proposed for temporal origins of
some fungal lineages are tentative at present. These dates need to be verified or
perfected with more studies and improved methodologies. Using a molecular clock
to date major fungal evolutionary events or diversification may be inaccurate with-
out appropriate calibration (Hipsley and Müller 2014). Thus, clear justification and
proper usage of molecular clock calibration are crucial to accurately determine
dates in the evolutionary history of fungi (Hipsley and Müller 2014). Despite these
limitations, research in this area has made significant progress in the last two
decades with assistance from the new dating techniques for fossils and the advance-
ment of molecular clock methods. These developments have provided insight into
how fungi evolved. Pirozynski (1976), Taylor and Krings (2005), and Taylor et al.
(2015) have published thorough reviews on fungal fossils, using early and more
recent literature, respectively. In this chapter, the author will mainly cover the
advances in fungal fossil discoveries made in the last decade. For molecular dating,
the focus is on the studies which used either fungal fossils or geological events to
calibrate their molecular clock.
11 Evolution of Fungi and Update on Ethnomycology 239
Fossil Evidences and Molecular Clock Dating
The divergence of animals from fungi was estimated at ca. 965 Ma (Doolittle et al.
1996). Oberwinkler (2012) recently drew a figure to show the evolutionary tree of
true fungi based on a number of key literature citations published in the last 10 years.
In his figure, he indicated the diversification of the fungal taxonomic groups and
animals at ca. 850 Ma, calibrated using fossil records. Preceding land plants, fungi
possibly colonized the land during the Cambrian (542–488.3 Ma) (Brundrett 2002;
Berbee and Taylor 2010). Fungi were first traced back to the Silurian period, about
408–438 Ma in the Paleozoic era based in fossil records (Alexopoulos et al. 1996).
Studies conducted in the last two decades showed that this dating was rather conser-
vative. Fossilized hyphae and spores recovered from the Ordovician of Wisconsin
(460 Ma) resemble modern-day Glomeromycota and existed at a time when the land
flora likely consisted of only nonvascular bryophyte-like plants (Redecker et al.
2000). Glomeromycota is a much older evolutionary lineage than those of
Ascomycota and Basidiomycota (Schüßler and Walker 2011). The first zygomyce-
tous fungi were estimated to appear on earth during the Precambrian, ca. 1.2–1.4 Ga
ago using molecular clock (Heckman et al. 2001; Blair 2009; Krings et al. 2013).
However, more conservative estimates dated the divergence at ca. 800 Ma (Berbee
and Taylor 2001). The conflicting results indicated that dating the divergence of
zygomycetous fungi is far from resolved and needs further studies. Krings et al.
(2014) recently published the first fossil record of a fungal “sporocarp” of
Mycocarpon rhyniense from the Lower Devonian Rhynie chert ca. 410 Ma ago. The
taxonomic placement of Mycocarpon rhyniense remains unclear, but the authors
hypothesized that the fossilized fungus might belong to zygomycetous fungi and
predates the oldest evidence of fungal “sporocarps” by ca. 80 Ma (Krings et al.
2014). Dating analysis by Gryganskyi et al. (2012) showed that the
Entomophthoromycota (formerly considered as zygomycetous fungi) originated
405 ± 90 Ma using molecular clock models. Based on ancestral state reconstruction,
the ancestor of all Entomophthoromycota was suggested to be morphologically
similar to species of Conidiobolus. The authors suggested that entomopathogenic
lineages in Entomophthoraceae probably evolved from ancestors of saprobes or fac-
ultative pathogens during or shortly after the evolutionary diversification of the
arthropods. Unfortunately, fossil evidence of these fungi is extremely rare. Strullu-
Derrien et al. (2014) reported two new endophytes Palaeoglomus boullardii
(Glomeromycota) and Palaeoendogone gwynne-vaughaniae (Mucoromycotina) in
Horneophyton lignieri dating back to ca. 407 million years old from the Rhynie
chert. Krings et al. (2013) suggested that some “sporocarp” types from fossils repre-
sented by mantled zygosporangia and zygomycetous fungi probably were important
in terrestrial paleoecosystems by the Carboniferous period (359–299 Ma). Fungal
fossils were infrequent and controversial before the early Devonian (416–359 Ma),
when fungal fossils including Chytridiomycota, Ascomycota, Glomeromycota, and
Peronosporomycetes became abundant in the Rhynie chert, mostly as Glomeromycota
(formerly reported as Zygomycota) and Chytridiomycota (Brundrett 2002; Taylor
and Taylor 1997; Redecker et al. 2000; Taylor et al. 2005; Krings et al. 2007).
240 D.-W. Li et al.
Considerable uncertainties exist in the timing of the origins of land-plant fungal

symbioses due to the fact that paleontological data and molecular clock date esti-
mates are often greatly different (Field et al. 2015). A recent study provided some
evidence that the earliest mycorrhizal fungi are the Mucoromycotina which predate
the Glomeromycota (Bidartondo et al. 2011). This report called into question the
ancestral position of Glomeromycota in fungal evolution and in forming symbioses
with land plants, since the study by Bidartondo et al. (2011) did not calibrate their
molecular clock using any available methods. Further research will be necessary to
confirm this study in the future. Strullu-Derrien et al. (2014) reported fossil evi-
dence Palaeoendogone gwynne-vaughaniae (Mucoromycotina) from Rhynie chert
from 407 Ma (Scotland, UK).
Whether Glomeromycota, zygomycetes, and Chytridiomycota are paraphyletic
or monophyletic, phylogenetics remain to be determined (Berbee and Taylor 2007).
Fossil evidence indicated that these fungi were present in the Devonian, early in the
history of vascular plants (Berbee and Taylor 2007).
In the latest study, Beimforde et al. (2014) studied all of the oldest Ascomycete
fossils available from amber (Albian to Miocene) and chert (Devonian and
Maastrichtian) representing four major ascomycete lineages (Lecanoromycetes,
Laboulbeniomycetes, Dothideomycetes, and Eurotiomycetes) and used a multi-
gene data set from 145 taxa representing all main taxonomic groups of the
Ascomycota to estimate divergence times for Ascomycota. The authors demon-
strated that Ascomycota started its initial diversification in the Ordovician (485–443
Ma), followed by repeated diversification of lineages throughout the Phanerozoic,
and this continuous diversification was not affected by mass extinctions (Beimforde
et al. 2014). A recent study was conducted using molecular dating and fossil cali-
bration to estimate the divergence times of most of the major groups of Ascomycota
(Prieto and Wedin 2013). The results from this study suggested that the origin and
diversification of the Pezizomycotina occurred in the Early Cambrian (531 Ma).
The main lineages of lichen-forming Ascomycota originated at least as early as the
Carboniferous, with successive radiations in the Jurassic and Cretaceous generating
the diversity of the main modern groups (Prieto and Wedin 2013). The origin of
Pezizomycotina was dated at ca. 419 Ma with a calibrated molecular clock and two
fossil records (Yang et al. 2012a), while the results of Prieto and Wedin (2013) sug-
gested it be ca. 530 Ma.
The lichen-like fossils discovered in marine phosphorite of the Doushantuo
Formation from South China, involving filamentous hyphae closely associated with
coccoidal cyanobacteria or algae, were dated back ca. 600 Ma ago. These fossils
showed that fungi developed symbiotic relationships with photoautotrophic organ-
isms prior to the evolution of vascular plants (Yuan et al. 2005). A fossil of a
perithecial fungus with both sexual and asexual states was found from the early
Devonian (400 Ma) several years ago in the cortex just beneath the epidermis of
aerial stems and rhizomes of the vascular plant Asteroxylon (Taylor et al. 2005). A
fossil of a perithecial sordariomycete was reported from the Lower Cretaceous (ca.
133 Ma) on Vancouver Island, British Columbia, Canada (Bronson et al. 2013).
Lichen fossils were found from early Devonian (415 Ma) (Honegger et al. 2013),
Lower Cretaceous (ca. 133 Ma) (Matsunaga et al. 2013), and Eocene (56 to 34 Ma)
(Rikkinen and Poinar 2008). The Pezizomycotina lichen fossils from the early
Devonian represent the oldest known record of lichens with symbionts (Honegger
et al. 2013). Takamatsu (2013) indicated that the powdery mildews originated in the
Late Cretaceous (66–100 Ma) and the first diversification of the major lineages
occurred at the Cretaceous/Paleogene boundary according to molecular clocks
using the 28S rDNA D1/D2 and ITS and fossil records. He stated that ancestral
powdery mildews may have first initiated diversification on broad-leaved deciduous
trees in the high latitudes of the northern hemisphere, and the cradle of four genera,
Blumeria, Golovinomyces, Leveillula, and Neoerysiphe, may have been distributed
in the areas from Central/West Asia to the Mediterranean (Takamatsu 2013).
A recent study linked the spores of the ascomycete genus Potamomyces with the
fossil form taxa Mediaverrunites (Schlütz and Shumilovskikh 2013). The authors
concluded that the genus Potamomyces evolved at least 25 Ma at the inception of
the younger Tertiary according to the fossil findings for Mediaverrunites. Schmidt
et al. (2014) indicated that new fossil evidence dated the fossil record of sooty
molds to ca. 100 Ma, from the early Miocene (17 Ma) to the Early Cretaceous
(Albian, ca. 100–113 Ma). Mesozoic and Cenozoic ambers from different regions
of the world contained sooty molds from eight northern hemisphere amber deposits.
Fragments of superficial subicula identical to those produced in the Metacapnodiaceae
(Capnodiales) were recorded since the Albian. The fossil fungi demonstrated that
capnodialean sooty molds had occupied their specialized niches since the time
when early angiosperms first appeared in the fossil record.
Berbee and Taylor (2001) estimated 500 Ma as a minimum age for
Basidiomycota. The Ascomycota and Basidiomycota diverged approximately
452 Ma (Taylor and Berbee 2006). However, this dating was adjusted to 430 Ma
(Berbee and Taylor 2007). The later dating was supported by fossils reported by
Krings et al. (2011). Taylor and Krings (2005) stated that all major fungal lin-
eages except the Basidiomycota were present in the Rhynie chert (ca. 410 Ma),
based on the reports on fossil fungi. All modern major taxonomic groups of fungi
were present by the Late Carboniferous (Pennsylvanian, 318–299 Ma) (Blackwell
et al. 2012; Alexopoulos et al. 1996). Fossil records of Palaeancistrus martinii
R. L. Dennis with clamp connections found in PA used to be the oldest direct
fossil evidence to date Basidiomycota to the middle Pennsylvanian period (ca.
305 Ma) (Dennis 1970; Taylor and Krings 2005). A recent study on the fossil fili-
calean fern Botryopteris antiqua preserved in a late Visean chert (Mississippian;
ca. 330 Ma) from Esnost (Autun Basin) in central France showed that this fossil
contained fungi with clamp connections and predated Palaeancistrus martinii;
consequently, it is the oldest direct fossil evidence of Basidiomycota (Krings et al.
2011). A genomic study by Padamsee et al. (2012) based on their phylogenetic
analysis of 71 proteins showed that Wallemia sebi, a microfungus, is the earliest
diverging lineage of Agaricomycotina. The time of this evolutionary diversion
was not dated in their study.
Hibbett et al. (1995) reported fossilized mushrooms from mid-Cretaceous (90–
94 Ma) in amber collected from New Jersey, USA, and the mushrooms were later
242 D.-W. Li et al.
described as Archaeomarasmius leggetti Hibbett et al. (1997). Fossilized basidi-

omata of several Fomes spp. had been discovered in southwest Idaho dating back to
the Tertiary (66–2.6 Ma) (Brown 1940). A new fossil basidioma of Ganodermites
libycus A. Fleischmann et al. (Polyporales, Ganodermataceae) was reported from
the Lower Miocene (Neogene) (23–2.6 Ma) of Jebel Zelten in the northern part of
the Libyan Sahara, North Africa, which represents the earliest convincing fossil
evidence for polypores. The fossil fungus is closely related to the modern genus
Ganoderma and showed a variety of characters present in Ganoderma sp.
(Fleischmann et al. 2007).
The hyphomycete in the Early Eocene Indian amber (48–56 Ma) collected from
the Tarkeshwar Lignite Mine of Gujarat State, Western India, is Monotosporella
doerfeltii Sadowski et al. (Sordariomycetes), which is attached to its substrate, and
is likely the degraded thallus of a cladoniform lichen (Sadowski et al. 2012).
Conidiophores of Aspergillus collembolorum Dörfelt and A. R. Schmidt growing on
a springtail were found in Eocene amber from the Baltics (50–35 Ma) (Taylor et al.
2015; Dörfelt and Schmidt 2005). The first fossil Aspergillus was found from
Dominican amber, and it was estimated to be from Lower Miocene-Oligocene (ca
40 Ma) (Thomas and Poinar 1988).
Evolution of Ecological Functions of Fungi
Yang et al. (2012a) revealed that fungal carnivorism diverged from saprophytism
ca. 419 Ma according to molecular clock calibration with two fossil records at the
same time period. Fossil evidence of mycoparasitism and hypermycoparasitism was
reported in Early Cretaceous Burmese amber (100 Ma). Palaeoagaracites antiquus
and R. Buckley (an agaric) was parasitized by another fungus, Mycetophagites
atrebora Poinar and R. Buckley, which was parasitized by Entropezites patricii
Poinar and R. Buckley, a hyperparasitic fungus (Poinar and Buckley 2007).
Paleoophiocordyceps coccophagus G. H. Sung et al. from the Early Cretaceous
(Upper Albian) is the oldest fossil record of a fungal parasite of an animal (Sung
et al. 2008).
Krings et al. (2007) studied the Rhynie chert plant Nothia aphylla Lyon ex
El-Saadawy and Lacy (400 Ma) and found that three fungal endophytes were pres-
ent in prostrate axes of N. aphylla as narrow hyphae producing clusters of small
spores, large spherical spores/zoosporangia, and wide aseptate hyphae that form
intercellular vesicles.
Ducousso et al. (2004) suggested that a single origin for the ectomycorrhizal
status of the common ancestor of Dipterocarpoideae and Sarcolaenaceae was dated
prior to Madagascar’s separation from the India-Seychelles block (ca. 88 Ma).
Their report, thus, predated the previous oldest ectomycorrhizal fossil (50 Ma)
(LePage et al. 1997). Moyersoen (2006) studied the South American ectomycor-
rhizal dipterocarp Pakaraimaea and suggested that a Gondwanaland evolution of
the ectomycorrhizal habit is at least 135 Ma. Hibbett and Matheny (2009) stated that
ectomycorrhizae have multiple origins. Tedersoo et al. (2010) proposed that the
ectomycorrhizal habit had evolved at least 66 times. The origin of freshwater asco-
mycetes was dated at 390 Ma using a Bayesian relaxed-clock method (Vijaykrishna
et al. 2006).
Update on Ethnomycology
There is much that we can learn from our ancestors regarding the utilization of fungi
for various purposes. In the last three decades, the science of ethnomycology has
made significant advancements. A comprehensive review of information on ethno-
mycology was covered in a book Conspectus of World Ethnomycology by Dugan
(2011). However, the ethnomycological studies remain incomplete in some geo-
graphic areas. A number of facts contribute to this phenomenon. One of the major
hurdles is the language skill to read ancient texts, such as ancient Chinese. Another
one is insufficient archaeological studies to verify and date writing records. It is
crucial to conduct more ethnomycological studies in the future. If the update in this
chapter can initiate more interest and research in this area, our intentions will be
served.
Microfungi in Human History
The utilization of fungi by human beings as food, for fermented beverages, medi-
cine, and other purposes such as ritual ceremonies, has been traced to prehistoric
and preliterate periods when human beings were hunter-gatherers. Domestication of
plants and animals led to the rise of agriculture and allowed food surplus and fer-
mentation possible for making alcoholic beverages and baking. Since there were no
written records during that period, archaeological evidence and molecular clock
dating are used to study the relationships between human beings and fungi.
Fungi have played and are still playing an important role in human history. We
often underestimated the ability of our ancestors to utilize fungi and are often sur-
prised to recognize the significance of fungi in the evolutionary history of human
beings. The archaeological discoveries and molecular technologies made in the last
three decades have greatly improved our understanding of how human beings use
fungi to their benefit. In early stages, the major purpose of human use of fungi was
for survival. When food became sufficient and food surpluses were possible, fungi
turn to being used to improve the quality of human life.
Mildews (microfungi) of cereals and vine crops were reported in the Old
Testament (c. 750 BC) (Agrios 2005). Theophrastus, the Greek philosopher (c.
300 BC), was the first one to write about diseases including rusts of cereals, legumes,
and trees (Agrios 2005). This topic has been intensively covered in plant pathology
books. Therefore, it will not be covered further here.
244 D.-W. Li et al.
Fungi, especially yeasts, were utilized for wine making, baking, medicinal use,
and other fermentation purposes in China dating back to several thousand years ago.
Liquor and wine making in China goes back to 6000–7000 years ago (Yu and
Zhuang 2006). Guo (1976) (aka Kuo Mo-jo) found that during the Yangshao culture
period (5000–3000 BC), there are a good number of bronze wine-ware vessels exca-
vated in China from the Zhou and later dynasties. Some of these historical relic
pieces are on display in museums not only in China, but also in Europe and North
America, as well as other areas. The earliest bronze wine cup, excavated in 1975,
dated back to the Xia dynasty (c. 2070–c. 1600 BC) (Luoyang Museum 2014).
Fermented Food and Condiments
In ancient Egypt, Egyptians considered fermentation a gift from the god Osiris,
whereas ancient Romans ascribed the emergence of mushrooms and truffles to
lightning bolts cast to the earth by Jupiter (Alexopoulos et al. 1996).
A number of microfungi have been used to produce fermented food and condi-
ments. Yeasts are the most important ones. Fermented food can be found in all
geographic areas and continents in the world. Different ethnic groups from different
continents all make significant contributions to fermented foods or condiments.
The earliest archaeological evidence showed that plant food processing and pos-
sibly the production of flour dated to ca. 30,000 years ago in Europe (Revedin et al.
2010). Fermented foods dated to ca. 10,000 BC to the Middle Ages for preserving or
salvaging food surpluses (Liu et al. 2013). Emmer wheat was domesticated in the
Middle East about 8000 BC (Gornicki and Faris 2014). The development of leav-
ened bread dated to 7000 BC (Liu et al. 2013), but the earliest archaeological evi-
dence of leavened bread is from ancient Egypt dated to ca. 2000–1200 BC (Samuel
1996, 1999). Scanning electron microscopy detected yeast cells in several ancient
Egyptian loaves and indicated that these bread loaves (emmer wheat (Triticum
dicoccum Schübl.)) were leavened (Samuel 1996).
Shevchenko et al. (2014) reported direct evidence that the Subeixi sourdough
bread excavated in Subeixi cemetery (500–300 BC) in Xinjiang, China, was made
from barley and broomcorn millet leavened using baker’s yeast and lactic acid bac-
teria based on the geLC-MS/MS proteomics analysis.
The Chinese used not only yeasts but also several microfungi for various pur-
poses. Aspergillus was used for fermented pasta and Mucor for making preserved
tofu (soy cheese); Monascus purpureus Went was used in an additional recipe for
making wine and Neurospora for fermented soybean crumb. Soy sauce originated
from China approximately 2000 years ago, and it is fermented in a two-step process
using yeast and bacteria (Lioe et al. 2012).
Dairy products were associated with the domestication of milk-producing ani-
mals in human history. The domestication of cattle, sheep, and goats had already
taken place in the Near East by 8000 BC or before, and among these animals, sheep
(Ovis aries L.) were the first to be domesticated from mouflon (Ovis orientalis
Gmelin) between 11,000 and 9000 BC in Iran (Krebs 2003; Clutton-Brock 1999;
Garrard et al. 1996).
Cheese making was suggested as dating to the same era as bread making (7000
BC) (Liu et al. 2013). However, that date probably is the earliest archaeological
evidence for milk use reported by Evershed et al. (2008). The earliest archaeologi-
cal evidence of cheese making dated back to Neolithic (5500 BC) from Kujavia,
Poland, where strainers with milk fat molecules were discovered (Salque et al.
2013). Cheese making was suggested as discovered accidentally by storing milk in
a container made from the stomach of an animal where the milk was turned into
curd and whey. By Roman times, cheese had become a daily food. Yeasts are pres-
ent in cheeses, and Debaryomyces hansenii (Zopf) Lodder and Kreger, Yarrowia
lipolytica (Wick., Kurtzman, and Herman) Van der Walt and Arx, Saccharomyces
cerevisiae Meyen ex E. C. Hansen, Kluyveromyces lactis (Dombr.) Van der Walt,
and K. marxianus (E. C. Hansen) Van der Walt are the predominant ones (Gori et al.
2011). However, the exact role played by yeasts in cheese making remains unsolved.
Maturation and aroma formation in Camembert were suggested by a number of
researchers (Gripon 1999). Penicillium roqueforti Thom is crucial to blue cheese
making and it was used as a secondary starter culture (Gori et al. 2011). Penicillium
camemberti Thom (Penicillium candidum Link) is used to produce white mold
cheeses, such as Brie and Camembert (Gori et al. 2011).
At present, cheese is one of the major agricultural products in the world, and
there are over 500 varieties in the market. Worldwide production of cheese in 2010
was over 20 million tons according to the “FAO Statistical Yearbook 2013: World
Food and Agriculture” (FAO 2013).
Fermented meat is suggested to relate to preservation of the meat leftover from
large animals killed by our hunter-gatherer ancestors before the beginning of agri-
culture. However, there is no archaeological evidence dating back to that era to
prove this hypothesis at present. Fermented meat is suggested to date back to ca.
1500 BC (Liu et al. 2013). Debaryomyces hansenii is the dominant yeast in fer-
mented meat products (Asefa et al. 2009). Several hyphomycetes, including
Penicillium commune Thom, P. nalgiovense Laxa, and P. solitum Westling, occur on
fermented meat products and cheeses (Asefa et al. 2009; Filtenborg et al. 1996;
Nout and Aidoo 2011).
The greatest varieties of fermented food are present in Asian countries (Nout and
Aidoo 2011). Shurtleff and Aoyagi (2011a) indicated that Douchi, a black soybean,
is the earliest known fermented or processed soyfood based on the fact that fer-
mented black soybeans were unearthed from the Han Tomb no. 1 (dated to about
165 BC) at Mawangdui near Changsha, Hunan province, in China. Yokotsuka (1985)
indicated that China is the birthplace of fermented vegetables and the use of
Aspergillus and Rhizopus microfungi to make processed food. The book called Shu
Jing or Shu Ching written in the Zhou dynasty in China (1121–256 BC) refers to the
use of “qu” or “chu” (koji in Japanese) a fermented grain product (Yokotsuka 1985).
Qu (koji) is also mentioned in the Zhouli [Rites of the Zhou dynasty] (300 B.C.E.)
in China (Shurtleff and Aoyagi 2012). Qu is a culture prepared by inoculating either
Aspergillus oryzae or Monascus purpureus microfungi onto cooked grains and/or
246 D.-W. Li et al.
soybeans in a warm, humid place. Discovery of qu making was a milestone in

Chinese food technology, as it is the basis for three major fermented soyfoods:
jiang/miso, soy sauce, and fermented black soybeans, grain-based wines (including
Japanese sake), and jiuniang (or Japanese amazake) (Shurtleff and Aoyagi 2012).
Tempeh is a very popular fermented food that originated on the Java island in
Indonesia and the only soyfood that did not originate from China or Japan (Shurtleff
and Aoyagi 2007, 2011b). It is produced by fermenting dehulled cooked soybean
cotyledons with Rhizopus oligosporus. Its history was not well documented. It was
suggested that Indonesians had been making tempeh for several centuries, probably
beginning in the early 1600s (Shurtleff and Aoyagi 2007, 2011b), but it has been
speculated that this food may have originated over 2000 years ago (Sastroanijoyo
1971). The earliest known reference to tempeh appeared in 1815 in a classic
Javanese literature, the Serat Centhini (see Volume 1, p. 295) (Shurtleff and Aoyagi
2011b) and in 1875 in European literature (Gericke and Rooda 1875). At present, it
has been introduced to other countries and commercially produced in Japan,
Netherlands, and the USA (Shurtleff and Aoyagi 2007). Soy tempeh contains 19.5
% protein (Shurtleff and Aoyagi 2007).
Wikipedia (2015) listed over 110 fermented foods from around the world. It is an
incomplete list. These fermented foods originated from different geographic areas
and countries. Some of them were fermented using microfungi. Without fermenta-
tion supplied by microfungi, our foods would be much less tasty. For additional
information, Industrial Applications, The Mycota X, 2nd edition by Nout and Aidoo
(2011) is an excellent reference.
Yeasts and Alcoholic Beverages
Yeasts are phylogenetically microfungi, with 1500 species currently described

(Kurtzman and Fell 2006) and estimated to be 1.5 % of all described fungal species.
Yeasts have been used for thousands of years by human beings for fermenting food
and beverages and preserving food (Samuel 1996). Fermentations in the Neolithic
times probably originated from natural inoculation by yeasts in the nature. It remains
unknown when human beings started to intentionally add selected yeast to make
beer, wine, steamed buns, or bread. Samuel (1996) indicated that fermentation with
selected yeasts led to speciation of new species in the Saccharomyces sensu stricto
complex by interspecies hybridization or polyploidization.
Guerra-Doce (2014) indicated that alcoholic fermentation might have been dis-
covered long prior to the domestication of plants and animals during the Neolithic.
The earliest alcoholic drink in the world at present was fermented from wild grapes
(Vitis sp.), or hawthorn fruit (Crataegus sp.), rice, and honey uncovered at the early
Neolithic village of Jiahu, in the Yellow River Valley in Henan Province, China, ca.
7000–6600 BC according to chemical analysis of traces absorbed and preserved of
ancient pottery jars (McGovern et al. 2004; Guerra-Doce 2014). The earliest wine
making dated to 6000 BC in Georgia (Vouillamoz et al. 2006). Archaeologists have
found similar evidence from residues in two ceramic vessels uncovered at the site
of Hajji Firuz Tepe in the Zagros Mountains in Mesopotamia of northwestern Iran,
ca. 5400–5000 BC during the Neolithic. Biochemical analysis showed that these
vessels contained a resinated wine (McGovern et al. 1996). The earliest evidence of
beer brewing was found from a ceramic vessel recovered at the cave of Can Sadurní,
Barcelona, in the early Neolithic Europe dated to 5000 cal BC (Blasco et al. 2008).
At the same time, evidence of malting, one of the key steps in beer brewing, was
found on two grinding stones (Blasco et al. 2008). Archaeological evidence of alco-
holic beverages was found in other areas around the world also. Evidence of alco-
holic beverages was found in Babylon and dated to 5000 BC (Battcock 1998), 2000
BC in pre-Hispanic Mexico (Battcock 1998), and 1500 BC in Sudan (Dirar 1993;
Pederson 1979).
Evidence of alcoholic beverages brewed with Saccharomyces cerevisiae was
found dating from 3150 BC in Egypt (Cavalieri et al. 2003). White wine was exca-
vated from the tomb of King Tutankhamun, an Egyptian pharaoh of the 18th dynasty
(ca. 1332 BC–1323 BC) (Guasch-Jané et al. 2006). The authors sequenced ITS from
the residue inside one of the earliest wine jars recovered from the tomb of King
Scorpion I in Egypt, and the result of sequencing showed that the fungus responsi-
ble for alcoholic fermentation was Saccharomyces cerevisiae.
Archaeologists excavated unique cereal alcoholic beverages preserved as liquids
inside sealed bronze vessels of the Shang and Western Zhou dynasties (ca. 1600–
1046 BC and ca. 1046–722 BC, respectively), and the actual alcoholic beverages still
remained in liquid state when the bronze vessels were unearthed after over 3000
years in the ground (McGovern et al. 2004; Anyang Archaeological Team 2004;
Henan Institute of Cultural Relics and Archaeology 2000). These findings provide
direct evidence for fermented beverages in ancient Chinese culture, which were of
considerable social, religious, and medical significance, and helped elucidate their
earliest descriptions in the Shang dynasty oracle inscriptions (McGovern et al. 2004).
Archaeologists unearthed a whole set of wine making gear from a tomb of the
Dawenkou culture (4100–2600 BC, the Neolithic period) in Lingyinhe, Ju county,
Shandong, in 1979 (Bao 2008). Wang (1987) speculated that the tomb occupant
could have been a professional wine maker.
Archaeologists excavated a fully equipped winery consisting of basins for wine-
presses, fermentation vats, storage jars, drinking bowls, and remains of domesti-
cated grapes from a cave complex of Areni-1, a Chalcolithic site in southeast
Armenia dated to around 4000 BC.
Quantities of carbonized grape berries (Vitis vinifera) in pots dated to 4200 BC
were unearthed from the prehistoric site of Dikili Tash in eastern Macedonia,
Greece. The significance of the discovery is that the grape berries had been pressed
which indicated the extraction of juice from grapes and suggested wine making
(Valamoti et al. 2007).
Since Neolithic times, alcoholic drinks have not been essential to the survival of
human beings. What is the reason that our human beings discovered alcoholic bev-
erage usage prior to domestication of plants and animals? Does this behavior con-
tribute any fitness to human beings during evolution?
248 D.-W. Li et al.
Edible Mushrooms as Food and Risk
Human beings collect mushrooms in the wild as food and for medicinal purposes,
probably dating back to the prehistoric period. Mushrooms were used as a delicacy,
food, and medication for therapeutic properties and often in religious ceremonies
during the early civilizations of the Chinese, the Fertile Crescent (including
Egyptians and others), Greeks, Indians, Latin Americans, and Romans (Miles and
Chang 2004). Roman emperors prohibited ordinary people from eating mushrooms
so that the mushrooms could be strictly reserved for nobility. Mushrooms were
highly regarded as luxuries and delicacies by Romans. Recipes for mushrooms sug-
gested by Diphilus of Siphnos dated to 300 BC (Dalby 2003; van Rossenberg 2005).
Classical Greek authors considered mushrooms as famine food, similar to acorns.
The discovery of a bowl of mushrooms collected from the wild in a Bronze Age
house near Nola in Italy is the first evidence that mushrooms were used as food in
prehistoric Europe (van Rossenberg 2005).
Chinese ancestors had started to collect edible mushrooms for the table dating
back to 4000 BC based on archaeological studies which excavated mushrooms along
with rice, Crataegus hupehensis Sarg., and other foodstuffs from an ancient site,
Hemudu culture, Yuyao in Zhejiang, China, in 1977 (Hemedu Site Museum 2015).
Collecting mushrooms for direct consumption always has some risks. Mycologists
often say that bad fungal taxonomy kills. In history, Charles VI, Holy Roman
Emperor, was killed by eating death cap mushrooms Amanita phalloides (Fr.) Link
in 1740 (Wasson 1972). His death led to a war and a diversion of European history.
It is possible that the death of Pope Clement VII in 1534 was the result of eating this
mushroom also (Wasson 1972). Every year, there are some isolated cases of human
death caused by eating poisonous mushrooms due to misidentification around the
world. Some cities set up hotlines for mushroom poisoning.
A mysterious epidemic occurred in Yunnan, China, in 1978. Some local villag-
ers suddenly died from an unknown cause. It was reported that victims even dropped
dead in the middle of a conversation (Stone 2010). Thus, this mysterious epidemic
was referred as sudden unexplained death syndrome (SUDS). From 1978 to 2010,
SUD had caused over 400 deaths from sudden cardiac arrest and several dozen
nonfatal cardiac cases in the area. The victims are children, adults, and senior people
(Zhou et al. 2012; Stone 2010). Prior to 2005, the Chinese CDC had sent scientists
to the area to study SUDS several times, but failed to determine the cause. In 2005,
a new research team was formed including cardiologists, epidemiologists, mycolo-
gists, medical examiners, and medicinal chemists. The “little white mushroom” or
“nail-like mushroom” thought to be edible by locals in that area was suggested by
the team to be the cause of a proportion of these deaths to local villagers after 5
years of research. SUDS caught attention around the world after (Stone 2010). Two
years later, the scientists put the pieces together and determined that the little white
mushroom was the cause. They found three mycotoxins including two new ones
(amino acids 2R-amino-4S-hydroxy-5-hexynoic acid (1) and 2R-amino-5-hexynoic
acid (2) and the known toxin g-guanidinobutyric acid (3)) extracted from fruiting
bodies of the little mushroom were responsible for SUDS and found two unusual
toxins that killed mice with an LD50 of 71 and 84 mg kg−1, respectively (Zhou et al.
2012). This little white mushroom was found to be new to science and described as
Trogia venenata Zhu L. Yang et al. (Cantharellaceae) (Yang et al. 2012b).
Hypoglycemia induced by Trogia toxin is suggested to be the mechanism. Is this
over 30-year mystery fully deciphered? It seems not so. At present, there is a dis-
agreement whether these toxins are the sole culprit. The exact toxin(s) produced by
Trogia venenata and responsible for SUDS remain not fully established, and the
associated mechanism has not been fully confirmed (Graeme 2014; Stone 2012).
However, since the public campaign by Chinese CDC to advise local villagers of
stopping consumption of Trogia venenata in 2009, no new SUDS cases have been
reported, and as a public health threat, the case is closed (Stone 2012).
Microfungi as Food
Microfungi play very important roles in fermented food and beverages. Two micro-
fungi are used directly as vegetable/food. Both are smut fungi belonging to Ustilago.
Jiaobai is a vegetable widely cultivated and consumed in China and several other
Asian countries (Terrell and Batra 1982; Guo et al. 2007; Chung and Tzeng 2004;
Zhang et al. 2012). The vegetable is Zizania latifolia Turcz. (Manchurian ricegrass,
broad-leaved wild rice, Asian wild rice, and water oat) infected by Ustilago escu-
lenta Henn., which causes host stems to hypertrophy for consumption (Chan and
Thrower 1980). Zizania latifolia was planted as one of the six major grain crops in
China since the Zhou dynasty (from 771 to 221 BC) (Guo et al. 2007). Since Jiaobai
was cultivated, Zizania latifolia was no longer cultivated as a grain crop. This fun-
gus is federally regulated and quarantined in the USA due to its potential risk to
native wild rice (Zizania aquatica L.) in North America (Yamaguchi et al. 1990).
Thus, the cultivation of this vegetable is banned in the USA.
Another smut-related food is huitlacoche (corn smut, Ustilago maydis), which is
a delicacy in Mexico. Huitlacoche consumption originates from Aztec cuisine. Only
immature galls are harvested for cooking purposes. Fully mature galls lose their
cooking value. It is consumed as a filling in quesadillas and other tortilla-based
foods and soups. Since the mid-1990s, farms in Florida and Pennsylvania have been
permitted by the US Department of Agriculture (USDA) to produce huitlacoche due
to demand from high-end restaurants (Pataky and Chandler 2003).
A number of microfungi, Fusarium spp., are notorious for producing mycotox-
ins in cereal crops that have detrimental effects on human and animal health if they
enter the food chain as food or animal feeds. However, Fusarium venenatum
Nirenberg is an exception. One of its strains (IMI 145425, ATCC PTA-2684) has a
high protein content and does not produce mycotoxin. The potential utilization of
this strain to produce mycoprotein has been studied since the 1960s and has been
commercially used for the production of the single-cell protein, mycoprotein (Wiebe
2002). Its product was approved for sale as food in the UK in 1984 (Wiebe 2002).
250 D.-W. Li et al.
The mycoproteins are marketed under a brand name, Quorn, and are currently sold
in 13 countries including the USA with 90 products ranging from steak strips to
burgers to fillets (Marlow Foods Ltd 2014).
Microfungi as potential protein producers or resources as alternatives to animal
meats should be studied further.
Medicinal Fungi
Fungi with medicinal properties are an important resource to modern medicine.

Medicinal fungi have been used for a long period of time. Unfortunately, medicinal
fungi are often overlooked and it leads to a deficiency in research on medicinal
fungi. Only a very small number of medicinal fungi have been utilized in modern
medicine (Li 2011). Li (2011) estimated that there are about 1500–2000 medicinal
fungi. A majority of them are macrofungi (polypores and mushrooms) and some
microfungi (Ying et al. 1987; Li 2011). In China alone, over 540 fungi have been
used in Chinese medicine (Li 2011). There are over 220 fungi both having medici-
nal properties used as medicinal herbs and being edible consumed as food in China
(Li 2011).
The discovery of Ötzi the Iceman, a well-preserved natural mummy (c. 3300 BC)
discovered from a glacier in Ötztal Alps, near Hauslabjoch on the border between
Austria and Italy in 1991, was big news, not only for archaeology and anthropology,
but also other fields of science, such as mycology. The iceman was carrying three
items of two polyspores, among other artifacts with him (Wikipedia 2014; Fowler
2001). The two polypores were later identified as Piptoporus betulinus (Bull.)
P. Karst. (for medicinal purposes) and Fomes fomentarius (L.) Fr. (for fire initia-
tion) (Peintner et al. 1998).
DNA sequencing from ancient archaeological specimens of polypores discov-
ered by archaeologists in the early Neolithic village of “La Marmotta” (Anguillara
Sabazia, Rome) (ca. 7000 years ago) showed that the fungus is Daedaleopsis tri-
color (Bull.) Bondartsev and Singer (Bernicchia et al. 2006). The authors suggested
that the fungus was collected for use during ritual functions or for their pharmaco-
logical properties. Montroux and Lundstrom-Baudais (1979) discovered Fomes in
swampy areas of the Neolithic sites of Clairvaux and Charavines in France that
dated between 2900 and 2399 BC.
Ophiocordyceps sinensis (Berk.) G. H. Sung et al. have been used for at least
2000 years (Shrestha et al. 2010) for its believed abilities to treat many diseases
related to lungs, kidney, and erectile dysfunction (Li 2011; Ying et al. 1987). Li
Shi- Zhen (1578) described over 30 medicinal fungi in his historical work on
Chinese traditional medicine Compendium of Materia Medica including macro-
fungi, Auricularia auricula (L.) Underw., Ganoderma lingzhi S. H. Wu, Y. C. and
Y. C. Dai (reported as Ganoderma lucidum (Curtis) P. Karst prior to 2012),
Morchella spp., Omphalia lapidescens Schroet., Phellinus igniarius (L.) Quél.,
Polyporus umbellatus (Pers.) Fr., Poria cocos (Schw.) Wolf., Termitomyces albu-
minosus (Berk.) R. Heim., Tremella fuciformis Berk., Trametes robiniophia Murr.,

and microfungi, Aecidium mori (Barclay) Dietel, Ophiocordyceps sinensis (Berk.)
G. H. Sung et al. (Cordyceps sinensis), Tilletia hordei Körn., Ustilago crameri
Körn., etc. (Li 1578). Ying et al. (1987) reported 272 medicinal fungi in details from
China in his monographic work Icons of Medicinal Fungi from China.
In addition to the medicinal microfungi described by Li (1578), there are a num-
ber of other microfungi that are often used in Chinese medicine. Use of Monascus
purpureus dates back over a thousand years in the North Song dynasty in China (Shi
and Pan 2011). In addition to use in rice wine making, food fermentation, and indus-
trial applications, Monascus purpureus has been used as a medication in Chinese
medicine (Ying et al. 1987). This fungus produces several secondary metabolites
such as dimerumic acid, pigments, polyketide monacolins, and γ-aminobutyric
acid. Studies showed that this fungus is effective for lowering blood cholesterol and
the management of diabetes, blood pressure, obesity, and Alzheimer’s disease and
has anticancer properties (Shi and Pan 2011; Ying et al. 1987; Lee et al. 2006).
Hypocrella bambusae (Berk. and Broome) Sacc. is used in Chinese medicine to
treat stomach problems and rheumatoid arthritis (Ying et al. 1987). Hypocrellin A
produced by H. bambusae showed promising antifungal and antileishmanial activi-
ties (Ma et al. 2004). Shiraia bambusicola Henn. is a pathogen to bamboo, primarily
in the southern provinces of China, and its stromata are commonly used in Chinese
medicine to treat a variety of disorders in humans (Yang et al. 2009). It also pro-
duces a number of perylenequinoid pigments, including hypocrellin A and hypo-
crellin B, and the hypocrellins showed excellent light-induced antitumor and
antiviral activities (Estey et al. 1996; Deininger et al. 2002; Zhang et al. 2004).
Several microfungi causing smut diseases have been used for their medicinal
properties, such as Ustilaginoidea virens (Cooke) Takah., Sphacelotheca sorghi
(Ehrenb. ex Link) G.P. Clinton (Sporisorium sorghi Ehrenb. ex Link), and several
more species of Ustilago: Ustilago esculenta Henn., Ustilago maydis (DC.) Corda,
Ustilago nuda (C.N. Jensen) Rostrup., Sclerotinia sclerotiorum (Lib.) de Bary,
Rhizoctonia sp., and Beauveria bassiana (Bals.-Criv.) Vuill. (Ying et al. 1987; Li
2011).
Claviceps purpurea (Fr.) Tul., a notorious mesofungus, causes both plant dis-
eases in a number of host grasses and human ergotism (aka ignis sacer, holy fire, or
St. Anthony’s fire). It also has some useful medicinal properties, which has been
known to humans for ca. three millennia (Ainsworth 1986). However, there are
conflicting reports on early fungus (ergot) description, its medicinal properties, and
epidemics of ergotism. The current brief review on ergot and ergotism is to clarify
or verify several contradictory reports in the literature. Among the hosts, rye, Secale
cereale L., is very susceptible to C. purpurea. The fungal pathogen will develop
ergot or spurred rye in the infected rye inflorescences. The earliest record of ergot
is probably an inscription to Gudea on a Babylonian tablet ca. 2500 BC with a text
“the women who gather noxious grasses and who were expelled from the city with
the exorcists and mutterers of charms” (Wellcome 1908). Another early record pos-
sibly referring to ergot is the “noxious pustule in the ear of grain” on an Assyrian
cuneiform tablet ca. 600 BC (Lapinskas 2007; Wellcome 1908; Van Dongen and de
252 D.-W. Li et al.
Groot 1995). Written reports of ergotism had not appeared until 857 in the Annales
Xantenses “a Great plague of swollen blisters consumed the people by a loathsome
rot, so that their limbs were loosened and fell off before death” (Barger 1931).
Ergotism is caused by ingesting ergot-contaminated grain products made from rye
infected by C. purpurea. Ergotism is the earliest known mycotoxicosis in human
history (Schlegel 2013). It is a well-studied fungus. A literature search using its
scientific name, Claviceps purpurea with Google Scholar, yielded 12,800 hits and
ergot, 71,800 hits, respectively.
The epidemics caused by C. purpurea were not only reported in the scientific
literature, but also captured in fine artworks (Pokorny 2003; Musée du Louvre
2015). Pieter Bruegel the Elder (ca. 1525–1569) reflected ergotism epidemics in his
several works. The most famous one is an oil painting “The Beggars (The Cripples)”
in 1568, which is in the possession of the Louvre in Paris (Fig. 11.1). Richard et al.
(2003) opined that this painting actually portrayed the gangrenous ergotism victims
of the tragic epidemics of the era. This is the reason the painting was chosen as the
cover of the book Mycotoxins: Risks in Plant, Animal and Human Systems (Richard
et al. 2003). In the description of the painting in the webpage of the Louvre, there
are two sentences that attracted our attention: “[t]he underlying significance of the
scene, the meaning of the foxtails in particular, remains unclear” and “[m]any
hypotheses have been put forward to interpret this painting, particularly addressing
the question of what the foxtails, hanging from the garments worn by the beggars,
Fig. 11.1 The Beggars by Pieter Bruegel the elder (ca. 1525–1569) in 1568. Copyright of Réunion
des Musées Nationaux, Louvre, Paris, France
are meant to symbolize” (Musée du Louvre 2015). It is rather possible that the inflo-
rescences hanging from garments in the painting may not be foxtails, but rye, which
was a staple crop in the Middle Ages and the sixteenth century in Europe. It seems
that Bruegel may have painted ergots to several of the seed heads in his painting
(Fig. 11.2). The seed heads of two plants are rather similar in morphology when
comparing them to the inflorescences of rye in the illustration of ergots (Secale
luxurians) in Caspar Bauhin’s Theatri Botanici in 1658 (Fig. 11.3). Secale luxurians
was the pharmaceutical named by Caspar Bauhin as one of the synonyms for ergot,
not for its host plant rye. However, there is no evidence to show whether Pieter
Bruegel the Elder had suspected the relationship between ergot and ergotism.
Caspar Bauhin’s illustration was considered the first one on ergot (Ainsworth 1986;
Van Dongen and de Groot 1995). However, the first illustration of ergot should actu-
ally go to Adam Lonicer (Adam Lonitzer or Adamus Lonicerus (1528–1586)), a
German physician/botanist with a drawing of ergotized rye with a caption of
“Hamelkorn” in Germany (Fig. 11.4) and giving a Latin name “Clavi siliginis” for
ergot in “Kräuterbuch” possibly in 1569 (Lonicer 1557–1577). Lonicer is also the
first one in the west to describe the medicinal properties of ergot and as a medication
used by midwives for assisting childbirth (Hofmann 1978; Ainsworth 1986).
Several authors had studied ergotism portrayed in a number of masterpieces of fine
arts from the Middle Ages, such as “Isenheim Altarpiece” painted for the Monastery
of St. Anthony by Matthias Grünewald and “St. Anthony Triptych” by Hieronymus
Bosch (ca. 1450–1516) to understand the ergotism epidemics and the roles played
by the monasteries of St. Anthony and apothecaries in the Middle Ages from medi-
Fig. 11.2 Detail from

“The Beggars” showing
possibly ergots by the
arrows
254 D.-W. Li et al.
Fig. 11.3 Drawing of

ergots (Secale luxurians)
and rye, the host plant by
Caspar Bauhin in Theatri
Botanici (1658)
cal, social, artistic, and religious perspectives (Kierulf 1982; Dixon 1984; de
Yébenes and de Yébenes 1990; Morán Suárez 1996; Battin 2009) due to confusing
interpretation of these works (Morán Suárez 1996).
Since the Middle Ages, rye was widely cultivated in Central and Eastern Europe.
Rye bread became the main staple food in most areas east of the French-German
border and north of Hungary (Schlegel 2013). Epidemics of ergotism were common
occurrences every 5–10 years in Europe from the Middle Ages to the nineteenth
century (Barger 1931). Wellcome (1908) opined that the ergotism epidemics that
occurred in 857 AD in France is the first report.
There are two kinds of ergotisms: gangrenous ergotism and convulsive ergotism
(Bar ger 1931), which occurred in different geographic areas in Europe (Eadie
2003). Between 1085 and 1927, epidemics of “convulsive ergotism” were wide-
spread in the east of the Rhine in Europe, while gangrenous ergotism occurred
mainly west of the Rhine (Wellcome 1908; Eadie 2003). Gangrenous ergotism—
ischemia—was the result of a restriction in blood supply to tissues or vasoconstric-
tive effects. Its symptoms included nausea, limb pain, limbs turning black,
impairment of sensation, and mummification, causing infected extremities to spon-
taneously break off or amputate. Gangrene was sometimes complicated by second-
ary infection, and the mortality rate was high (Eadie 2003). This type of ergotism is
Fig. 11.4 The earliest

illustration of ergotized rye
by Adam Lonicer in page
525 of “Kräuterbuch”
(1557–1577). The caption
is “Hamelkorn” in
Germany
characterized by the burning sensation in the extremities that led to the name “ignis
sacer”, i.e., holy fire (Wellcome 1908). Convulsive ergotism (St. Vitus Dance)
resulted in a nervous dysfunction, where the victim is twisting and contorting their
body in pain, trembling and shaking, and wryneck, painful seizures, spasms, con-
vulsions, confusions, and delusions. Hallucinations, mania, or psychosis may occur.
The discovery of the cause of gangrenous ergotism in 1630 is attributed to a
French physician, Dr. Tuillier (or Thuillier, a later spelling), via his experimentation
(Barger 1931). In several literature citations, this date was reported as 1670 or 1676
(Caporael 1976; Schumann 2000; Miedaner and Geiger 2015). However, the con-
troversy about the cause of convulsive ergotism continued to 1800 in Germany
(Wellcome 1908; Barger 1931). However, Garrison (1929) opined that the medical
faculty at Marburg in 1597 reported the cause of ergotism to be consumption of
bread made from spurred rye. It could be the first report to link the disease to the
cause. At present, we are not sure whether this report was supported by any
experimentation.
The epidemics of ergotism decreased with increased knowledge of the fungus
and the mycotoxins it produced, implementation of regulations, and advances in
milling procedures (Belser-Ehrlich et al. 2013). Since 1900, ergotism outbreaks in
humans have been very infrequent, but not eradicated. Several outbreaks have
occurred since then. During 1926–1927, a severe ergotism outbreak occurred in
Southern Russia with 10,000 cases of convulsive ergotism (Kent and Evers 1994).
256 D.-W. Li et al.
In 1927, Manchester in England suffered an outbreak with >200 cases of gangre-

nous ergotism (Robertson and Ashby 1928; Kent and Evers 1994). An infamous
outbreak occurred in Pont St. Esprit, France, in 1951 with ca. 150 people sick and
four fatalities due to muscular spasm and cardiovascular collapse for most cases and
one case with moist gangrene at the toes (Gabbai 1951). However, several alterna-
tive theories were later proposed to explain this epidemic, such as mercury poison-
ing. At present, consensus on the true cause of the epidemics has not been reached
(Belser-Ehrlich et al. 2013). India suffered a number of outbreaks caused by other
members of the genus Claviceps with devastating results in 1956–1957 and 1975
due to consumption of C. fusiformis ergotized pearl millet, Pennisetum glaucum
(L.) R.Br. (Krishnamachari and Bhat 1976; Patel et al. 1958), and sorghum (Sorghum
bicolor (L.) Moench) caused by Claviceps sorghi P. Kulkarni et al. and C. africana
Freder. et al. in 2001 (Navi et al. 2002; Pažoutová et al. 2000). Outbreaks occurred
in Ethiopia in 1977 and 2001, respectively, by consuming barley containing ergot-
ized wild oats (Demeke et al. 1979; Urga et al. 2002).
The ergot sclerotia contain high concentrations (up to 2 % of dry weight) of the
alkaloid ergotamine (derivatives of 6,8-dimethylergoline and lysergic acid deriva-
tives) with biological activities affecting circulation and neurotransmission (Eadie
2003; Tudzynski et al. 2001). The first clear account of ergot and its poisonous
properties is provided by the Persian physician, Abu Mausur Muwaffak Harawi (or
Abu Mansur Muwaffaq Heravi) (?950 AD) in his Book of the Remedies (Kitab al-
abnyia ‘an Haqa’iq al-adwiya) (Wellcome 1908). Since his book was written
between 968 and 977 AD (Golzari et al. 2012), the date of 950 AD cited by Wellcome
is questionable. Ergotamine has been prescribed for various causes of headaches,
including migraines. Ergometrine is used to control postpartum hemorrhage
(Mahmud et al. 2014), and ergonovine causes contraction of the uterus (Balki et al.
2015). The knowledge that the ergot could be used for the latter was known since
the seventeenth century when midwives prepared extracts of ergots for child deliv-
ery. Ergotamine in extracts of ergot had first been used to treat migraine headaches
in Italy in 1862 (Eadie 2004). The use of ergot as an obstetrical remedy was known
to the Chinese (emperor of Zhou dynasty 1100 BC) (p. 123, reference 15) (Bove
1970; Schiff 2006). Natural ergot alkaloid and its derivatives have been used to
assist child delivery and treat postpartum hemorrhage; diseases of the ears (includ-
ing inner auditory tubes), eyes (including cornea), lips, nose, tongue, and skin, as
well as hyperthyroid; autonomic nervous regulation; and motion sickness in China
(Ying et al. 1987; Li 2011).
At present, clinical ergotism due to ingestion of contaminated cereal grains is
rare (Ayarragaray 2014). However, it may occur by medications related to ergot
alkaloids administered for treating other medical conditions. An HIV-positive indi-
vidual was diagnosed with ergotism due to taking ergotamine tartrate for 2 weeks to
treat his severe migraine headache (Fröhlich et al. 2010), and a recent case was
reported for the same reason (Adam et al. 2014). Pharmaceutical properties of ergot
have been used to treat several medical conditions. Its alkaloids are still in use as an
oxytocic drug and used to treat migraines and Parkinson’s disease (Houghton and
Howes 2004; Gizzo et al. 2013; Tepper 2013). The gene for technological biosyn-
thesis of ergot alkaloids recently attracted attention (Li and Unsöld 2006). At pres-
ent, research has been conducted to identify cellular and molecular factors which
determine the response of cancer cells to six ergot alkaloids and their possibility for
tumor therapy (Mrusek et al. 2015). Ergoflavin isolated from Claviceps purpurea
showed cytotoxicity against five human cancer cell lines (Deshmukh et al. 2009).
Ergotamine produced by Claviceps purpurea was considered a potential biosecurity
threat (Paterson 2006; Wilson and Ho 2015). The genome of Claviceps purpurea
(strain 20.1) had been sequenced being 32.1 Mb in size (Schardl et al. 2013). The
authors who studied the dynamic of alkaloid loci of Clavicipitaceae with multi-
genome analysis found that the fungi including C. purpurea are under selection for
alkaloid diversification.
Medicinal properties of microfungi are great sources for the modern medicine
and pharmaceutical industry. Since the discovery of penicillin by Alexander
Fleming (1922), a number of microfungi have been used to produce medicinal
drugs, such as antibiotics, antifungals, immunosuppressants, etc. Using fungi to
produce medicinal drugs has been well covered in the literature. However, the
microfungi, which have been widely used in some traditional (alternative) medicine
since ancient times, are less studied. Some mechanisms of medicinal properties of
these fungi remain unclear.
A recent study on the endophytic fungi of 29 traditional Chinese medicinal plants
found 31 fungal groups at different taxonomic levels and 73 morphospecies (Huang
et al. 2008). Some phenolic compounds coexisted with certain endophytic fungi in
the same hosts. The results raise a question whether the endophytic fungi and their
secondary metabolites play any role in the medicinal properties of these plants.
Endophytic fungi are the new sources of anticancer bioactive compounds (Shukla
et al. 2014; Kumar et al. 2014; Deshmukh et al. 2014). The endophytic fungi of
medicinal plants and their pharmaceutical properties should be studied in the future.
At present, we have a better understanding of when and how the major fungal
phyla and some of their functions evolved. However, there are still some discrepan-
cies in dating fungal evolutionary events. It is clear that fungi have played a signifi-
cant role in human history and evolution. There is so much we can still learn from
the usage of fungi by our ancestors. Fungi are great resources to the human race
which should be better studied, conserved, and utilized.
Acknowledgements The authors are appreciative to Dr. Di Lu, Nanjing Agricultural University,
and Yale University Library for their assistance to obtain some literature which otherwise will not
be available to the authors.
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Chapter 12
Phylogenetic Diversity of Fungi in the Sea
including the Opisthosporidia
Ka-Lai Pang and E.B. Gareth Jones
Introduction
Kohlmeyer and Kohlmeyer (1979) stated that the ocean is a stable environment
with little fluctuation of temperature and salinity and a lack of growth substrates,
which exerts little selection pressure for the evolution of diverse lineages of marine
fungi. This opinion may have been true prior to molecular sequencing of biodiver-
sity, but modern techniques have revealed a much greater diversity (Nagahama
et al. 2003; Arfi et al. 2012). The open ocean has more or less constant physical
conditions in terms of salinity and temperature, and there is a general lack of
growth substrates for fungi. However, the majority of marine fungi found so far
have been reported from the terrestrial/ocean interface where the physical condi-
tions can change considerably seasonally or even daily. For instance, salinity of
mangrove water varies from 5 ‰ in Mai Po Mangrove, Hong Kong (Jones 2000),
to 46 ‰ in mangroves of the Red Sea (El-Sharouny et al. 1998). In addition, plen-
tiful organic materials are available for fungal colonization including wood; her-
baceous plants such as mangrove leaves, sea grasses, and palm fronds; dead
animals; and algae (Luo and Pang 2014). A recent documentation of over 1000
marine fungi (Jones et al. 2015) and an estimated number of 10,000 (Jones 2011)
further suggest that there is a high fungal diversity in the sea. Marine fungi mostly
K.-L. Pang, Ph.D. (*)

Institute of Marine Biology and Center of Excellence for the Oceans, National Taiwan Ocean
University, 2 Pei-Ning Road, Keelung 20224, Taiwan (R.O.C.)
e-mail: klpang@ntou.edu.tw
E.B.G. Jones, Ph.D.
Botany and Microbiology Department, College of Science, King Saud University,
Riyadh 1145, Saudi Arabia
e-mail: torperadgj@gmail.com

DOI 10.1007/978-3-319-29137-6_12
268 K.-L. Pang and E.B.G. Jones
Fig. 12.1 Marine fungi. (a) A deliquescing ascus. (b) Nimbospora effusa with large oil globules,
sheath, and appendages. (c) Halocyphina villosa on mangrove wood. (d) A section of the discomy-
cete Dactylospora vrijmoediae. (e) A filamentous ascospore of Lulworthia sp. (f) A muriform
ascospore of Aigialus grandis. Scale bar: a–b, f = 10 μm; c = 2 mm; e = 30 μm
belong to the Ascomycota and asexual forms with only a few basidiomycetes and
chytrids (Jones et al. 2009a). Many marine fungi, especially in the family
Halosphaeriaceae, have evolved special morphological features as an adaptation
to a marine life, including deliquescing asci (Fig. 12.1a) for aquatic dispersal of
ascospores in water, large oil globules (Fig. 12.1b) in ascospores for flotation, and
elaborate ascospore appendages/sheaths (Fig. 12.1b) for attachment to substrates
in the sea (Pang 2002). The diverse morphology of ascospore appendages has pro-
vided key characters for the delineation of genera and species in the
Halosphaeriaceae and other groups (Jones 1994).
Early studies in the field of marine mycology were descriptive, with mostly
diversity studies of various substrata (Kohlmeyer and Kohlmeyer 1979) and taxo-
nomic studies to classify marine fungi based on morphology at light and electron
microscopic levels (Jones and Moss 1978; Jones 1995). The taxonomic outline of
marine fungi by Johnson and Sparrow (1961) listed 2 orders, 5 families, and 65
genera. Later, Kohlmeyer and Kohlmeyer (1979) classified the marine fungi into 7
orders, 17 families, and 61 genera. With the advent of phylogenetic inference using
sequence data, many previously thought to be closely related taxa have been shown
to be distantly related and subsequently transferred to other orders and families
(Suetrong et al. 2009; Jones et al. 2014). New orders and families have also been
established for taxa forming independent lineages, e.g., Lulworthiales (Kohlmeyer
et al. 2000), Savoryellales (Boonyuen et al. 2011), and Dyfrolomycetales
12 Phylogenetic Diversity of Fungi in the Sea including the Opisthosporidia 269
(Hyde et al. 2013). A recent update by Jones et al. (2009a) documented 530 marine
fungi belonging to 29 orders, 56 families, and 209 genera. This observation sug-
gests that many morphological characters of marine fungi are possibly gained
through convergent evolution and therefore are not reliable taxonomic characters. It
also shows the high phylogenetic diversity of marine fungi in the sea.
In this chapter, we provide an updated account of the phylogenetic diversity of
fungi in the sea. We also include current knowledge of the Cryptomycota, the
Microsporidia, and the new phylum Aphelida in the sea, taxa which form a branch
at the basal position to the rest of the fungi (Karpov et al. 2014a).
Marine Fungi: More Evolved Branches
At least eleven major lineages have been discovered in the kingdom fungi (Blackwell
2011). Marine fungi are predominantly ascomycetes and asexual fungi with an
unknown taxonomic position, while only a few basidiomycetes have been reported
from the marine environment (Jones et al. 2009a). Also, few chytridiomycetes and
other groups, including the Mucoromycotina, have been reported from marine habi-
tats (Jones and Pang 2012). Isolation of the zygomycetes (Mucoromycotina,
Kickxellomycotina), marine yeasts, and predacious fungi may require special isola-
tion techniques, e.g., dilution plating of sediment or seawater or inclusion of nema-
todes for the discovery of predacious fungi (Shearer et al. 2007; Statzell-Tallman
et al. 2010; Swe et al. 2011). Marine zygomycetes have been reported from sponges
(Holler et al. 2000; Passarini et al. 2013), sediments (Bubnova 2010), and wood
(Rämä et al. 2014) or found associated with marine arthropods (Lichtwardt 2012).
Most marine basidiomycetes belong to the Agaricomycotina, while
Flamingomyces ruppiae and Parvulago marina are in the Ustilaginomycotina (Jones
et al. 2009a). The few marine Basidiomycota are not monophyletic, suggesting indi-
vidual lines of evolution from terrestrial environment to the sea (Hibbett and Binder
2001; Binder et al. 2006). Calathella mangrovei, Halocyphina villosa (Fig. 12.1c),
Nia vibrissa, Physalacria maipoensis, and Mycaureola dilseae are in the euagaric
clades; C. mangrovei, H. villosa, and N. vibrissa cluster in the Nia clade with
Lachnella spp., Flagelloscypha spp., Cyphellopsis anomala, Merismodes fascicu-
lata, and Dendrothele acerina, while P. maipoensis and M. dilseae form a separate
clade with Gloiocephala, Armillaria, and Flammulina velatipes (Binder et al. 2006).
Many marine Agaricomycetes are intertidal, with minute, enclosed basidiomata on
wood, marsh plants, and red algae, and have lost ballistospory, morphological adap-
tations for an aquatic life (Binder et al. 2006; Jones et al. 2009a). Marine basidiomy-
cetes, such as common mangrove species Halocyphina villosa and Calathella
mangrovei, are mainly saprobic, but Mycaureola dilseae is parasitic on the red alga
Dilsea carnosa (Binder et al. 2006). Marine basidiomycetous yeasts contribute fur-
ther to marine fungal diversity; Jones et al. (2015) document marine yeasts:
Ascomycota, 138 species (in 35 genera), and Basidiomycota, 75 species (in 26 gen-
era), many reported from deep water including many new species of the genera
Fig. 12.2 Phylogenetic lineages of fungi in the sea. Topology is based on Schoch et al. (2009) and
Karpov et al. (2014a)
Cryptococcus, Rhodosporidium, Rhodotorula, and Sporobolomyces (Kutty and

Philip 2008; Fell et al. 2011).
The Ascomycota constitutes roughly 80 % of the total marine mycota, and
they belong to different classes including Eurotiomycetes, Laboulbeniomycetes,
Lecanoromycetes, Leotiomycetes, Lichinomycetes, Arthoniomycetes, Sordario-
mycetes, and Dothideomycetes (Fig. 12.2), with the majority in the last two classes
and many with unknown taxonomic positions, such as the genera with cleisto-
thecial ascomata: Biflua, Crinigera, Dryosphaera, and Eiona (Jones et al. 2009a;
Jones 2011). Many of these genera have restricted distribution and were infrequently
collected, thus preventing a molecular study. Dothideomycetes and Sordariomycetes
were also found to be the dominant classes of fungi occurring on the mangrove trees
Avicennia marina and Rhizophora stylosa using tag-encoded 454 pyrosequencing of
the 18S and ITS regions of the rRNA gene (Arfi et al. 2012). With the increased
mycological research activity in Asian mangroves in recent years, many (rare)
marine fungi have been recollected after their original descriptions, which enabled
a systematic evaluation. An example is Manglicola guatemalensis, a species origi-
nally described on Rhizophora in Guatemala (Kohlmeyer and Kohlmeyer 1971), but
recollected in Thai mangroves (Suetrong et al. 2010). This enabled its classification
in the Jahnulales, an order previously not represented in marine habitats.
Laboulbenia marina is the only species in the Laboulbeniomycetes, and it was
found on a beetle in the Laminaria zone in France (Kohlmeyer and Volkmannn-
Kohlmeyer 2003). The Laboulbeniomycetes are predominantly terrestrial and fresh-
water species, so the marine occurrence of this fungus was questioned by Jones
et al. (2009a).
Marine Dactylospora species were classified, based on morphological observa-
tions, in the Lecanorales (Lecanoromycetes), along with their terrestrial
Dactylospora counterparts. Recently, Schoch et al. (2009) and Diederich et al.
(2013), based on sequence analyses, found that marine Dactylospora (D. man-
grovei, D. haliotrepha) formed a separate lineage with Sclerococcum sphaerale in
the Eurotiomycetes and well separated from their terrestrial counterparts and unre-
lated to the Lecanoromycetes. Pang et al. (2014) further confirmed these observa-
tions and described a new marine species, D. vrijmoediae (Fig. 12.1d). Other marine
Eurotiomycetes include Eupenicillium and Gymnascella, both infrequently reported
(Jones et al. 2009a), which probably will have a much wider occurrence with greater
emphasis on molecular techniques.
Amylocarpus encephaloides is the only species sequenced in the marine
Leotiomycetes, an assignment suggested by a phylogenetic analysis of the 18S
rDNA gene (Landvik 1996). This species is likely to be related to taxa of the
Erysiphales as suggested by a number of studies (Landvik 1996; Berbee 2001;
Hambleton and Sigler 2005). No sequences are available for Vibrissea nypicola (but
terrestrial species are assigned to the Helotiales, Leotiomycetidae, and Leotiomycetes)
and Laetinaevia marina. Lichina (Lichinaceae, Lichinomycetes) is a lichenized
genus with two marine species, L. confinis and L. pygmaea (Jones et al. 2009a), that
group with Pseudopaulia tessellata (Schultz and Büdel 2003). Halographis is a
marine lichenoid species in the Arthoniomycetes and placed in the Roccellaceae,
Arthoniales (MycoBank), but no sequence is available for this species. The most
specious marine lichens are members of the Verrucariales with the genera:
Collemopsidium (7 species), Hydropunctaria (6 species), Mastodia (1 species),
Wahlenbergiella (3 species), and Verrucaria (16 species) (Jones et al. 2015). The
placement of Melaspilea mangrovei in the Melaspileaceae, Arthoniales,
Arthoniomycetidae, and Arthoniomycetes is unresolved as it has not been sequenced.
This species has been reported from mangrove wood and is probably saprobic
(Vrijmoed et al. 1996).
Most marine Sordariomycetes belong to the Halosphaeriaceae, Microascales

(Pang 2002; Jones et al. 2009a; Sakayaroj et al. 2011). This family includes pre-
dominantly marine fungi from wood, but several genera can also be found in fresh-
water habitats, such as Aniptodera, Halosarpheia, and Nais (Pang 2002). The
taxonomic position of the freshwater genus Fluviatispora in the Halosphaeriaceae
requires a molecular study. The same is true for Chadefaudia and Trailia, which are
found on marine algae (Jones et al. 2009a). Spatafora and Blackwell (1994) were
the first to include a sequence of the Halosphaeriaceae (Halosphaeriopsis mediose-
tigera) and its assignment to the Microascales. Over the last two decades, many
taxa of the Halosphaeriaceae have been collected, isolated and sequenced, and
shown to be a monophyletic group (Spatafora et al. 1998; Sakayaroj et al. 2011).
However, the phylogenetic relationships between genera and species are not well
resolved, and Jones (1995) suspected there has been a rapid evolutionary radiation.
Spatafora et al. (1998) concluded that members of the Halosphaeriaceae were prob-
ably evolved from terrestrial environments over a short period of time. Adaptive
features include: development of wide range of ascospore appendages for attach-
ment to comparatively scarce substrata in the sea and deliquescing asci as a means
for spore dispersal (Jones 1994, 2006). Pang et al. (2003) speculated that unfurling
ascospore appendage type in genera such as Halosarpheia and Saagaromyces,
which can be commonly found in mangrove environments, may be an intermediate
form between unornamented species and those with complex ascospore appendage
morphology and ontogeny, e.g., Corollospora and Naufragella. Sakayaroj et al.
(2011) further suggested that unfurling ascospore appendages might have been
gained and lost several times during evolution. Consequently, mangrove environ-
ment may be a stepping stone for oceanic taxa to evolve from terrestrial habitats.
Kohlmeyeriella, Lindra, and Lulworthia (Fig. 12.1e) were originally placed in
the Halosphaeriaceae based on their morphological characteristics, although the
latter two genera produce filiform ascospores (Jones 1995). Spatafora et al. (1998)
first suggested that Lindra and Lulworthia were unrelated to the Halosphaeriaceae
and formed a new lineage in the Sordariomycetes, the Lulworthiaceae (Lulworthiales)
(Kohlmeyer et al. 2000). Kohlmeyeriella, Rostrupiella, Haloguignardia, and
Spathulospora were later added to this new order (Inderbitzin et al. 2004; Campbell
et al. 2005; Koch et al. 2007), although they share few morphological features in
common. The validity of the Spathulosporaceae requires further studies as this fam-
ily did not form a distinct lineage in the Lulworthiales. Spathulospora is a parasite
of the red algal genus Ballia, near the Antarctic region, and has been rarely reported
(Kohlmeyer and Kohlmeyer 1975; Jones et al. 2009a). Other members of this order
are mainly saprobic on wood or algae. Pontogeneia, a parasitic genus on brown
algae, and Koralionaste from corals (Koralionastetales) have recently been shown
to form a dichotomy with the Lulworthiales (Campbell et al. 2009). Members of
both orders differ in morphology, including the presence of paraphyses and periphy-
ses, and the lack of apical mucus-containing chambers or gelatinous sheaths of the
ascospores in the Koralionastetales (Campbell et al. 2009).
Other new lineages of marine fungi include the Savoryellales and the new fami-
lies established for the taxa formerly known as the “TBM clade” (Schoch et al.
2006). Savoryella is an aquatic genus with freshwater, brackish, and marine species
(Jones et al. 2009a) and morphologically similar to the Halosphaeriaceae (Read
et al. 1993) and Sordariales (Jones and Hyde 1992). As a result, the taxonomy of
Savoryella had been unresolved and was referred to various orders or Ascomycota
incertae sedis (Jones et al. 2009a). Vijaykrishna et al. (2006), in a phylogenetic
analysis of the 18S rRNA gene, firstly suggested that Savoryella is unrelated to the
Halosphaeriaceae nor the Sordariales, but rather a group of taxa in the Hypocreales.
In a multigene phylogenetic analysis (18S, 28S, 5.8S rDNA, rpb1, rpb2, tef1),
Boonyuen et al. (2011) discovered that Savoryella is closely related to two freshwa-
ter genera Ascotaiwania and Ascothailandia and they together constitute a new lin-
eage, the Savoryellales, in the Hypocreomycetidae.
Swampomyces is characterized by forming a clypeus on wood, cylindrical asci
with an apical structure, septate paraphyses, and hyaline ascospores (Kohlmeyer
and Volkmann-Kohlmeyer 1987). Torpedospora radiata, on the other hand, pro-
duces coriaceous ascomata, deliquescing asci, and ascospores with several armlike
appendages (Meyers 1957). Both genera formed a monophyletic, yet unknown, lin-
eage in the Hypocreomycetidae, based on a phylogenetic analysis using 18S and
28S rDNA (Sakayaroj et al. 2005). This observation was further confirmed by
Schoch et al. (2006) who used a wider representation of taxa (Juncigena adarca,
Etheirophora spp.) and genes (18S and 28S rDNA, rpb2) and tentatively named it
as “Torpedospora/Bertia/Melanospora (TBM) clade” but no taxonomic change was
proposed. Recently, Jones et al. (2014) demonstrated that the marine fungus
Chaetosphaeria chaetosa and the terrestrial genus Falcocladium also belong to the
assortment of species in the TBM clade and formally established new families:
Juncigenaceae, Etheirophoraceae, Falcocladiaceae, and Torpedosporaceae to
accommodate these taxa.
Marine fungi can also be found in other orders of the Sordariomycetes:
Hypocreales, Diaporthales, Sordariales, Ophiostomatales, Xylariales,
Magnaporthales, and Phyllachorales (Jones et al. 2009a). However, many orders
are only represented by a single marine species, while many others have not been
subjected to a molecular study. Xylariaceous taxa are common on wood and the
brackish water palm Nypa fruticans, for example, Halorosellinia oceanica and
Nemania maritima were confirmed to be members of the Xylariaceae (Smith et al.
2003; Pinnoi et al. 2010). Pedumispora rhizophorae, a taxon previously assigned to
the Diaporthales, was recently found to be related to the Diatrypaceae, Xylariales,
based on a phylogenetic analysis of the 28S and ITS regions of the rDNA, although
the deliquescing asci and filiform ascospores with polar hooklike end cells do not fit
the morphological delimitation of other diatrypaceous genera (Klaysuban et al.
2014). Diatrypasimilis is another member of the Diatrypaceae recently reported
from decaying Rhizophora wood collected in Australian mangroves (Chalkley et al.
2010), with features more typical of the family. Marine Hypocreales, including
Kallichorma spp., Heleococcum japonense, and Emericellopsis maritima, are
referred to the Bionectriaceae (Rossman et al. 2001; Zuccaro et al. 2003), while
Sedecimiella taiwanensis from mangroves of Taiwan and China is the sole member
of the Niessliaceae (Pang et al. 2010).
Marine Dothideomycetes predominantly belong to the subclass

Pleosporomycetidae, which have been mainly collected from mangrove substrata
(Jones et al. 2009a). These fungi occupy the upper tidal zone with occasional sea-
water submergence and have active spore discharge mechanisms (Suetrong et al.
2009). A significant change in the taxonomic classification of a number of marine
Dothideomycetes has been observed in the last few years with the extensive collec-
tion and isolation of mangrove fungi in Thailand and other Asian countries. As a
result, a number of new orders and families have been established, including
Dyfrolomycetaceae (Dyfrolomycetales), Halojulellaceae, Trematosphaeriaceae,
and Manglicolaceae, and this may suggest the wide morphological diversity
expressed by the marine Dothideomycetes (Hyde et al. 2013).
Few marine species in the subclass Dothideomycetidae have been sequenced.
Mycosphaerella eurypotami and Scirrhia annulata grouped with other terrestrial
Mycosphaerella spp. in the Mycosphaerellaceae, Capnodiales (Suetrong et al.
2009), while M. pneumatophorae is distantly related to this group and grouped with
Phaeotrichum benjaminii, Tyrannosorus pinicola, and Venturia inaequalis (Schoch
et al. 2009).
Considerable advancement has been made in the classification of the marine
Pleosporomycetidae in the last decade. Suetrong et al. (2009) identified 17 families
in the Pleosporales, with many marine taxa forming lineages outside these recog-
nized families resulting in the introduction of four new families (Jones et al.
2012a). Fifteen families (Aigialaceae, Amniculicolaceae, Didymellaceae,
Didymosphaeriaceae, Halojulellaceae, Leptosphaeriaceae, Lenthitheciaceae, Lophio-
stomataceae, Massarinaceae, Morosphaeriaceae, Phaeosphaeriaceae, Pleospo-
raceae, Sporormiaceae, Testudinaceae, Trematosphaeriaceae) of the Pleosporales
include marine taxa (Suetrong et al. 2009; Jones et al. 2012a; Ariyawansa et al.
2013). Many marine taxa in these families are represented by a single/few species,
further proving their terrestrial origin: Neomassariosphaeria typhicola predomi-
nantly from marsh plants in the Amniculicolaceae, a family of freshwater ascomy-
cetes (Zhang et al. 2009a); Didymella fucicola from brown algae in the Didymellaceae
(Suetrong et al. 2009); Lentithecium phragmiticola and Keissleriella rarum from
saltmarsh plants in the new family Lentitheciaceae (Zhang et al. 2009b);
Lophiostoma scabridisporum and Paraliomyces lentiferus in the Lophiostomaceae;
Tremateia halophile on Juncus roemerianus in the Didymosphaeriaceae;
Loratospora aestuarii and Phaeosphaeria species (Ph. albopunctata, Ph. olivacea,
Ph. spartinicola, Ph. typharum) in the Phaeosphaeriaceae (Suetrong et al. 2009;
Zhang et al. 2009b); Paradendryphiella spp., Decorospora gaudefroyi, and
Alternaria maritima in the Pleosporaceae (Suetrong et al. 2009; Zhang et al. 2009b;
Inderbitzin et al. 2002; Suetrong et al. 2009; Zhang et al. 2009b); Amorosia littora-
lis in the Sporormiaceae (Mantle et al. 2006); and Massarina ricifera and Verruculina
enalia in the Testudinaceae (Suetrong et al. 2009). Many marine Pleosporales still
await phylogenetic analysis before they can be properly taxonomically assigned,
e.g., marine Massarina spp., and Leptosphaeria spp. (Jones et al. 2012a).
Suetrong et al. (2009) investigated the phylogeny of Aigialus spp. (Fig. 12.1f),
Ascocratera manglicola, and Rimora (Lophiostoma) mangrovei and found they
formed a robust monophyletic group in a basal position of the Pleosporales, sepa-
rating from other known families of the order, so a new family, the Aigialaceae, was
established for this group. These genera have similar ascomatal morphology and
asci, while they mainly differ in ascospore morphology: muriform and dark-colored
in Aigialus and hyaline in Ascocratera and Rimora. A new family, the
Morosphaeriaceae, was proposed to accommodate Morosphaeria velatispora,
M. ramunculicola, Helicascus spp., and Kirschsteiniothelia elaterascus (Suetrong
et al. 2009). There is great morphological variation in this group, especially the
ascospores. Subsequently, K. elaterascus was transferred to Morosphaeria
(Boonmee et al. 2012). Kirschsteiniothelia is a polyphyletic genus with K. mari-
tima, another marine species, grouping with Mytilinidiales (Suetrong et al. 2009).
The marine mangrove fungi Halomassarina thalassiae and Falciformispora ligna-
tilis constituted a monophyletic group with Trematosphaeria pertusa, the type of
species of the newly proposed family Trematosphaeriaceae that was reinstated
(Suetrong et al. 2009). However, these fungi have few morphological characters in
common. Whether the marine Trematosphaeria species affiliate with this group will
require further study.
Julella avicenniae was described from intertidal wood of Avicennia but can also
be found on the more terrestrial part of the tree (Borse 1987; Hyde 1992; Jones et al.
2009a). Julella avicenniae differs from J. buxi (type species: Didymosphaeriaceae,
Pleosporales) in that the ascus contains eight ascospores with an apical apparatus.
Therefore, a new genus Halojulella was proposed for J. avicenniae in the new fam-
ily Halojulellaceae based on morphological and sequence data (Ariyawansa et al.
2013; Ariyawana et al. 2014). The taxonomic placement of J. herbatilis, on Juncus
roemerianus, requires sequence data to confirm its position in the Halojulellaceae
and its relationship with H. avicenniae.
Asexual marine fungi are fairly common and constitute about 15 % of the total
marine fungi (Jones et al. 2009a). Abdel-Wahab and Bahkali (2012) listed 143 spe-
cies of asexual marine fungi belonging predominantly to the Dothideomycetes and
the Sordariomycetes (Ascomycota). Asexual marine fungi can also be found in other
classes, including the Eurotiomycetes, the Leotiomycetes, and the Orbiliomycetes
(Abdel-Wahab and Bahkali 2012). Halenospora varia, a cosmopolitan species on
wood, was found to be unrelated to Zalerion, an earlier assignment based on mor-
phology (Bill et al. 1999; Jones et al. 2009a). Other asexual genera have also been
proven to be polyphyletic, e.g., Cirrenalia and Sigmoidea (Jones et al. 2009b;
Abdel-Wahab et al. 2010), which suggests that conidial morphology is unreliable in
classifying asexual marine fungi. Sequences for many asexual marine fungi have
been made available, allowing their formal taxonomic classification. Asexual fungi
of the Orbiliomycetes are nematode-trapping fungi in the genera Arthrobotrys,
Dactylella, and Monacrosporium, while Aspergillus sydowii, found on the sea fan
Gorgonia spp., belongs to the Eurotiomycetes. Species of Aspergillus and Penicillium
have been repeatedly isolated from marine sediments and marine animals
(Khudyakova et al. 2000; Zhang et al. 2012) and whether some of them are marine
would require a detailed investigation at the molecular level.
Within the Ascomycota, most asexual marine fungi belong to the Dothideomycetes
(Pleosporales) and the Sordariomycetes (Halosphaeriaceae, Lulworthiales), which
corroborate the observation that most marine sexual fungi are in the same two
classes and that teleomorphic and anamorphic phases of the same fungus occupy a
similar niche (Alexopoulos et al. 1996). Many sexual/asexual relationships have
been linked from culture observations, e.g., Nereiospora cristata/Monodictys
pelagica and Halosphaeriopsis mediosetigera/Trichocladium achrasporum
(Mouzouras and Jones 1985; Shearer 1986), while others can only be referred to a
genus, e.g., Halosigmoidea marina and H. parvula (putative stage in Corollospora),
or are unknown, especially those in the Lulworthiales, e.g., Cumulospora, Hydea,
and Matsusporium (Abdel-Wahab et al. 2010; Abdel-Wahab and Bahkali 2012).
Sequence data can only be used to classify asexual fungi rather than linking asexual/
sexual relationships due to the incomplete representation of reference sequences in
the GenBank.
Marine chytrids have been poorly studied, few have been documented in recent
years, and most marine texts do not include them (Kohlmeyer and Kohlmeyer 1979;
Jones et al. 2009a). However, their importance as parasites of marine animals and
their retrieval from sediments in hydrothermal vents suggest that they may have an
important role in the marine environment (Nagano et al. 2010; Gleason et al. 2012;
Hatai 2012). Currently marine Chytridiomycota species include members of the
orders Chytridiales (two families, four genera, 12 species), Cladochytriales (one
genus, one species), Lobulomycetales (one family, one genus, one species),
Rhizophydiales (two families, two genera, two species), and incertae sedis (five
genera, 10 species) with a cumulative number of 26 species (Jones et al. 2015).
Another zoosporic group of organisms the Blastocladiomycota is represented by
one species in the genus Catenaria (Blastocladiales, Catenariaceae).
Marine Fungi: Basal Lineages
The new superphylum Opisthosporidia was recently established to accommodate

the three phyla, Cryptomycota, Microsporidia, and Aphelida, which have been
found to be related to the kingdom Fungi but form a basal branch to other fungi
(Karpov et al. 2014a). The three phyla are united by (1) primarily phagotrophic
mode of nutrition, (2) formation of cysts with penetrative apparatus, and (3) pres-
ence of tubular/flat mitochondrial cristae (Karpov et al. 2014a). Marine Microsporidia
and Aphelida are mainly parasites of marine organisms (Karpov et al. 2014a;
Stentiford et al. 2013), while marine Cryptomycota have been detected in the se
diment, in the form of environmental sequences of small subunit ribosomal RNA
gene (van Hannen et al. 1999; Lara et al. 2010; Jones et al. 2011a).
Pseudaphelidium drebesii is the only marine species represented in the Aphelida,
parasitic on the marine diatom Thalassiosira punctigera (Karpov et al. 2014b). The
life cycle of P. drebesii involves attachment of zoospores to the host leading to

cysts, penetration of host cells via infection tubes, and formation of a plasmodium
cleaving into amoeboid cells which encyst and are released to form a new genera-
tion of zoospores (Karpov et al. 2014b).
Microsporidia are obligate intracellular parasites of eukaryotic hosts, which can
be found in terrestrial, freshwater, and marine environments. The life cycle of the
Microsporidia varies and it generally involves infection using a polar tube, intracel-
lular replication as meronts, and differentiation into spores (Kearney and Gleason
2014). In marine environment, the Microsporidia infect a wide variety of hosts
including crabs (Stentiford et al. 2014), nematodes (Sapir et al. 2014), fish (Matthews
et al. 2013), copepods (Jones et al. 2012b), lobsters (Stentiford et al. 2010), shrimps
(Brown and Adamson 2006), and amphipods (Dunn et al. 2001). The classification
of the Microsporidia is based on their ecology, i.e., classes Terresporidia,
Aquasporidia, and Marinosporidia for microsporidia infecting terrestrial, freshwa-
ter, and marine hosts, respectively (Vossbrinck and Debrunner-Vossbrinck 2005).
Marinosporidia is polyphyletic, suggesting the unreliability of this criterion
(Stentiford et al. 2013). Site of infection (tissue type) to the hosts may be more
phylogenetically relevant (Stentiford et al. 2013).
Cryptomycota was first described as a group of sequences (LKM11) closely
related to fungi derived from denaturing gradient gel electrophoresis (van Hannen
et al. 1999). Subsequently, other studies also detected the presence of sequences
related to LKM11 in different terrestrial, freshwater, and marine habitats (Jones
et al. 2011a; Livermore and Mattes 2013). In particular, LKM11 was detected in
marine anoxic sediment (Dawson and Pace 2002), deep-sea sediment (Nagano
et al. 2010), deep-sea methane cold-seep sediments (Nagahama et al. 2011), and
marine upper-water column (Livermore and Mattes 2013). Lara et al. (2010) was
the first study to show that LKM11 was related to Rozella allomycis with a good
support using 18S rRNA gene sequence and named the group Rozellida. Jones
et al. (2011b) formally named this group the Cryptomycota due to the cryptic
nature of the group (discovered only by environmental sequences). No culture is
available for LKM11 but by using tyramide signal amplification fluorescence in
situ hybridization (TSA-FISH) coupled with monoclonal antibody TAT116 against
α-tubulin, cells of the Cryptomycota are ovoid, 3–5 μm in size; one or more cells
are attached to diatoms; and a flagellum is present in at least one stage of their life
cycle (Jones et al. 2011a). These characters can also be found in Rozella spp.,
which are parasites of Oomycota, Blastocladiomycota, and Chytridiomycota
(Gleason et al. 2014). However, no chitin or cellulose was detected in cells of the
Cryptomycota by Calcofluor white (Jones et al. 2011a); in R. allomycis, chitin or
cellulose was present in young resting spores (James and Berbee 2012). One
marine species, R. marina, was reported as a parasite of Chytridium polysiphoniae,
which in turn, is parasitic on brown algae (Sparrow 1936; Glockling et al. 2012).
Zoospores of R. marina are formed inside the sporangium of C. polysiphoniae
(Johnson 1966).
Concluding Remarks
The paper by Barghoorn and Linder (1944) is generally regarded as the stimulus for
the study of marine fungi. However, progress was slow until the 1960s when many
studies reported on marine Sordariomycetes growing on submerged wood and drift-
wood. The discovery of marine fungi, predominantly Dothideomycetes, on man-
grove substrata led to a dramatic increase in our knowledge during 1980s to late
1990s, with many new taxa described from the tropics, especially Asia. From 2000,
the number of species described has dropped with mycologists focusing on deter-
mining the phylogenetic affinity of taxa already described. However, the study of
marine fungi has proceeded along two paths: the mycelial forms (circa 899 species)
and yeasts (circa 213 species), with little interaction between the two groups of
mycologists studying them. Other taxa have not been critically studied, especially
the so– called marine-derived fungi and facultative marine fungi, taxa mainly docu-
mented by those interested in the discovery of compounds with new bioactivity.
The last decade has seen greater awareness of the “fungal-like” organisms once
referred to the Phycomycetes. This interest has been stimulated by the realization
that many are important pathogens of marine organisms, their source of polyunsatu-
rated fatty acids (PUFAs), and their report from deep sea hydrothermal vents. Few
publications have focused on bringing these different approaches together, so that
we have a better understanding of the marine mycota and related organisms (Jones
and Pang 2012). Marine lineages of fungi are summarized in Fig. 12.2.
The next decade will be dominated by the development of new molecular tech-
niques for their study, e.g., tag-encoded 454 pyrosequencing of the ribosomal rDNA
and rRNA and fluorescence in situ hybridization (FISH). These will bring different
challenges, especially dealing with the vast amount of data generated and in their
interpretation. Also better documentation of sequences generated from these studies
and correct identification of data deposited in world data banks enable a better
understanding of the phylogenetic relationship of the different taxa. Currently, the
number of marine fungi is circa 1112, while Jones (2011) predicts a figure of
11,000–12,500 species (Jones et al. 2015).
Acknowledgments K.L. Pang would like to thank the Ministry of Science and Technology,
Taiwan, for financial support (NSC101-2621-B-019-001-MY3).
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Chapter 13
Biology and Ecology of Freshwater Fungi
Clement K.M. Tsui, Christiane Baschien, and Teik-Khiang Goh
Introduction
Freshwater fungi complete at least one part of their life cycle in water and distribute
propagules (spores, conidia, sporangia) in or above water. It has been estimated
that there are more than 3000 species of fungi occurring in the aquatic habitats
(Abdel-Aziz 2008). Taxonomically, aquatic fungi comprise taxa from all fungal
phyla (Cryptomycota, Chytridiomycota, Blastocladiomycota, Mucoromycotina,
Glomeromycota, Dikaryomycota).
Fungal morphology in freshwater ranges from zoospores and nonmotile single
cells of Cryptomycota, Chytridiomycota, Blastocladiomycota, yeasts, and asep-
tate and septate hyphae up to interwoven hyphae in more or less complex plec-
tenchyma in higher fungi. Sporangiophores and sporangia (Chytridiomycota,
Blastocladiomycota, and Mucoromycotina), ascomycetous and basidiomycetous
Our work is dedicated to John Webster (1925–2014) for his pioneer and influential work on
freshwater fungi.
C.K.M. Tsui, Ph.D. (*)
Department of Pathology and Laboratory Medicine, University of British Columbia,
Vancouver, BC, Canada, V6T 1Z4
e-mail: clementsui@gmail.com
C. Baschien, Ph.D.
Department of Microbial Ecology and Diversity, Leibniz Institute
DSMZ-German Collection of Microorganisms and Cell Cultures,
Inhoffenstr. 7 B, 38124 Braunschweig, Germany
T.-K. Goh, Ph.D.
School of Biological Sciences, Faculty of Integrative Sciences and Technology,
Quest International University Perak, No. 227, Plaza Teh Teng Seng (Level 2),
Jalan Raja Permaisuri Bainun, 30250 Ipoh, Perak Darul Ridzuan, Malaysia
e-mail: teikkhiang.goh@qiup.edu.my

DOI 10.1007/978-3-319-29137-6_13
286 C.K.M. Tsui et al.
yeasts, conidiophores, and a high diversity of more or less conspicuous conidia

(Ascomycota and Basidiomycota derived) are also visible under the microscope. In
addition, complex structures such as acervuli, pycnidia, and ascocarps and basidio-
carps as well as lichens can be found on substrates in freshwaters. Over the past few
decades, various morphological and ecological groups of water-associated fungi
have been identified (Fuller and Jaworski 1987; Goh and Hyde 1996; Hyde et al.
1997; Ingold 1975; Jones 1981). These include the zoosporic fungi, the aquatic
ascomycetes, the “Ingoldian fungi,” the aero-aquatics, and a great diversity of mito-
sporic fungi (asexual Ascomyceta) occurring on submerged plant materials.
Worldwide, freshwater comprises diverse habitats such as groundwater, streams,
rivers, canals, and lakes but also includes amphibious habitats, such as ditches,
peats, and swamps (Shearer et al. 2007). Generally, most freshwater fungi are asso-
ciated with organic matter derived from decaying plants and animals. However,
there are many microhabitats in and adjacent to freshwater, which provide space
and possibilities for different fungal life strategies. These microhabitats include
roots and other parts of submerged and riparian plants, the canopy, the ambient soil,
and sediments. Indeed, freshwater fungi are recorded from the tree canopy (Ando
1992; Ando and Tubaki 1984a, b; Sridhar et al. 2009), rainwater (Gönczöl and
Révay 2004), and soil (Park 1974). Also leaf litter in treeholes (Gönczöl and Révay
2003), dew drops (Tubaki et al. 1985), and honey (Magyar et al. 2005) have been
identified as locality for spores of freshwater fungi. Fungi have been observed
within cooling towers (Eaton and Jones 1971a, b), groundwater (Krauss et al. 2005),
and potable water distribution systems (Doggett 2000; Nagy and Olson 1982)
including tap water (Heinrichs et al. 2013).
Who Are the Members of Freshwater Fungi?
Traditional taxonomy and identification of fungi is mostly based on fruiting bodies

and spores. Freshwater fungi are divided into various morphological and eco-
logical groups (Shearer et al. 2007; Goh and Hyde 1996). The different groups
require specialized methods to examine their biodiversity, taxonomy, distribu-
tion, population dynamics, and ecological functions. Traditionally, it has been a
challenge to characterize all the different groups of freshwater fungi within a
freshwater habitat.
The first group is (I) the aquatic hyphomycetes (more than 300 described mito-
sporic fungi) (Figs. 13.1 and 13.2), also known as “the Ingoldian fungi” in honor of
the pioneer mycologist Prof. C. T. Ingold, which comprises conidial states of mainly
Ascomycota and a few Basidiomycota (Shearer et al. 2007; Jones et al. 2014). Ingold
(1942) discovered the conidia in the foam of streams and showed the connection to
their associated mycelia on submerged leaves. Most aquatic hyphomycetes produce
conspicuous stauroconidia (e.g., tetraradiate [Alatospora, Articulospora] or other
branched forms [Varicosporium]) or scolecoconidia (sigmoid, curved, or straight
[Anguillospora, Flagellospora]). The conidial shape is an adaptation to survival and
13 Biology and Ecology of Freshwater Fungi 287
Fig. 13.1 Ingoldian fungi. (a, b) Triscelophorus acuminatus. Conidia production seen at the edge
of submerged leaf. Each conidium has four arms. (c, d) Triscelophorus monosporus. Conidia with
three arms. (e) Lunulospora cymbiformis. Conidia which are sickle-shaped and distinctly bent
more or less at right angle. (f) Anguillospora crassa. Typical sigmoid-shaped conidium. (g, h)
Helicomyces roseus. Typical coiled conidia. (i) Helicomyces roseus. Conidium which has uncoiled
and become more or less sigmoid shaped in water. Scale bars: a = 50 μm; b − i = 20 μm
dispersal in aquatic habitats discussed below (Dix and Webster 1995; Webster 1959).
Aquatic hyphomycetes are known as important decomposers in the turnover of leaf
litter in woodland streams [overview in (Gessner et al. 2007)]
The second group is called the (II) aero-aquatic fungi (mitosporic ascomycetes)
(~90 species described) (Fig. 13.1g, h), which are found on submerged plant litter
and wood in flat lentic waters, such as woodland ponds and ditches, which may have
periodical levels of water. In contrast to aquatic hyphomycetes, the aero-aquatic
fungi release their propagules aerially. Therefore, they produce conspicuous air-
trapping dispersal units, which can flow on the water surface; for example, some
spores are helicoid in more than one plane (Fig. 13.1g, h) (e.g., Helicoma, Helicoon)
or spiral into a sphere or a net [e.g., Candelabrum, Spirosphaera (Voglmayr 2004,
2011; Voglmayr and Delgado-Rodriguez 2003; Voglmayr et al. 2011)]. The aero-
aquatic life strategy was first described by Agathe van Beverwijk (1951).
Fig. 13.2 Lunulospora curvula. Conidiogenesis of crescent-shaped unbranched conidia.

Bar = 25 μm
The third group is named (III) freshwater ascomycetes (~600 species described
meiosporic fungi), which comprise the sexual states of phylogenetically heteroge-
neous ascomycete fungi occurring worldwide in freshwater habitats on herbaceous
and woody substrates (Shearer et al. 2007, 2009; Goh and Hyde 1996; Shearer
1993a; Vijaykrishna et al. 2006). In addition, they are also collected from sub-
merged dead macrophytes (Shearer 1993a; Fallah and Shearer 2001). The morphol-
ogies in asci and ascospores are very diverse and noticeable. Asci of freshwater
ascomycetes are either deliquescent, with apical apparatus (e.g., Massarina,
Jahnula), or fissitunicate (ectoascus and coiling endoascus, e.g., Kirschsteiniothelia
(transferred to Helicascus)) (Shearer 1993b). Many freshwater ascomycetes pro-
duce ascospores with appendages, which facilitate attachment to substrates (Shearer
et al. 2007; Shearer 1993a; Wong et al. 1998). Gelatinous gel-like sheaths and/or
thick-walled hyphae (Shearer et al. 2009; Ingold 1955) are thought to enhance
attachment and adhesion to plant materials (Digby and Goos 1987; Ingold and
Chapman 1952). However, spores borne with gelatinous sheath and the active dis-
charge of ascospores are also present in strictly terrestrial ascomycetes. In compari-
son to aquatic and aero-aquatic hyphomycetes, freshwater ascomycetes seem to be
less exclusively adapted to life in aquatic habitats (Vijaykrishna et al. 2006).
The fourth group of freshwater fungi is the (IV) zoosporic fungi (Fig. 13.3),
which produce flagellate zoospores as part of their life cycle. They are known for
Fig. 13.3 Oomycetes. (a) Baiting of oomycetes, using sesame seeds. (b) Baiting of oomycetes,
using insect carcasses. (c) Saprolegnia sp., zoosporangium releasing zoospores. (d) Saprolegnia
sp., oogonium, with attached antheridia, and many encysted primary zoospores. (e) Achlya sp.,
zoosporangium showing discharged and encysted zoospores at the apical pore. (f) Dictyuchus sp.,
a dictyoid zoosporangium in the process of zoospore discharge. Scale bars: a, b = 5 mm; c = 10 μm;
d−f = 20 μm
their parasitic life strategies but they are also involved in the decomposition of
organic matter (Sparrow 1960). Historically, they have been known as the members
of “phycomycetes,” a functionally defined group. They have also been traditionally
termed the “lower fungi” which basically comprises species without a septate
hyphal system. With the advent of molecular studies and recent taxonomic treat-
ments based on phylogenetics, the “phycomycetes” is in fact heterogeneous, com-
prising of members from the Eumycota (“true fungi”) and the Chromista (Oomycetes
and Labyrinthulomycetes). The biology, taxonomy, and phylogenetic relationship of
these aquatic organisms have been well documented (Fuller and Jaworski 1987;
Beakes 2003; Bowman et al. 1992; Buczacki 1983; Powell 1993).
The freshwater zoosporic fungi belong mostly to the Chytridiomycetes and the fun-
gal-like Oomycetes, which are microscopic organisms not producing any fruiting bod-
ies visible to the naked eye. Members of the Chytridiomycetes are generally called
chytrids, whereas those belonging to the Oomycetes are usually called water molds.
They usually reproduce asexually by means of zoospores, but in Oomycetes, sexual
reproduction may occur by means of oogamy, resulting in the formation of oospores,
which are survival structures generally resistant to adverse environmental conditions.
The chytrids produce uniflagellate haploid zoospores, whereas those of the water
molds are biflagellate and diploid. These taxa are associated with dead and living plant
materials but also with algae, cyanobacteria (Sonstebo and Rohrlack 2011), inverte-
brates, fish, and amphibians (Powell 1993; Longcore et al. 1999; Powell et al. 2013).
The main orders of Chytridiomycetes are Chytridiales, Spizellomycetales,
and Monoblepharidales. Furthermore members of Neocallimastigomycota
and Blastocladiomycota occur in aquatic habitats (Powell and Letcher 2014).
Members of Chytridiales and Blastocladiales are more frequently encountered
in freshwater systems. The main orders of Oomycetes found in aquatic environ-
ments are the Saprolegniales, Leptomitales, and Peronosporales. Common genera
of Saprolegniales frequently isolated from the aquatic systems are Saprolegnia,
Achlya, and Dictychus.
Apart from chytrids, Cryptomycota is a recently discovered (Jones et al. 2011)
phylum with endoparasitic lifestyle (James et al. 2013; Lazarus and James 2015).
They differ from other filamentous fungi in that they lack chitinous cell walls in the
trophic stage. Based on molecular markers, they have been detected in lakes and
wetlands (Ishii et al. 2015; Wurzbacher et al. 2014).
The fifth group (V) belongs to aquatic–terrestrial hyphomycetes (mitosporic
ascomycetes) (Figs. 13.4, 13.5 and 13.6) that comprise a huge morphological diver-
sity of conidial (mitosporic) fungi growing on decaying plant material (Shearer
et al. 2007; Hu et al. 2014) and capable of sporulating underwater (Bärlocher et al.
2008; Baschien et al. 2009). They are distinguished based on the features of conidia,
conidiophores, and the conidiogenesis. For example, common fungi include
Dactylaria, Dictyochaeta, Canalisporium, and Sporoschisma; some of these fungi
are linked with their corresponding sexual relatives using cultural or molecular
studies. Some studies also demonstrate the presence of fungi of terrestrial origin,
including saprobes from plants and soil, such as Cladosporium, Alternaria, and
Penicillium species. Fungi on leaves from the canopy (Gönczöl and Révay 2006)
may fall into the water.
Fig. 13.4 Lignicolous hyphomycetes. (a) Ellisembia adscendens. Colony on submerged wood.
Conidia are long and projecting upward on the wood surface. (b) Phaeoisaria clementidis. Colony
on submerged wood. Conidiophores are in the form of long synnemata projecting upward on the
wood surface. (c) Vermiculariopsiella sp. Colony on submerged wood. Conidiophores are in the
form of dense sporodochia on the wood surface, surrounded by long setae and producing conidia
in the form of white slimy mass. (d) Monotosporella setosa. Colony on submerged wood.
Conidiophores are long, erect, and projecting from the wood surface. (e−g) Monotosporella setosa.
Conidiophores producing conidia at their apex. (h) Ellisembia adscendens. Conidia. (i)
Canalisporium elegans. Conidia, which were produced in the form of sporodochia on the surface
of submerged wood. (j, k) Dictyosporium elegans. Conidia. Scale bars: a−d = 200 μm; e−h, j,
k = 20 μm; i = 50 μm
Fig. 13.5 Lignicolous hyphomycetes. (a) Beltrania africana. Conidiophores and conidia from
submerged wood. (b) Cryptophialoidea secunda. Part of setiform conidiophore showing the fertile
region, bearing unilateral phialidic conidiogenous cells, producing falcate conidia. (c)
Pseudobotrytis terrestris. Conidiophore bearing umbellately arranged polydenticulate conidioge-
nous cells, producing two-celled conidia. (d) Sporoschisma uniseptatum. Conidiophore which has
a swollen venter, producing endogenous conidia, usually in basipetal chains. (e) Sporoschisma
mirabile. Conidia, initially borne on chains, have become disarticulated in water mount. Scale
bars: a = 20 μm; b = 10 μm; c, d = 20 μm; e = 50 μm
Fig. 13.6 Nawawia quadrisetulata. (a) Conidia, bearing 4–5 setulae at the corners of the distal
end. (b−d) Conidiophores showing sequential development of conidia. (e, f) Scanning electron
micrographs of setulate conidia produced at the apex of conidiophores. (g) Conidiophores showing
the terminal phialides. Scale bars: a−d = 20 μm; g = 5 μm
How Are They Adapted to an Aquatic Lifestyle?
Spores are the dispersal propagules of fungi. Aquatic fungi need to produce spores
that can be dispersed in water, and then become entrapped to, and subsequently
colonize new substrates. The completion of the life cycle and hence the survival of
the species is reliant on the spores.
The zoosporic fungi release motile spores that can actively disperse/swim in the
lentic aquatic environments (e.g., ponds, lakes, ditches, pools, and swamps). They
are usually chemotactic, being sensitively attracted to their host or substrates that
release specific sugars and amino acids (Fuller and Jaworski 1987). Usually the
zoospores swim for a few minutes to hours before they stop and encyst. At the initial
stage of zoospore encystment, depending on the species, the flagella are either
retracted or shed before the zoospore assumes a spherical shape. An important prop-
erty of the zoospores in general is their ability to stick firmly to the surfaces of the
substrates upon which they settle down to encyst. Adhesion ensures that the zoo-
spore, upon reaching a favorable substrate, accidentally, or through specific tactic
responses, would remain firmly attached. Evidently, this behavior has great ecologi-
cal value for both saprobes and parasites as it establishes a permanent contact
between the fungus and its potential food source. Swimming zoospores are for dis-
persal and thus are not adhesive but become so in the initial stage of encystment
before a cyst wall is made. The adhesive phase lasts only about 30–60 s (Sing and
Bartnicki-Garcia 1972). Apparently, the timing of the adhesive phase, coinciding
with the change from motile to sessile form, provides obvious ecological advantage
to the fungus. Once the cysts mature, i.e., after a well-defined wall is made, the cells
lose their ability to attach themselves to solid surfaces. After successful adhesion
and encystment of the spore, germination of the cyst leads to penetration and colo-
nization of the substrate.
Besides the zoosporic fungi, most aquatic fungi are saprobes that grow/colonize
on submerged wood or waterlogged leaves in freshwater. The spores of freshwater
ascomycetes and Ingoldian fungi are not motile in the aquatic environment. These
fungi usually occur in lotic habitats (e.g., streams, creeks, rivers, brooks), and thus
they possess strategies to survive in turbulent water. Their propagules are released
from conidiophores growing out of substrates and thus are able to detach from the
surface of the substrates, disperse (float) in water, and eventually become entrapped
(settled) or attached (adhere) to new substrates, which they can colonize by pene-
tration of a germ tube. The mechanisms of fungal adhesion and the role of muci-
lage in fungal attachment in the aquatic environment have been well documented
(Wong et al. 1998; Au et al. 1996; Ingold 1966; Jones 2006). An excellent review
of the adaptations for dispersal in filamentous freshwater fungi is given by Goh and
Hyde (1996).
Many freshwater ascomycetes have ascospores with various sheaths, append-
ages, or wall ornamentations, which probably function in dispersal and/or attach-
ment of the spores. Shearer (1993a) provided a list of ascomycete species possessing
ascospores surrounded by mucilaginous sheaths. There are ascospores that possess
unfurling mucilaginous appendages, which uncoil in water to form long viscous

threads. Several unique appendage types have now been shown to exist in freshwa-
ter ascomycetes, which account for their success in the aquatic environments (Jones
2006; Wong and Hyde 1999).
The Ingoldian fungi have been known for decades to have sigmoid or tetraradiate
spores (Ingold 1975; Goh 1997). Sigmoid conidia often become attached at their sticky
poles and straighten in the direction of the water current so that they are less likely to
be washed away in a lotic habitat (Webster and Davey 1984). Tetraradiate and variously
branched spores act as an anchor and allow their entrapment to the substrates or in
surface foam (Ingold 1966). Tetraradiate spores can also effectively attach to the sub-
strate with three “legs” forming a strongly adhesive tripod (Webster and Descals 1981).
Adhesive mucilaginous material is also produced at each arm of the tetraradiate spore
in contact with a surface and attaches them firmly to the substrate.
The aero-aquatic hyphomycetes, such as Beverwykella, Cancellidium, and
Clathrosphaerina, are normally found on decaying plant materials in slow-flowing
streams and stagnant ponds (Gessner et al. 2007; Voglmayr and Delgado-Rodriguez
2001, 2003; Voglmayr and Krisai-Greilhuber 1997). Their propagules are beautiful,
extraordinary, and interesting as they possess a special floatation device, usually
composed of intricate branching hyphal network and thus air trapping, enabling
these fungi to be dispersed from one static water habitat to another. Conidia of
Helicoon species are composed of tightly coiled filaments, which assume a barrel
shape, and can trap air in water for floating (Goh and Hyde 1996). For further dis-
cussion of the adaptation strategies in the aero-aquatic fungi, see Fisher (Fisher
et al. 1977) and Webster and Descals (1981).
A great diversity of dematiaceous hyphomycetes have been discovered from
wood submerged in freshwater (Goh and Tsui 2003). Among these lignicolous
hyphomycetes, many possess long mononematous stiptate conidiophores, which
stand erect from the submerged substrates and bear masses of conidia at the apices.
Examples of such hyphomycetes are Acrogenospora, Cryptophiale, and Spadicoides.
Others produce erect synnemata (e.g., Bactrodesmium longisporum, Nawawia den-
droidea, and Phaeoisaria clematidis) or occur as sporodochia (e.g., Dictyosporium,
Canalisporium, and Yinmingella). Producing easily detachable conidia at the apex
of long, erect conidiophores, or having conidia produced in dense masses from spo-
rodochia, may be adaptation strategies conducive to effective spore dispersal in the
aquatic habitats. Moreover, certain features observed in some of the commonly
found hyphomycetes from submerged wood are worth mentioning, because they
may represent special adaptation features for dispersal in the aquatic habitats. For
example, Chalara, Sporoschisma, and Sporoschismopsis (Goh et al. 1997) produce
long chains of conidia from erect conidiophores, which eventually disarticulated in
water for ease of dispersal. The muriform conidia of Canalisporium have distinct
pores or canals in the septa (Goh et al. 1998) which enable air to trap inside the
conidia for floating. The various spore types of these lignicolous hyphomycetes in
freshwater also are provided with modified appendages, mucilaginous sheaths, setu-
lae, or arms (Goh 1997), and these are functionally comparable to those of aquatic
ascomycetes and Ingoldian fungi.
How Did They Evolve?
The absolute age of fungal phyla is an area of active research, and only broad
estimates exist for the oldest clades (Taylor and Berbee 2006). Currently, the diver-
gence of younger phyla from the Cryptomycota, Chytridiomycota, and
Blastocladiomycota clades—and the loss of the zoospore stage—is thought to have
occurred during the Neoproterozoic, approximately 600–800 Myr (Stajich et al.
2009). The first divergence of terrestrial fungi (Endogonales and Glomales) has
been estimated to have occurred about 600 Myr or even earlier, while Ascomycota
and Basidiomycota separated 500 Myr (Berbee and Taylor 2001). The origin and
radiation of Euascomycetes (whose groups commonly produce conidia adapted in
aquatic habitats) may have taken place in the Mesozoic, about 240 Myr. This puts a
lower limit to the earliest appearance of aquatic ascomycetes and relatives. Did the
majority of the taxa appear in a brief burst of rapid radiation, or was this spread out
over an extended period of time? Are new species still invading running waters
today (Schlütz and Shumilovskikh 2013)?
Fungi in freshwater are grouped ecologically and morphologically, and it is not
surprising that they have evolved independently from multiple lineages. Shearer
(Shearer 1993a) first proposed the multiple origins of freshwater ascomycetes,
which are present as endophytes, pathogens, or saprobes on plants, and have
become adapted to aquatic environment when these plants invaded water. 18S
rRNA data (Vijaykrishna et al. 2006; Baschien et al. 2006; Belliveau and Bärlocher
2005) showed that freshwater fungi (sexual and asexual ascomycetes) evolved
from terrestrial fungi in (more than) four different classes, Sordariomycetes,
Dothideomycetes, Leotiomycetes, and Orbiliomycetes. Many freshwater fungi
have terrestrial relatives, supporting the fact of secondary adaptation to the fresh-
water environment. A comprehensive study of 84 fungi of described and unde-
scribed freshwater Dothideomycetes and 85 additional ascomycetes representative
of the major orders and families of Dothideomycetes based on ribosomal genes
also confirmed the polyphyletic origins of freshwater ascomycetes (Shearer et al.
2009). Apart from these, molecular studies of fungi on submerged leaves using
denaturing gradient gel electrophoresis [DGGE; (Kelly et al. 2010; Nikolcheva
et al. 2003)] and the analysis of terminal restriction fragment length polymorphism
[TRFLP; (Nikolcheva et al. 2003; Nikolcheva and Bärlocher 2005)] and of clone
libraries (Clivot et al. 2014; Harrop et al. 2009) provided evidence for the presence
of fungi of terrestrial origin.
Many genera of freshwater fungi (ascomycetes and their asexual relatives) are
also not monophyletic. The polyphyletic origin of aquatic hyphomycetes has been
reinforced by additional studies (Baschien et al. 2006, 2013; Campbell et al. 2006,
2009). For example, Ingoldian fungi are assigned to four classes: Sordariomycetes
(~11 spp.), Dothideomycetes (~10 spp.), Pezizomycetes (1 sp.), Orbiliomycetes (3–5
spp.), and Leotiomycetes (>75 spp.). The morphology of tetraradiate and sigmoid
conidial shape has been recognized as convergent development in unrelated aquatic
hyphomycete taxa (Ingold 1966; Webster 1980). Molecular studies of ribosomal
genes also reassured the convergence of the conidial shape (Baschien et al. 2006,
2013; Campbell et al. 2006; Belliveau and Bärlocher 2005). Also, the helicosporous
aero-aquatic fungi have evolved from multiple lineages within the ascomycetes
(Tsui and Berbee 2006).
The molecular studies mentioned above showed that many ascomycetous and
basidiomycetous freshwater fungi are closely related to terrestrial fungi. A few spe-
cies of the genera Varicosporium, Tetracladium, Filosporella, and Anguillospora
have been isolated as endophytes from aquatic or terrestrial roots (Fisher et al. 1991;
Kohout et al. 2012; Nemec 1969; Sati and Belwal 2005; Watanabe 1975). The step
back from terrestrial to aquatic life cycles could have been aided by an endophytic
lifestyle (Selosse et al. 2008). Furthermore, the localization of freshwater fungi
inside roots or other amphibious plant parts may function as temporary reservoir
during different stages within a life cycle. The production of different conidial
shapes and synanamorphs may also be an adaptation to shifts between aquatic,
semiaquatic, and terrestrial habitats (Baschien et al. 2013). Kohout and co-workers
(Kohout et al. 2013) proposed the scenario that terrestrial ancestors of recent aquatic
plants interacted with different root-associated fungi (RAF). The aquatic hyphomy-
cetes could have evolved from non-mycorrhizal RAF that once entered aquatic
habitats together with their host plants.
What Are They Doing in Freshwater Habitats?
The primary ecological role of fungi in aquatic habitats is to decompose dead plant
material—both woody and herbaceous debris. When Kaushik and Hynes (1971)
demonstrated the crucial role of fungi in the decomposition of plant materials to
detritus in streams, limnologists recognized the important ecological function of
aquatic hyphomycetes. Bärlocher and Kendrick (1974) showed that aquatic hypho-
mycetes condition leaf material for aquatic invertebrates (Plecoptera, Trichoptera,
Coleoptera, Crustacea, Gastropoda) by increasing the palatability using exoen-
zymes [cellulases, pectinases, laccases; (Abdel-Raheem and Shearer 2002; Chamier
1985)]. Most aquatic fungi have the ability to decompose a wide range of organic
substrates, although a few species may be limited to one or a few types of substrates.
For example, Gulis (2001) showed that wood/twig substrates bear fungal communi-
ties distinct from those on leaves. In general, aquatic ascomycetes and basidiomy-
cetes are thought to be responsible for the decomposition of woody debris while
Ingoldian fungi decompose either leaves or herbaceous debris. Through decomposi-
tion, freshwater fungi can facilitate the transfer of nutrients and energy between
trophic levels in the food web (Gessner et al. 2007).
Wood is a complex substance and its major chemical constituents contain cellu-
lose, hemicellulose, and lignin. The degradation mechanisms of wood are well
known in terrestrial fungi, and it is assumed that similar mechanisms are present in
freshwater fungi. Cellulose hydrolysis is achieved by endoglucanases and cello-
biohydrolases, collectively termed cellulases (Eaton and Hale 1993). Hydrolysis of
hemicellulose, a mixed polymer, occurs via the action of hydrolytic xylanases,

mannanases, and possibly other hydrolases with broad substrate specificity (Eaton
and Hale 1993). The degradation of lignin involves two peroxidases, lignin peroxi-
dase and Mn-dependent peroxidase, and a polyphenol oxidase, laccase, known as
lignin-modifying enzymes (LMEs) (Pointing 2001). The vast majority of freshwa-
ter ascomycetes, regardless of eco-climate, habitat, and substrate distributions, are
capable of breaking down cellobiose, hemicellulose, and xylan and starch, which
are important carbon compounds in plant-based debris (Bucher et al. 2004; Simonis
et al. 2008; Yuen et al. 1998).
Three fungal wood decay types are recognized on the basis of whether or not
they can degrade cellulose and lignin or just cellulose alone, namely, soft rot, white
rot, and brown rot (Eaton and Hale 1993). Soft rots occur in wood that has an unusu-
ally high level of moisture, which is often the case for woody substrates in aquatic
environments. Most of the freshwater fungi are capable to form soft rot decay and
to produce cellulases (Bucher et al. 2004; Abdel-Raheem and Shearer 2002). White-
rot fungi normally have the enzymes to degrade both cellulose and lignin simultane-
ously. The residual material that is left behind has a somewhat fibrous appearance
and is very pale in color, looking as if it had been bleached. In contrast, members of
brown-rot fungi can degrade cellulose. After decomposition, wood is brown in color
and tends to be broken up into cubical fragments that quickly disintegrate into a
powdery brown residue. It has been questionable whether or not freshwater ascomy-
cetes can form white rot or brown rot because the breakdown of lignin in woody
debris is primarily accomplished by basidiomycetes that do not seem common in
the aquatic habitat. However, Junghanns et al. (2005) showed that the aquatic
hyphomycete Clavariopsis aquatica produced laccases. Five putative laccase genes
(lcc1 to lcc5) identified in C. aquatica were differentially expressed in response to
the fungal growth stage and potential laccase inducers (Sole et al. 2012). Recently,
Kerr and co-workers (2013) showed the oxidation of lignin in leaf litter by undeter-
mined aquatic fungi using high spatial resolution infrared microspectroscopy. The
modification of lignin by freshwater fungi degrades lignin into carbohydrate-
depleted recalcitrant carbon, which may influence the carbon pool in aquatic
environments.
Leaves and other nonwoody plant parts (e.g., fruits and seeds) represent a different
type of substrate than wood and bark. In general, angiosperm leaves are readily
decomposed. When a leaf falls from a tree from the riparian zone, it would be colo-
nized by soil-inhabiting fungi—those already present in the layer of litter at the soil
surface or various aquatic fungi when the leaf becomes submerged. Most aquatic
hyphomycetes can degrade cellulose, various hemicelluloses, and pectin (Chamier
1985; Abdel-Raheem and Ali 2004; Chandrashekar and Kaveriappa 1991; Zemek
et al. 1985). Many studies have examined the enzymatic activities of freshwater
fungi and reported amylase, β-glucosidase, β-xylosidase, endoglucanase, endoxyla-
nase, lipase, pectinase, and protease activity (Chamier 1985).
To which extent aquatic—terrestrial—hyphomycetes play an important role in
aquatic systems is still unclear, although they are common on leaves and wood. It is
generally assumed that the so-called aquatic–terrestrial fungi are not able to compete
against aquatic hyphomycetes and hence are removed after the first 2–3 days of the
decomposition process (Harrop et al. 2009; Bärlocher and Kendrick 1974;
Nikolcheva et al. 2005; Perez et al. 2012). However, aquatic–terrestrial fungi are
present on longer-exposed leaves in streams [e.g., (Hameed et al. 2008; Smither-
Kopperl et al. 1998)]. Kelly et al. (2010) studied fungal communities on decompos-
ing maple and aspen leaves, and his group reported that the majority of operational
taxonomic units (OTUs) represented terrestrial Cladosporium species, whereas
aquatic hyphomycete sequences were not observed. Indeed, several aquatic–terres-
trial hyphomycetes are able of decomposing leaf litter (Bucher et al. 2004; Singh
et al. 2014). For example, the terrestrial leaf litter ascomycete Torula herbarum,
isolated from a tropical stream, has been found to be able to break down lignin
(Bucher et al. 2004).
Few aquatic fungi form a symbiotic mycorrhizal relationship with the roots of
trees and other plants and macrophytes. This association is mutually beneficial to
both the plant and the fungus. The fungus enables the plant to take up nutrients
that are unavailable, and the plant provides nutrition for the fungus. There are two
fundamentally different types of mycorrhizal associations—ectomycorrhizal
(usually involving a basidiomycete) and endomycorrhizal (most often involving a
member of the Glomeromycota). In the former, the fungus produces a covering of
hyphae (called a sheath, Hartig net, or mantle) around the outside of smaller root-
lets of the host plant. Other hyphae invade the cortex of the rootlet but do not
disrupt the individual cells. In endomycorrhizal associations, no sheath is formed
and hyphae of the fungus actually invade cells of the cortex of the rootlet. Perhaps
80 % of all vascular plants form mycorrhizal associations with fungi. First aquatic
vesicular–arbuscular mycorrhiza was discovered in Littorella uniflora, Lobelia
dortmanna, and Isoetes lacustris by Sondergaard and Laegaard (1977). Vesicular–
arbuscular mycorrhizas (VAM) in Isoetes plants were later also observed by
Sudova et al. (2011).
Furthermore, dark septate endophytes (DSE) and fungal root associates (RAF)
from aquatic habitats were reported (Kohout et al. 2012; Seena et al. 2008).
Mycorrhiza and some DSE are known to be mutualistic. On the other hand, endo-
phytism can be the balance between rather antagonistic relationships between fun-
gus and host which may even develop into a pathogenic outcome (Schulz and Boyle
2005). Several species of aquatic hyphomycetes were isolated as endophytes from
aquatic or terrestrial plants (Sati and Belwal 2005). Some aquatic hyphomycetes
were also found in roots of terrestrial habitats [e.g., (Tedersoo et al. 2007)]. However,
there are few reports about the transmission of freshwater fungi either vertical to the
next generation of the same host or horizontal between hosts. It is not known if
endophytic aquatic hyphomycetes have a harmful or beneficial effect on the hosts.
Further thorough studies are needed to elucidate the aspects of endophytic life of
aquatic hyphomycetes.
For instance, all the described Minutisphaera spp. (M. fimbriatispora, M. japon-
ica, M. aspera, and M. parafimbriatispora) have been isolated from submerged
wood in freshwater habitats so far (Ferrer et al. 2011; Raja et al. 2013), suggesting
that they play an ecological role in nutrient cycling and organic matter decomposition
in freshwater habitats (Pointing 2001). A recent BLAST search (Altschul et al.

1990) of newly sequenced ITS strains of M. aspera and M. parafimbriatispora in
GenBank identified two ITS sequences from endophytes (“Pleosporales sp. 39 g,”
JX244063, and “Didymosphaeria sp. TS_04_050,” HQ713763) as the top BLAST
matches with high percent identity values and coverage. Based on the uncorrected
p-distances calculated in PAUP*, the two ITS sequences from endophytes were
identical to each other and shared 99 % sequence similarity with ITS sequences of
M. aspera. The high ITS sequence similarity between these fungal endophytes and
M. aspera could imply that Minutisphaera may have a dual mode of lifestyle as
saprobes on submerged wood as well as fungal endophytes inside the roots of trees.
However, additional studies are warranted to test this ecological hypothesis (Selosse
et al. 2008).
In contrast to the aquatic ascomycetes (meiosporic and mitosporic fungi), which
generally colonize organic matter, which are comparatively larger in size, such as
fallen leaves, submerged wood, or aquatic plants, the zoosporic fungi are colonizers
of smaller substrates. They are particularly fond of substrates which contain chitin,
keratin, or cellulose (Wong et al. 1998). In case of small particles such as algae, pol-
len grains, seeds, and zooplankton carcasses, and other temporarily available sub-
strates decomposition is achieved by the much smaller chytrids (Chytridiomycetes)
and water molds (Oomycetes), rather than the aquatic hyphomycetes. This is because
the zoosporic fungi are relatively simpler in structures. They do not depend on mac-
roscale hyphal networks and thus are capable of very fast responses to changes in
their environment. Being actively motile, the zoospores of these aquatic fungi
actively search for adequate substrates using chemotaxis. Once a suitable substrate
has been reached, the zoospore encysts. An appressorium or a penetration tube is
formed and the food particle (substrate) is invaded by tiny rhizoids tapping the inter-
nal nutrient reservoirs. Zoosporogenesis can occur in a short time, producing a pro-
lific number of zoospores from zoosporangia. Their life cycle can be completed in
days, either endobiotic, epibiotic, endophytic, or ectophytic, depending on the rela-
tionship of the thallus with the host or organic substrate.
Like other aquatic fungi, the chytrids and water molds are heterotrophs. Most of
the species are benign saprobes, but they often exist as parasites, sometimes as sym-
bionts, and of course as decomposers. The aquatic systems harbor a wealth of
organisms that can serve as suitable hosts for the parasitic zoosporic fungi: algae
from different phyla, cyanobacteria, protists, zooplankton, fish, birds, mussels, eggs
of liver flukes, nematodes, crayfish, mites, insect larvae, amphibians, mammals,
plants, and other aquatic fungi. For the decomposers, resources of organic matters
derived from animals include fish scales, fish eggs, carcasses, feathers, and hair,
while plant-derived resources include pollen, spores, seeds, small fruits, and plant
debris (Cole et al. 1990).
Chytrids are surprisingly abundant on filamentous algae, phytoplanktons, and
diatoms, and some species are known to severely deplete local populations of
their algal hosts. The abundance of chytrids in aquatic systems are considered
much higher than traditionally thought (Kagami et al. 2014). The phytoplanktons
infected with chytrid zoospores could become an excellent food source for zoo-
planktons in terms of size, shape, and nutritional quality, and the nutrients from
within the planktons can be transferred to the zooplanktons through the

“mycoloop” pathway in the aquatic ecosystems (Kagami et al. 2014). Also pollen
deposited in lakes could be consumed by saprotrophic chytrids, rather than para-
sitic relatives (Masclaux et al. 2013).
Ibelings and co-workers studied the host–parasite interactions between freshwa-
ter phytoplankton and chytrid fungi and found that algal population was naturally
regulated by the parasitism of these fungi (Ibelings et al. 2004). Encounters with
these parasitic chytrids can be fatal to algae, particularly if their defense mechanism
is breached by the fungal parasites. When the alga is attacked by the fungus, it
undergoes a “suicide” response, which is a controlled hypersensitive reaction. This
hypersensitivity is regarded as a common defense mechanism in algae. If this con-
trolled “suicide” progress is initiated at the right moment during fungal infection, it
results in the successful interruption of the fungal infection cycle, because the para-
site’s ability to reproduce via spore production is inhibited. This mechanism is con-
ducive to maintaining a healthy algal population because it reduces the abundance
of the deadly fungal parasite in the environment. If such control is unsuccessful,
however, the parasitic chytrid prevails and thus resulting in mass mortality of the
algal species. The ecological relevance of this negative interaction between the two
parties is obvious. The failure of the algal “suicide” mechanism in response to fun-
gal infection can lead to shifts in the algal community composition in a given aquatic
system.
In rare, but important cases, some zoosporic fungi cause severe damage to larger
aquatic organisms. They infect frogs, shrimps, fishes, or fish eggs (Chukanhom and
Hatai 2004; Noga 1993) and thereby exert strong population pressure. Such damage
is of great importance for aquaculture and often demands antifungal treatments.
Some oomycetes, especially species of Aphanomyces and Saprolegnia, are aggres-
sive pathogens of fish and crustacea. For example, Aphanomyces astaci causing the
crayfish plague has driven the European crayfish population to the edge of extinc-
tion (Reynolds 1988). The most notorious parasitic chytrid is Batrachochytrium
dendrobatidis, which cause worldwide extinction of several known and unknown
species of frogs (Berger et al. 1998; Skerratt et al. 2007). Aquatic plants are also
greatly affected by some oomycetes. For example, Pythium phragmites has been
found to cause reed decline (Nechwatal et al. 2005). For more discussion on dis-
eases of freshwater fishes caused by zoosporic fungi, see Willoughby (2003).
Distribution and Biodiversity
Distribution Pattern
Regarding the distribution pattern of freshwater fungi, some may be restricted to

tropical, temperate, or cold water habitats, while others are cosmopolitan. The geo-
graphical distribution of Ingoldian mitosporic ascomycetes (=anamorph or asexual
fungi) are relatively well studied compared to those of the freshwater ascomycetes.
The Ingoldian fungi most commonly occur on shed leaves in streams and rivers, and
they are documented by stream biologists. Some are cosmopolitan and some are
restricted in distribution. Also the phylogeographic pattern varies among and within
a species. Molecular barcoding (ITS) of 130 isolates of six Ingoldian fungi revealed
significant genetic differentiation between continents within a single fungal species
(Duarte et al. 2012). The knowledge on the distribution pattern of freshwater asco-
mycetes is accumulating even though most investigations are concentrating in
tropical and subtropical Asia, North America, as well as the neotropics.
Some studies have reported shifts in fungal community composition by latitude
and temperature (Arnold and Lutzoni 2007). Such spatial shifts/turnover in com-
munity is also expected in freshwater fungi. Wood-Eggenschwiler and Bärlocher
(1985) used distribution data obtained from the literature (Webster and Descals
1981) for over 150 species of Ingoldian mitosporic fungi and they concluded, “on a
worldwide scale, temperature together with its influence on vegetation in different
climatic regions is the major factor in determining distribution patterns of Ingoldian
mitosporic fungi.” Wood-Eggenschwiler and Bärlocher (1985) discovered that there
was a higher similarity in species composition of Ingoldian fungi between geo-
graphically distinct tropical locations (South America, West Africa) than between
tropical and temperate regions that were located on the same continent, either
African or North and South American. Raja et al. (2009) also reported a change in
species composition of freshwater ascomycetes along the temperate–subtropical
latitudinal ecotone in Florida, USA.
Apart from the macroclimatic factors, microenvironmental factors also affect the
distribution and abundance of freshwater fungi. Chauvet (1991) studied the distribu-
tion of Ingoldian mitosporic fungi at 27 stations in France, and he concluded that the
most important environmental factors are altitude, pH, temperature, and season,
although the relationship between species composition and each environmental fac-
tor is hard to establish. Longitudinal distribution patterns in freshwater fungi along
a river and stream are reported for both leaf litter and woody substrates (Gönczöl
1989; Shearer and Webster 1985a, 1991; Tsui et al. 2001a). Tsui et al. (2001a)
reported changes in fungal communities and taxonomic compositions from upstream
to downstream in responses to salinity and riparian vegetation. Shearer and Webster
(1985a) reported that Ingoldian mitosporic fungi communities in headwater streams
were distinctly different from the downstream communities in the River Teign.
Using water filtration, leaf pack baiting, and collection of naturally occurring sub-
strates, lower species diversity with a lower frequency occurrence of species was
observed in the headwaters (Shearer and Webster 1985a). Using molecular data of
DGGE, Miura and Urabe (2014) also demonstrated that taxonomic composition and
richness of epilithic fungal assemblages change along the longitudinal gradient of
the river, according to the water temperature, and the spatial variation in abundance
and composition of dissolved organic matter and nutrients. While species diversity
could change spatially, the genetic variability within a species does not vary locally.
Using eight microsatellite markers, Anderson and Shearer (2011) revealed small
genetic differentiations among populations of Tetracladium marchalianum from
Wisconsin and Illinois, USA. They concluded that the fungal populations may be
highly connected in local habitats.
Substrate Preference
Freshwater fungi demonstrate substrate specialization, even though they are saprobes.
For instance, of the 548 species of freshwater ascomycetes reported up to 2009
(http://fungi.life.uiuc.edu/), 60 % are reported only from submerged woody debris
and about 30 % are reported only from herbaceous substrates, while only about 10 %
species are reported from both submerged wood and herbaceous substrates [reviewed
in (Raja et al. 2009)]. During the substrate distribution pattern investigation of fresh-
water ascomycetes in the Florida Peninsula (Raja et al. 2009), the results implied
substrate preference among freshwater fungi. Of the 132 fungal taxa collected in
freshwater habitats, 100 were reported only on woody debris, 14 species occurred
exclusively on herbaceous debris, and 18 species were found on both woody and
herbaceous debris (Raja et al. 2009). Cai et al. (2003) also reported substrate prefer-
ences in freshwater fungi during an investigation on the biodiversity of freshwater
fungi on submerged bamboo and submerged wood in Liput River in the Philippines.
Fifty-eight and 38 fungal taxa were collected on bamboo and wood, respectively, but
only 16 among them were in common on both substrates (Cai et al. 2003).
Thomas et al. (1992) observed Alatospora acuminata more frequently on Acacia
leaves, while Tetrachaetum elegans was more common on Eucalyptus leaves. The
authors suggested five possible reasons: First, different substrates have different
nutrients, favoring the growth of some fungi over others. Second, different sub-
strates contain different inhibitory chemicals, for example, tannin, impacting sporu-
lation and spore germination. Third, variations in the gross physical structure of
substrates affect differentially the impaction efficiency of various fungal spores.
Fourth, variations in fine physical structure of substrates affect penetration and colo-
nization by fungi. Fifth, different substrates vary in their decay rate—durable sub-
strate has a much longer exposure time to the spores and to fungal colonization.
Gulis (2001) investigated five different substrate types from 92 watercourses of
Belarus for aquatic hyphomycete colonization (52 species). He found specific fun-
gal assemblages correlating with leaf litter types which suggests possible substrate
preferences of aquatic hyphomycetes triggered in particular by lignin content.
Seasonal Variation
Seasonal occurrence with fluctuations in conidial numbers has been reported in

many parts of the world (Thomas et al. 1989). Although most species can be col-
lected throughout the year, their relative abundance (measured in terms of conidial
production) is influenced by the seasonal availability of substrates, which is in
agreement with seasonal input of deciduous tree litter in temperate regions
(Bärlocher 1992). For instance, the conidial peak in summer in Australia was also
highly correlated with the leaf fall of eucalypt forests in summer (Thomas et al.
1989), and the timing of conidial maxima in New Zealand streams was well
correlated with the prevailing litter fall patterns (Aimer and Segedin 1985). Recent
studies employing DGGE illustrated seasonal changes in fungal communities in a
lake in Japan (Ishii et al. 2015).
Human Disturbance
Most freshwater habitats are vulnerable to human disturbance such as agriculture,

urbanization, and industrialization. Any perturbation around the riparian environ-
ment affects significantly the in-stream fungal communities and the biogeochemical
cycles through runoff processes. Organic pollution caused by the mass discharged
of domestic or agricultural wastes reduces the amount of oxygen in the water. Most
freshwater fungi cannot survive in such anoxic and polluted environments. For
example, organic pollution reduced substantially the diversity of aquatic hyphomy-
cetes (Raviraja et al. 1998) and ascomycetes (Tsui et al. 2001b). Toxic metal enter-
ing the rivers as a result of mining and industrialization also impact negatively the
fungal communities. Spore production, biomass, and fungal diversity are severely
depleted under high concentrations of coal, copper, zinc, and cadmium (Krauss
et al. 2001; Niyogi et al. 2009; Sridhar et al. 2005). Similarly, the application of
chemical pesticides can also change the fungal communities. For example, the anti-
gen and biomass production of Neonectria (Heliscus) lugdunensis was influenced
by the herbicide mecoprop (Bermingham et al. 1998). Recent pyrosequencing
(metagenomics) data also showed declining fungal diversity in most eutrophic
streams (Duarte et al. 2014). However, previous studies demonstrated the oppo-
site—anthropogenic nutrients stimulated fungal spore production and mycelial
biomass on leaves (Gulis and Suberkropp 2003).
Functional Biodiversity/Outlook: Who Is Doing What?
The most prominent ecological function of freshwater fungi is the decomposition

of allochthonous organic matter in aquatic systems. The research of freshwater
fungal ecology has been focused on stream-inhabiting aquatic hyphomycetes
mostly in temperate low-order streams (Bärlocher 2010). Properties of ecosystem
functions, such as fungal biomass, fungal productivity, and fungal impact on the
decomposition process (Lecerf and Richardson 2010a), have been investigated in
hundreds of field and laboratory (microcosm) studies [meta-analyzed in (Ferreira
et al. 2014)]. These studies were accompanied by inventory biodiversity (morpho-
logical and molecular) approaches of aquatic hyphomycetes [e.g., (Nikolcheva
et al. 2003; Seena et al. 2008; Duarte et al. 2014; Casas et al. 2011; Pascoal et al.
2005; Shearer and Webster 1985b)]. However, the results of studies investigating
the functional consequences of biodiversity changes often remained unpredictable
or inconsistent [summarized in (Graça et al. 2015)]. Within the last decade, it has
become apparent that species traits and functional diversity may be better corre-
lated with ecosystem function than taxonomic identity (Gessner et al. 2010; Lecerf
and Richardson 2010b).
Nowadays we have the methods at hand to even more thoroughly elucidate
species assemblages and, furthermore, investigate the possible and active traits of
freshwater fungi. The progress in sequencing and annotating fungal genomes will
soon shed light on the genetic diversity and metabolic potential of freshwater
fungi from all fungal phyla. Transcriptomes [e.g., using RNA-seq; (Wang et al.
2009)] from single cultures or microcosm studies of freshwater fungi will enable
us to study the metabolism during decomposition and/or degradation processes.
For the identification of metabolic pathways, the quality of genome annotation is
crucial (Kuske et al. 2015). The optimal study design is to have very well-anno-
tated reference genomes, the transcriptome (the expressed genes), and the corre-
sponding proteome (the produced enzymes) as shown by Hori and co-workers
(2014). Freshwater fungal communities are complex assemblages of mostly fila-
mentous fungi, single-celled chytrids, and yeasts. They are members of food webs
and mediators of biogeochemical pathways for the energy transfer between differ-
ent trophy levels. Future methodical improvements will hopefully ease the chal-
lenges of meta-analyses to understand the “why-is-who-doing-what” in the
ecology of freshwater fungi.
Acknowledgments The authors wish to thank the following students of Professor T.K. Goh, for
providing the pictures of freshwater fungi used in this chapter: Bao Yee Loh (Oomycetes), Huey
Kei Lee (Ingoldian fungi), and Wee Kee Sim (lignicolous hyphomycetes). We are grateful to
Dr. Huzefa Raja for comments and suggestions. The authors thank the master student of Christiane
Baschien, Jens Schmidt for the picture of Lunulospora curvula conidiogenesis.
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Chapter 14
Dispersal Strategies of Microfungi
Donát Magyar, Máté Vass, and De-Wei Li
“Fungi cannot walk or run, but some can swim, most can soar,
a few can jump, and some must be carried” (Kendrick 1985).
Introduction
Spore dispersal is an extensively studied field in microfungi biology. Even in the

early 1900s, the knowledge accumulated as in some volumes of Buller’s book
(Buller 1909). Since that time many books and review papers have been dedicated
to this topic, and most informative papers were published in the 1960s. Among
them Ingold’s Fungal Spores is the most comprehensive work, in which all the four
major branches—fungal dispersal with air, water, plants, and animals—are covered.
In the last 40 years, however, no similar work was published—only from the studies
of aquatic hyphomycete dispersal and airborne fungi tended to become a distinct
lineage of mycology. Others, e.g., entomophilous dispersal, have been only
D. Magyar, Ph.D. (*)

Department of Air Hygiene and Aerobiology, National Public Health Center, Budapest, Hungary
e-mail: magyar.donat@gmail.com
M. Vass
Deptartment of Ecology and Genetics, Limnology, Uppsala University, Uppsala, Sweden
e-mail: mate.vass@ebc.uu.se
D.-W. Li, Ph.D.
e-mail: Dewei.Li@ct.gov

DOI 10.1007/978-3-319-29137-6_14
316 D. Magyar et al.
sporadically studied. In recent years, fungal spore dispersal has been studied with
increasing intensity due to their allergological, phytopathological, and ecological
potential, and a wealth of new information has been gained. Although some funda-
mentals in understanding spore dispersal should be provided, the aim of this chapter
is to review the last 40 year’s discoveries, updating the knowledge of the dispersal
of microfungi according to Ingold’s original concept, and show the most important
results and directions.
The Spore Dispersal Pathway
Many fungal species developed different strategies—adaptations or mechanisms to

aid dispersal, mainly by means of air currents, water, or animals. Most widely, air-
borne dispersal of spores is studied. The concept of “aerobiology pathway” involves
three stages (Edmonds 1979): (1) liberation, (2) transport, and (3) deposition. The
journey of the spores does not always finish here: a subsequent phase, resurfacing
(4) may also occur. Multiple resurfacing in airborne dispersal (re-aerosolization) is
important, giving a “second chance” to find the host. These steps are also present in
other dispersal strategies. Thus, it is reasonable expanding this concept to create the
new term “spore dispersal pathway.” The pathway and the dispersal strategies are
summarized in Fig. 14.1 [detailed description of the steps and mechanisms shown
in this figure exceeds the frame of this chapter; for further details, see (Gregory
1961; Ingold 1971; Kendrick 1990; Madelin 1994; Lacey 1996; Deacon 2006;
Lacey and West 2006; Elbert et al. 2007; Money and Fischer 2009)]. Dispersal steps
are often combined, e.g., airborne transport and deposition of the spore in the can-
opy are often followed by stemflow transport.
The number of spores produced in and emitted from a fungal colony is on a wide
scale (from one big spore of Valdensinia heterodoxa to 400,000,000 conidia of a col-
ony of Penicillium sp. 2.5 cm in diameter or even 5.4 trillion spores of Ganoderma
applanatum) (Ingold 1971). Even the world’s human population—approximately 7
billion—is insignificant as a number when compared to the number of spores that just
a single fungus may produce within its perennial fruiting body over a 6-month period.
This is also an important factor in fungal dispersal, concerning the K- or r-strategy of
the fungi. The r-selected organisms produce many spores, but with only a low chance
of reproductive success (e.g., Cladosporium, Rhizopus), while K-selected organisms
produce few spores, but with significant chance of establishment in a new environ-
ment (Andrews 1992). Concentration dynamics of the spores are strongly influenced
by each stage of the pathway (Ingold 1971; Lacey 1981, 1996).
Spores can also be classified as primary and secondary inocula. Primary inocu-
lum consists of propagules of a pathogenic fungus that start the disease cycle in a
new growing season of a crop. Secondary inoculum distributes the pathogen within
the main growing season, and this usually leads to the development of epidemics
(McGee 2003).
Spores are dispersed in different space and time scales. The microscale dispersal
is limited to rather short time and small space scales on the order of < an hour and
14 Dispersal Strategies of Microfungi
Fig. 14.1 The aerobiology pathway (liberation, transport, deposition, and resurfacing) modified after Edmonds’ concept (Edmonds 1979), including Lacey’s
317
(1991) classification of liberation mechanisms, vertical transport after Gregory (1961), horizontal transport after Gage (1999)
space scales up to several hundred meters. For mesoscale transport, the time and
space scales are days and several hundred km. Macroscale transport is the greatest
in time and space scales including global circulation patterns (Magyar 2007).
In recent years the importance of understanding the detailed dispersal strategy
has been enhanced by an unprecedented number of fungal diseases, which are con-
sidered a worldwide threat to food security and a cause of extinctions of wild plant
species (Fisher et al. 2012). Successfully controlling these emerging diseases
depends on whether their propagates can be controlled, thus it is important to know
the dispersal strategies of fungi. Many fungi have developed different active and
passive strategies to release, disperse, and colonize new environments (Incagnone
et al. 2014). In this chapter, these mechanisms are presented and discussed.
Dispersal Units of Fungi
Fungi may be present as propagules (spores, hyphal fragments, partial conidiophores,

pieces of mycelium, sclerotia) or attached to substrata (Fig. 14.2), but spores are the
main units of dispersal (Bärlocher 1992a). The transportation of each of these forms
results in the fungi widespread occurrence. Fungal spores occur in a fantastic scale
of shapes from the simple globose to the exceptionally complex. Morphology of
spores may be indicative of its ecology, dispersal, or sporogenesis as well.
Thick-walled, pigmented spores have higher resistance against drying and UV
radiation during aerial transport than thin-walled and hyaline ones. Spore size and
roughness affect the dispersal of spores by the rate at which spores fall through the
air and their ability to impact on stems and other obstacles or surfaces. The large-
spored leaf- and stem-pathogenic fungi appear to be specialized to impact on the host
plants (impactors), while the spheroid, minute spored molds (penetrators) appear to
be specialized to penetrate deeper into the vegetation (Gregory 1961). Spores could
be grouped according to their wettability, into hydrophobic dry spores (xerospores)
and readily wettable slime spores (gloiospores) (Fig. 14.1). Gloiospores are carried
within rain droplets, but xerospores are found on their surface. Certain fungi have
two or more morphologically different spore types, and each of them is adapted for
different dispersal circumstances. It is often observed in the anamorph–teleomorph
pairs (e.g., orbiliaceous fungi have penetrator-type ascospores and radiate, anchoring
conidia). Spores with appendages and mucilage on their surface are usually dispersed
by rainwater or animals. The function of these structures in Coelomycetes fungi are
summarized by Nag Raj (1993). The role of some structures on the spores is still
unclear, e.g., hornlike hyaline cells on some bark fissure-inhabiting fungi (Excipularia,
Oncopodiella, and Oncopodium). Cytoplasmic viscosity and the presence of ergos-
terol of airborne and waterborne fungal spores are significantly different between the
two spore types and correlated with spore survival. The mode of spore dispersal is
related to cellular composition (Van Leeuwen et al. 2010).
The different spore forms of rust fungi have different modes of dispersal: pycnio-
spores are released from their conidiogenous cells into a viscous liquid and locally
allocated by insects, splashing water, and contact among host plant organs (Littlefield
14 Dispersal Strategies of Microfungi 319
Fig. 14.2 Fungal dispersal units (spores). Drawn at different scales. Group outlined with a blue
line: dry spores with rough surface. Group outlined with a green line: spores with appendages or
associated with mucilage. Group outlined with a red line: radiate spores. a Aspergillus conidia; b
Aspergillus conidia in chain; c fungal mycelia inside an airborne pollen grain; d two xylariaceous
ascospores adhered on their flat side form a sphere minimizing air drag; e ascospore of the subter-
ranean fungus Tuber mesentericum; f Pilobolus, a discharged sporangium just landed on a glass
slide; g Ascobolus immersus (reported as Dasyobolus immerses) ascospore octade; h Pestalotiopsis
conidia, i Oncopodiella sp. germinated conidia from stemflow rainwater; j Metschnikowia reu-
kaufii yeast cells from floral honey; k Loramyces juncicola ascospore; l Pleospora scirpicola asco-
spore; m–n Alternaria sp. m with short rostrum and n with long rostrum; o–s Fusarium sp., o
macroconidium, p microconidium, q mesoconidium, r mycelia and conidia on airborne plant
debris, s chlamydospore; t Podospora fimicola; u Torula herbarum; v Urocystis spores surrounded
by sterile cells that possibly aid wind dispersal; w Tetracladium conidium from streamwater foam;
x Phragmidium mucronatum teliospore; y Aglaospora profusa ascospore; z Dendryphion nanum
conidium from the burrows of earthworms; a’ Leptosphaeria maculans ascospore and b’ conidia of
its anamorph, Phoma lingam; c’ Cordyceps militaris. d’ Cross-shaped conidia of Valdensinia het-
erodoxa turn to have a lacrymiform shape when discharged, which aerodynamically should make
the conidium travel further. g, f, l, k, t, x, and c’ were redrawn after Ingold (Ingold 1971, 1978) and
d’ after Zhao and Shamoun (2010)
1981). Aeciospores are produced in tightly packed chains, released by the dissolu-
tion of intercalary cells, and aerially disseminated (Littlefield and Heath 1979),
while the teliospores may remain attached to the host organ on which they were
produced (Littlefield 1981).
The distribution of infected plants in a population is useful information pointing

out the source of inoculum or the nature of the vector. Random distribution is rather
rare. It can be a result of airborne inocula, insect-borne inocula or seed-borne patho-
gens, via a long distant transportation. Diseased plants aggregated in clusters are more
common, and it indicates a random distribution of the inoculum. A disease dispersed
by aphids or a root fungal pathogen might produce a pattern of aggregation when the
inoculum spreads from the initial infected point. It is highly unusual to observe a regu-
lar distribution of diseased plants in the field. However, vegetatively propagated crops
could show such a pattern, if all planting materials were infected or if pathogens left
in the soil by a previous crop with even space were able to infect the next crop.
Soilborne pathogens, such as root fungal pathogens or nematodes, or diseases carried
by soil-inhabiting vectors show a patch distribution. Soil organisms usually spread
slowly, as do the diseases transported by these vectors, and consequently lead to
patchy distribution. A very common disease distribution pattern is a gradient.
Gradients typically indicate that the inoculum source is outside the field crop. The
gradient steepness is relative to the proximity of the source. The slope of the gradient
can be influenced by other factors, such as the way of the pathogen is spread (e.g.,
flying insects vs. crawling insects). A much greater effect on the spread of disease is
from vector movement than from vector population (McGee 2003).
Dispersal of spores is the major determining factor for the distribution and range
of fungi in nature. Despite the importance of the process of spore release in allergology
and plant pathology, it has not been fully studied in many fungi.
Liberation
Liberation mechanisms of spores vary greatly among different groups of fungi. These
mechanisms have been reviewed by Gregory (1961) and Ingold (1971). Based on
these works, the mechanisms are summarized and illustrated (Fig. 14.2), but the pres-
ent descriptions are focused on the knowledge gained in the past years.
At the interface of the solid objects, the air is stationary. Airflows around the
objects are moving slowly due to the drag created by the object’s surface. This is the
laminar boundary layer which surrounds all surfaces at a variable depth between
0.1 mm and 9.0 mm on a leaf surface (depending upon leaf size and wind speed), to
a meter or more that often exist on a woodland floor on calm days (Nobel 1991).
Above this boundary layer, the spores could be dispersed because the air becomes
progressively more turbulent in local eddies, until there is a net movement of the air
mass. In order to become airborne, spores must cross the laminar boundary layer to
reach a new site. This is the essential feature of spore dispersal, so microfungi
require different strategies to accomplish it. Release mechanisms could be classified
as active (utilizing the energy generated by the fungus) and passive (utilizing the
energy from the environment). Fungi that grow on more rigidly supported surfaces
can release the spores by active processes, and others involve adaptations of the
spore-bearing structures rather than of the spores themselves. Airborne dispersal
needs different strategies under dry and rainy conditions, and many fungi have
adapted to both circumstances (e.g., the Pleospora is liberated in wet, while its
anamorph, Alternaria, in dry weather) (Magyar 2005). Molecular techniques are
major alternatives or supplementary to morphological identification of fungi.
However, the latter is still raison d’etre in dispersal research, according to the differ-
ences in the ecological adaptations of different spore forms.
In windless conditions, gravity alone may overcome adhesive forces attaching
the spore, when it is elevated on a tall sporophore, stem or leaf. Mushrooms and
some myxomycetes are examples for this strategy. When the stalk (stipe) of a mush-
room elongates, the cap (pileus) reaches the layer of the turbulent air. The basidio-
spores fall from the gills or pores vertically into turbulent air and carried away by
wind. In calm days when higher temperature differences are present, spores could
be lifted up from cultures to the top of 10–12-cm glass cylinders by convection
alone (e.g., at 10 °C differences for Botrytis cinerea and Chrysonilia sitophila)
(Lynch and Poole 1984).
Wind plays a major role in spore release. Moderate wind speed [0.4–2.0 m s−1
(Gregory 1961)] is enough to detach spores from the colony of many fungal species,
but the maximum number of the removed spores varies among species (e.g., between
0.4 and 1.0 m s−1, Alternaria alternata (reported as Alternaria tenuis) (Rotem 1994);
between 0.5 and 1.0 m s−1, Erysiphe graminis (Aylor et al. 1981); at 0.5 m s−1,
Aspergillus fumigatus (Pasanen et al. 1991); between 1.8 and 2.3 m s−1, Puccinia
recondita f. sp. tritici; between 1.8 and 2.8 m s−1, Puccinia striiformis (Geagea et al.
1997); between 3.0 and 5.0 m s−1, Spilocaea pomi (sexual state, Venturia inaequalis
(Wiseman 1932). Fungal spores may be deflated by a higher wind speed from the
ground (3.0–5.4 m s−1) than from the phyllosphere (0.5–2.0 m s−1) (Jones and
Harrison 2004). The efficiency of deflation should be reduced when spores are
slimy or the substrate is wetted (Jones and Harrison 2004; Ward and Manners 1974).
In this case, spores are cemented to each other and to the surface (e.g., Zoberi 1961;
Pady et al. 1969; Cohen and Rotem 1970; Rotem 1994; Geagea et al. 1997). It has
been shown that especially intermittent wind and vibration is effective (Aylor 1990,
1993; Górny et al. 2001), because it provides a small but continuous stream of via-
ble airborne spores in the air, removing the highly mobile (dry and detached) part of
spore mass produced in the fungal colony. Gloiospores (e.g., Stachybotrys charta-
rum), however, remain attached on the surface and could not be removed by low
airspeeds [0.3–1.6 ms (Tucker et al. 2007)].
Dispersal of a high proportion of lichen-forming fungi is efficiently carried out
by vegetative symbiotic propagules: soredia, isidia, blastidia, and thallus fragments
(Honegger 2009). The predominant mode in reindeer lichens (Cladonia spp.) is
thallus fragments, which cover thousands of km2 area of arctic tundras. Soredia,
powdery propagules, are composed of fungal hyphae wrapped around green algae
or cyanobacteria. Large amounts of mycobiont-derived crystalline secondary
metabolites are carried by the soredia of a high number of lichens at their surfaces
and are highly hydrophobic. This phenomenon facilitates wind dispersal.
Colonies of some microfungi growing on leaf surfaces sometimes produce
chains of spores from a basal cell so that the mature spores are pushed upwards
through the boundary layer as more spores are produced at the base of the chain
(e.g., Blumeria graminis). The spores are then removed by air currents or, sometimes
more effectively, by mist-laden air (e.g., Cladosporium) (Harvey 1970). Wadia et al.
(1998) showed that spore counts of Passalora personata were decreased by steady
wind, but intermittent wind gusts caused a high concentration. Continuous wind in
3–6 days exhausted the colonies. Stronger wind gusts tear off the strongly attached
mycoparticles, sclerotia, and immature conidia as well. Spores should not be pro-
duced until new conidiophores develop and replace the broken ones (Langenberg
et al. 1977). During evolution, two groups of Alternaria spp. adapted different strat-
egies through the boundary layer by developing a beak, a unique conidiogenous
apparatus on conidia. The dispersal of small-spored Alternaria involved the cate-
nate proliferation of conidia emerging from the beak or the secondary conidiophore
of the precedent conidium. For the other group, the liberation of filament-beaked
Alternaria was facilitated by the elongated filamentous beak. Both strategies result
in the elevation of conidia through the boundary layer (Chou and Wu 2002). The
erosion of fungal colonies is stronger on the upper part of the plant canopy than near
the ground (Rotem 1994; Aylor 1990). Some spores hardly became airborne,
because their dispersal is not primary anemophilous, like coprophilic fungi.
Staurosporae, “giant spores” (Bipolaris, Podosphaera, Phragmidium, and
Tetraploa), fruiting bodies, sclerotia, and fungal particles aggregated with pollens
and plant debris could be also aerosolized by strong wind. Rotem (1964) observed
immature conidia of Alternaria solani in the windstorms of Negev desert and some
of them were attached on plant debris. Meredith (1966) mentioned that conidio-
phores with immature conidia, hyphal fragments, and plant debris with spores were
collected by a Hirst-type air sampler. Hyphal fragments of phylloplane fungi should
be dispersed due to scrubbing action of host plants which takes place during wind
currents (Tilak and Pande 2005). Airborne plant debris could carry the oospores of
Peronosclerospora sorghi (Bock et al. 1997).
Ballistospore discharge is a feature of basidiomycetes. The process of ballisto-
spory and the role of the droplets observed on the spores were poorly understood
until recently, because this process occurs so rapidly that the launch of the ballisto-
spore has never been visualized. Analyses show that ballistospores catapult into the
air at initial accelerations in excess of 10,000 g. Recent technological development
of ultrahigh-speed video cameras allowed to capture the fast motions during spore
discharge at camera speeds of up to 100,000 frames s−1 (Pringle et al. 2005; Noblin
et al. 2009). According to the records, just few seconds before discharge, fluid
begins to condense on the spore surface at two locations (Fig. 14.3; McLaughlin
Fig. 14.3 (continued) complex. This force puts the hilum under tension, which provides a coun-
teracting force that cannot exceed the fracture force (FB). Fourth, the hilum is fractured, thus
releasing the spore. (b) The corresponding stages in jumping. First, the center of mass is lowered
to allow the legs to do work on the substratum. At this stage, the gravitational force (Fg) and the
ground reaction force (FR) are balanced. Second, as the legs unfold, the moments at the joints (M)
are resisted by the substratum, thus providing the impulse (I) necessary to accelerate the center of
mass. Third, late in the jump, the fast-moving upper body starts to entrain the legs, which to this
point were moving slowing upward. Fourth, after takeoff all body parts are moving at similar
speeds and only gravity acts on the body (c) A typical basidium with four spores. (d) Structure of
the lower half of the spore. (e) Spore ejection in Auricularia auricula [a–c, e, pictures courtesy
from Xavier Noblin and Jacques Dumais; d, based on McLaughlin et al. (1985)]
Fig. 14.3 The four stages of ballistospore ejection. (a) First, the growth of the drop brings the center
of mass of the spore–drop complex closer to the end of the sterigma. Second, at the start of the
coalescence process, the drop and spore exert on each other forces of equal magnitude but opposite
direction (FD and FS). The expected downward displacement of the spore is prevented by the pres-
ence of the sterigma, giving rise to a reaction force FSt acting at the hilum. Third, in late coales-
cence, the momentum of the drop is transferred to the spore, which was immobile until then. The
transfer of momentum is equivalent to a force FSD applied at the center of mass of the spore–drop
et al. 1985; Noblin et al. 2009). (1) Over the punctum lacrymans of the hilar appen-
dix, the Buller’s drop starts to bubble. (2) On the adaxial drop on the adjacent spore
surface, another drop is present. Once initiated, Buller’s drop increases in diameter
rapidly for a few seconds and then suddenly merged with the adaxial drop. As the
drop redistribute mass from the hilum of the spore in the direction of the other end
of the spores, the spore is catapulted. Recordings demonstrated that coalescence
may result from the directed collapse of Buller’s drop onto the spore, but it may also
involve the movement of the spore toward the drop. The energy of spore discharge
is derived from the rapid, surface tension powered movement of Buller’s drop onto
the spore surface, to push spore off from the sterigma. The release of surface tension
energy at coalescence provides the kinetic energy and directional momentum to
launch the spore away from the fungus. This mechanism is responsible for launch-
ing basidiospores from mushrooms, but microfungi also utilize it in their dispersal,
such as basidiomycetous yeasts, rusts, and smuts. It was calculated that the relative
sizes of the spore and drop determine the distance of the launch. Increase in the
radius of the drop tends to catapult the spore over a greater distance (Money and
Fischer 2009). The results revealed a surprising similarity with the mechanics of
jumping in animals (Fig. 14.3b; Noblin et al. 2009).
Explosive discharge of Ascomycota is one of the fastest movements in nature.
These fungi, as small guns, shoot ascospores to 1- or 2-cm distance to break free of
the boundary layer. The mechanism of asci that act as small water cannons and
expel the spores into the air has long been thought to be driven by turgor pressure
within the extending ascus. In recent studies the pressures within the ascus were
measured. Such studies quantified the components of the ascus epiplasmic fluid
that contribute to the osmotic potential. Although few species have been examined
in detail, the results indicate diversity in ascus function that reflects ascus size,
fruiting body type, and the niche of the particular species (Trail 2007). Because of
their microscopic size, ascospores experience great fluid drag. It was shown that
ascospores are shaped to maximize their range in the nearly still air surrounding
fruiting bodies using numerical calculation of optimal spore shapes (shapes of
minimum drag for prescribed volumes). Analysis showed that spores are con-
strained to remain within 1 % of the minimum possible drag for their size, being
near their physical optima (Roper et al. 2008). Ingold (1971) opined that spores
would be shaped to maximize the force used by apical rings of the asci to push on
them. But, surprisingly, the individual geometric dimensions of spores and apical
ring critical to these hypotheses are either very weakly or not correlated (Fritz et al.
2013). There is a mechanism avoiding energy losses during spore’s ejection
through the apical ring. This mechanism is based on a physical principle discov-
ered 50 years ago in the study of elastomeric seals and O-rings used to control fluid
flow in engines, pipes, and other engineering applications (Dowson and Higginson
1959). The apical ring is an elastic seal and distorts significantly when the spore,
which is lubricated by a thin fluid layer, passes through it. Some apothecial fungi,
e.g., the plant-pathogenic Sclerotinia sclerotiorum, are dispersed by synchronizing
the ejection of thousands of ascospores, and this creates a blast of air that carries
spores through the laminar boundary layer (Fig. 14.4). High-speed imaging showed
Fig. 14.4 Synchronized ejection of ascospores creates a coherent jet of air, enhancing dispersal in
open and crowded environments. (a) Early documentation of synchronous spore release in a draw-
ing of Otidea cochleata (Bulliard 1791). (b) Spore jets originating from Sclerotinia sclerotiorum
apothecia travel much farther than individually ejected spores. (c–e) Mathematical modeling
shows how spores mobilize the air around the ascocarp. (c) Simulated jet profile for synchronous,
random ejection showing both (e) The flow of air that carries the spores and (d) spore motions. (e)
Average spore (black curves) and air (red curves) speeds on cross sections of the jet show how the
spores initially push upon the still air, and as the jet develops the spores are pulled upward by the
air that they set into motion. (f–i) If the spore jet impacts upon an obstacle, such as a glass slide,
then pressure gradients within the jet displace spores out and around the obstacle. Images are taken
at t = 0, 0.11, 0.32, 0.55 s after initial impact [pictures courtesy from Roper et al. (2010)]
that synchronization is self-organized and likely triggered by mechanical stresses.

Spores “cooperate” maximally to generate the favorable airflow with optimal
geometry (Roper et al. 2010). Some quantity of moisture is usually required for
spore discharge in different Ascomycetes. Consequently, the peak of ascospore
concentration could be measured some minutes after the start of rainfall or 5–10 h
later. Sometimes fog is enough to generate spore discharge (Gregory and Stedman
1958; Carter and Moller 1961; Hirst and Stedman 1962; Arseniuk et al. 1997).
Asci may be exhausted in rainfalls lasting for many days (Gregory and Stedman
1958; Turott and Levetin 2001).
Drying-induced hygroscopic movements exist in some fungi. When drying out,
their spore-bearing structures twist, leading to the release of the spores (e.g.,
Botrytis, Phytophthora infestans, and Peronospora). The reduction of R.H. is trig-
gered by various factors (temperature/solar radiation, wind). Results of several
laboratory experiments on spore discharge of fungal species under controlled
climatic conditions showed active spore discharged in case of decreasing relative
humidity (de Bary 1887; Meredith 1961, 1962, 1965, 1966; Ingold 1971).
Outdoor concentration of spores reaches its maximum at the mornings (Hirst 1953;
Panzer et al. 1957; Waggoner and Taylor 1958; Hamilton 1959; Sreeramulu 1959;
Dutzmann 1985).
Airborne concentration of ascospores (Chaetomium, Xylariaceae, etc.) may also
correlate positively with drying. In this case ascospores are not ejected into the air,
but a mass of spores (cirrus) is formed covering the surface of the fungal fruiting
body. After the cirrus is dried, spores are liberated. Incipient desiccation of the peri-
thecium of some ascomycetes (e.g., Claviceps purpurea) may also exert pressure on
its content so rate of discharge increases. The main mechanism involved in the dis-
persal of plant pathogens is rain splash which is the critical component in epidemic
development of many diseases (Madden 1992). A single raindrop falling on a
Botrytis colony in a leaf lesion could contaminate an area of 2.5 m2 (Weston and
Taylor 1948). Rain splash occurs when a raindrop falls onto a surface covered by a
thin film of water, forming a crater, with many (100–5000) secondary droplets pro-
duced at its periphery (see Edgerton’s iconic “milk drop coronet” photograph).
Madden (1992) and Huber et al. (2006) found a direct relation between the number
of spores released by the rain splash and physical characteristics of raindrops, e.g.,
kinetic energy, velocity, and Weber number. Weber number is a dimensionless, per-
tinent parameter for the description of splash droplet formation after impact. The
bigger the Weber number is the more the water drop interface is deformed and
potentially breaks into splash droplets (Saint-Jean et al. 2006). The literature of
hydrodynamic modeling and physics of splash dispersal was vast in the last decades,
especially in plant pathogens, like Botryosphaeria (Ahimera et al. 2004),
Colletotrichum (Peres et al. 2005), Fusarium/Gibberella (Hörberg 2002; Paul et al.
2004), and rusts (Sache 2000). Experiments were also conducted, such as splash
from single water drop impactions, spore transport with simulated rain over small
areas, and disease spread in the field with naturally occurring rain. Studies showed
that with increasing size of impacting raindrops (and hence increasing velocity and
kinetic energy), the number of splash droplets produced also increased, as well as
the number of spores disseminated and flight distance of the splash droplets. It was
found that transport distance is very short, generally <15 cm in each splash event.
This indicates that the deposition of spores on a potential infection site is a result of
continual re-splashing of droplets and spores in them (Madden 1997).
Raindrops or hail can also release dry spores on a surface, by “puff” and “tap”
mechanisms. Spores can be discharged by raindrops when the raindrops hit lesions
at terminal velocity after an unimpeded free fall from a height of several meters or
as secondary droplets at lower-velocity dripping from vegetation (Herwitz 2006). A
raindrop splotches sideways, when it falls on a rigidly supported surface, and the
consequential gust of air disturbs the boundary layer, leading to the dry spores to
become airborne. The first large drops of an intensive rainstorm increase the con-
centration of dry fungal spores (Hirst 1953; Ainsworth 1952; Harvey 1967; Rantio-
Lehtimäki 1977; Hjelmroos 1993; Kurkela 1997; Venables et al. 1997; Allitt 2000).
A study by Hirst and Stedman (1963) showed the possibility of the dry dispersal of
fungal spores by incident raindrops in case of some rust, smut, and conidial fungi.
The mature basidiospores of some puffballs (Basidiomycota) are enclosed in a

papery basidioma with an apical pore, so that raindrops “puff” the spores into the
air, like bellows. Ascospores of Venturia inaequalis are released from the lesions on
leaves following the impact of raindrops during rain. Dampened periodic vibrations
were induced by raindrops on leaf surfaces. Vibrations, linear, and/or oscillatory
flows of water on the leaves induced spore releases at low energy thresholds. The
fungal perceptibility of kinetic energy facilitated selective discharge of spores when
environmental conditions are most favorable to survival. Determining kinetic effects
of rain which reveal start, thresholds, and proportional distribution is significant for
research on disease prognosis, and at the same time, it may serve as a model for a
common fungal sensing mechanism (Alt and Kollar 2010).
Airborne Transport
It is unequivocal that wind plays an important role in transportation of fungal spores.

Airborne spores are primarily present in the troposphere. Dispersal of airborne
spores is closely related to the size, shape, roughness, density, and electrostatic
charging of individual spores and air viscosity, convection, layering, wind, turbu-
lence, wind gradients close to the ground, and patterns of atmospheric circulation.
When mixing of air masses is low, populations of spores should increase in still air.
Daily maximum of spore population should evolve in certain geographical areas,
where diurnal high winds occur on a regular basis (Gregory 1961; Ogden et al.
1969). Spore free winds or high wind velocity reduces the number of spores hover-
ing above the source (Hamilton 1959; Lopez and Salvaggio 1983; Giner et al. 2001).
According to the theories of eddy diffusion, turbulence dilutes clouds of spores.
Eddies dilute spore cloud as it travels downwind, spreading it both horizontally and
vertically, or physically move it. Air movements vary from small turbulent eddies to
large frontal systems (several thousand km in length and several hundred km in
width) and jet streams in the stratosphere and in the upper troposphere where spores
are speedily transported. Gregory (1961) classified distinct zones of troposphere
with regard to the dispersal of airborne fungi.
The laminar boundary layer is thickened at nighttime. The local eddy layer is
eddies resulted from small cup-shaped depressions or roughness on the surface. In
the turbulent boundary layer, diurnal changes are less pronounced. The transitional
or frictional turbulence layer attains up to 500–1000 m in height, in which turbu-
lence weakens with altitude. The top of this layer is the limit to which fungal spores
may be transported upwards by turbulence. The convective layer is a stratum in
which airborne particulates can be transported only by convection. During summer,
when the sun heats the ground, warm air bubbles carrying a large number of spores
may form in areas of 1.25 km2 every 6–15 min.
Small propagules like fungal spores may be dispersed by the wind over dis-
tances of thousands of kilometers. Long-distance dispersal of fungi was proved by
spore-trapping devices mounted on the outsides of aircrafts. One of the approaches
toward understanding the long-distance dispersal is trajectory analysis based on

circulation patterns as derived from worldwide synoptic observations. Long-
distance dispersal can be highly significant for plant disease epidemiology, espe-
cially when new pathogenic races or fungicide-resistant strains develop and are
spread across or between continents. In some fungi, asexual reproductive struc-
tures (mitospores) have been reported to disperse within [e.g., continental Europe
to Great Britain (Brown et al. 1991)] and between continents [e.g., from South
Africa to Australia (Watson and De Sousa 1983), Fig. 14.5]. Many other examples
of long-distance dispersal (LDD) are in evidence (Aylor et al. 1982; Brown and
HovmØller 2002; Frank et al. 2008), e.g., in India (Nagarajan and Singh 1976),
between Paraguay and Argentina (Waller 1979), from Angola to Brazil (Bowden
et al. 1971), across the Tasman Sea from Australia to New Zealand (Viljanen-
Rollinson and Cromey 2002), and between North Africa and Southeast Europe
(Tuboly and Vörös 1962). For additional examples, see (Taylor et al. 2006).
Meteorological data support the suggestion that Hemileia vastatrix urediospores
developed in a coffee rust epidemic in Angola in 1966 were transported across the
Atlantic at an altitude of 1500–2000 m in 5–7 days and deposited over the coffee
plantations in Bahia, Brazil (Bowden et al. 1971). Similarly, urediospores could
be easily transported from South Africa to Australia in <5 days traveling at 12,000-m
altitude (Nagarajan and Singh 1990).
Long-distance dispersal is an important ecological process. It can signifi-
cantly increase the range in which pathogen epidemics spread across a land-
scape, result in rapid spread of a disease to previously disease free areas, and
influence the spatial distribution of pathogen populations in fragmented land-
scapes. Spores released during the hottest part of the day are shown to be more
likely to undergo long-distance dispersal than those released at other times
(Savage et al. 2012).
A number of features of spores are important for long-distance dispersal: resis-
tance to desiccation resulted from hydrophobins in the walls, resistance to ultravio-
let radiation conferred by pigments in cell walls, or the shape and size of spores.
These characters may differentially influence long-distance dispersal among taxa
(Finlay 2002). For instance, Glomeromycota, which develop relatively large, nor-
mally non-wind-dispersed asexual spores, had the lowest average geographical range
(Tedersoo et al. 2014). The relatively delicate conidia of aquatic hyphomycetes are
not suitable for longer-distance dispersal (Duarte et al. 2012). Generally region-based
distribution patterns of fungi are, to some extent, incompatible with clustering of
plants and animals, where Holarctic lineages are deeply nested within larger tropical
groups (Sanmartín and Ronquist 2004). Overall, fungi have broader geographical
distribution than those of flowering plants, but even if an accepted hypothesis on the
distribution patterns of species on continents is present, it is a huge challenge to
elucidate these patterns on geographical or biological islands, especially on the
most remote and isolated ones (Incagnone et al. 2014; Whittaker and Fernández-
Palacios 2007).
Long-distance dispersal is a stochastic occurrence which may be exceptionally
significant in determining a population (Wingen et al. 2013), since the dispersal pat-
Fig. 14.5 Selected dispersal events of fungal pathogens. Red and blue arrows indicate invasions of new territories (first year recorded in brackets). Red arrows
indicate dispersal that probably occurred by direct movement of airborne spores. Blue arrows indicate pathogens that were probably transported to the new
territory in infected plant material or by people and spread thereafter as airborne spores. Orange circles indicate the worldwide spread of black Sigatoka
disease of banana; the first outbreak recorded on each continent is marked. Green arrows indicate periodic migrations of airborne spores in extinction–recolo-
nization cycles (from Fig. 14.1 from James K. M. Brown and Mogens S. Hovmøller, SCIENCE 297:537 (2002). Background World Map: © Luckinbeal,
Southern Connecticut State University, New Haven, Ct. Reprinted with permission from AAAS)
terns of fungal spores influence population structure, gene flow, and fungal
community structure (Lilleskov and Bruns 2005) and play an important role in most
conceptual models of community assembly. However, it is difficult to directly mea-
sure dispersal of fungal spores across a whole community at ecologically relevant
spatial scales. The passively liberated spores are less efficient on long distance than
actively liberated ones, and their dispersal distances are not associated with the mass
of the propagules (Jenkins et al. 2007). Microorganisms generally have a cosmopoli-
tan distribution (Incagnone et al. 2014). In the words of O’Malley (2008): “Everything
is everywhere, but the environment selects.” Wood-Eggenschwiler and Bärlocher
(1985) concluded that geological barriers or distance rarely restricts species occur-
rence. In contrast, a recent study showed that fungi largely exhibit strong biogeo-
graphic patterns that appear to be driven by dispersal limitation (Tedersoo et al.
2014). Temperature and its influence on vegetation are considered as the main factor
determining fungal distribution worldwide.
Spore Dispersal by Indoor Air
Air movement outdoors influences air movement indoors forcing air through
cracks on the windward side and sucking it out on the leeward. Wall openings and
gaps at structural joints (electrical outlets, cable boxes, etc.) are 1 mm wide in
average. Thus, through-wall gaps may serve as a route for dispersal of fungal
spores. Within a building, thermal rise or chimney effect transports spores upward
via elevator pits, staircases, or floor openings. Air circulates indoors as a result of
thermal convection. Thermal gradients between heaters, walls, and windows gen-
erate mixing (Daws 1967). Convection may very efficiently carry fungal spores
from the first- to fourth-floor halls in five min and into rooms in 20 min (Christensen
1950). Aerosols are translocated by heat sources, even by heat from a lamp. The
heat from the human body may be adequate to change the local convection system
moving air up from floor level (Lewis et al. 1969). Occupants generate turbulence
via their activities. Spores may be dispersed by bed-making, sweeping, and build-
ing repair work, resulting in spore concentrations 10–17 times greater than before
disturbance (Maunsell 1954; Reponen et al. 1992).
Ventilation systems could rapidly circulate spores or transport them from any
point source throughout entire buildings (Philips 1965). Concentration and viability
of the airborne spores could also be reduced by filtering and UV lamps installed in
air ducts (Kowalski 2006; Burroughs and Hansen 2008). Spore cloud may be
diluted, but not completely removed by artificial ventilation. Even without any
mechanical ventilation, dispersal of a spore cloud may be distributed all over a large
facility in a short period of time.
In grain warehouses, postharvest and plant-pathogenic fungi are common
because spores from the stored grain become airborne. It was suggested that Tilletia
teliospores spread readily inside grain warehouses, possibly by the activities of
transportation systems. Teliospore dispersal takes place rapidly between rooms and
floors via wall openings and spouts. Spouts are used for loading and emptying the
storage room to another room at another floor, generating airborne dust and turbu-
lent air movements (Halász et al. 2014).
Deposition
Airborne spores can be removed from the air in three key ways: sedimentation,
washout, or impaction. Spores are heavier than air and so tend to sediment, but
upward movements by convection and by turbulence impede this process. Gravity
certainly determines the range of the spore dispersal, even after high-speed launches,
but viscous drag from the air acts as a far greater brake, causing a rapid deceleration.
The heavier (larger) spores settle faster than lighter (smaller) spores. Stokes’ law
describes the relationships between terminal velocity (Vt) and size of smooth spheres
1–50 μm in diameter in viscous fluids. The relevant equation is Vt = 0.0121r. Vt is the
terminal velocity (cm s−1) and r, the spore radius (μm). The simplified equation is
appropriate to determine the Vt of many spores in the air. The sedimentation rates
agree closely with Stokes’ law for perfect spheres of unit density (1.0). Value of Vt
varies depending on spore shape (deviation from the ideal sphere), drag (by spore
surface roughening), degree of hydration, and aggregation of spores (with trapped
air between cells). The sedimentation rates for spores with unusual shapes are
adjusted with correction factors.
The effect of the direction of the launch is insignificant on the distance that a
spore is shot. The effect of gravity is indiscernible until the spore is slowed down by
air viscosity. When spores are launched horizontally, their typical traveling path is a
“Wile E. Coyote trajectory,” similar to the tragic coyote featured in Warner Brothers
cartoons falling off a cliff (Money and Fischer 2009).
Rain removes spores from air by impaction on raindrops, by capturing on
cloud droplets, or even by forming cloud nuclei. Impaction of spores on the wet
surfaces is increased by wind speed. In saturated air, spores gaining weight accel-
erate sedimentation (Weinhold 1955). Madelin and Johnson (1992) showed that
spores had larger aerodynamic diameters when aerosolized at 95–98 % R.H. than
the ones at 40 %.
In prolonged rain, droplets wash off the xerospore from air (Hamilton 1959; Ho
et al. 1995; Katial et al. 1997; Fernández et al. 1998; Lim et al. 1998). Gloiospores
(being wettable) become incorporated in the raindrops and spread as a film across a
wettable surface or drip from a nonwettable one. While nonwettable spores covered
with rodlets of hydrophobins remain on the surface of the raindrop, if this rolls
across a nonwettable surface, such as a leaf cuticle, it will leave a trail of spores
behind (Talbot 1997).
Dense vegetation efficiently facilitates spore removal from the air by both impac-
tion and reducing air current speed to allow sedimentation. An individual tree can
filter the 66–80 % of the airborne particulates passing through its canopy and amount
of dust removed may reach several hundred kg (Kovács 1985). Tree leaves after rain
can recuperate their original filtering capacity. The deposition of fluorescent powder
dispersed as an aerosol on different plant leaves (Ilex, Poa, Raphanus, and Solanum)
was studied by Hirst and Stedman (Hirst and Stedman 1971) in a small wind tunnel.
Deposition was greatest on the small plant parts, such as petioles, stems, and longer
spines of the holly leaves (Ilex). There was much less deposition on the larger blades
of the leaves, particularly when these leaves were oriented nearly parallel to wind
direction. Similar results were reported from experiments with fungal spores.
Impaction is most efficient for large spores blown fast toward small objects. When
airborne spores move toward an object (or vice versa), the air is deflected around the
object and inclines to bring spores with it. The momentum (mass × velocity) of a
spore inclines to take it along its current path for at least some distance. Three points
are associated with this phenomenon: (a) larger spores have a better chance of
impacting than smaller spores at any given air speed; (b) as the air speed increments,
so gradually smaller spores can impact; and (c) as the receiving object size incre-
ments, so the air deflection is greater, and this decreases the probability of impaction
(Carter 1965). Small fungal spores (4–5 μm in diameter) fail to impact on objects
only 1 mm in diameter at a wind speed of 2 m s−1 (the typical maximal wind speed in
vegetation). Filtering efficiency was higher in Rosa rugosa, and the efficiencies of
other studied plants are listed in a descending order: Acer campestre, Carpinus betu-
lus, Lonicera sp., and Ligustrum vulgare (Kovács 1985).
Carter (1965) found that spore deposition of Eutypa armeniacae is high on thin
stems of apricot. The spore clusters impacted best on the narrow leaf petioles of ca.
1–2 mm in diameter, less well on the thicker stems of young apricot, and worse on
the broader leaf blades at all wind speeds. It might be considered that the impaction
efficiencies were rather low, <3 % in all cases. Spores were present on the adaxial
surface of leaves, when sedimentation occurred under gravity. Spores impacted on
both abaxial and adaxial surfaces of the leaf surfaces when the impaction was due
to airflow. Wet and sticky surfaces covered by rainwater, plant sap, honeydew, etc.
act as excellent natural spore traps (Chamberlain and Chadwick 1972). Waxy cuti-
cle and trichomes on leaf surfaces may enhance the trapping efficiency and assist
retention of certain types of spores (Forster 1977), Hydrophobic characteristics of
the leaves reduce the efficiency of wet deposition of spores (lotus effect). Spores
incline to occur in preferred patterns associated with leaf anatomy. The most active
zone of the trees for spore impaction and resurfacing is the top of the canopies of
these woody plants (Aylor 1978).
Resurfacing
A large number of spores could resuspend into the air when adhesion diminished by
drying airflow. Not all spores that land become securely attached. Some are resus-
pended again by the agitation of strong wind or by rain splash. The demarcation of
the resurfacing phase is important to introduce the concept of multiple-stage disper-
sal of spores. Observations of Carter (1965) in Eutypa armeniacae become the clas-
sic description of multiple-stage dispersal. This fungus is a pathogen of apricot trees
and grapevines, naturally releases its spores as clusters of eight ascospores held
together in mucilage—a relatively large propagule with sufficient momentum to
impact on twigs at relatively low wind speeds. After impaction, the fugal pathogen
depends on secondary spread by water, either rain or irrigation water, which wash
off the mucilage and carries the separate ascospores down to any wound sites.
Ascospores of Pleospora herbarum discharged from the perithecia in wet
weather can be rediscovered in the air samples several months later, during dust
storms, when deposited spores are resuspended into the airstream. The possibility of
resurfacing of spores is scarcely considered; however, such events may be hazard-
ous and unexpected.
Dispersal in Water
Almost two-thirds of Earth’s surface is covered by oceans, rivers and streams and
reservoirs which represent a substantial amount of water. Despite of this huge water
mass, fungi with primary (Chytridiomycetes) or secondary adaption (aquatic hypho-
mycetes, yeasts) to aquatic environments (Ingold 1971; Jakucs and Vajna 2003;
Wurzbacher et al. 2010) account for only less than 2 % of the overall described
approximately 3000 taxa (Shearer et al. 2007). The main ontogenic stages of their
whole dispersal in water are similar to that of nonaquatic fungi: spore production,
liberation, transport, and deposition. The boundary layer is also present in water,
due to the flow of water around solids, but such layers are much thicker. However,
due to the different media, dispersal strategies are somewhat different from those of
terrestrial (aerial) ones (Ingold 1971) presented in the previous section of this chap-
ter. Furthermore, dispersal strategies are highly influenced by the two major types
of the aquatic environment (Sigee 2005): standing (lentic systems) and flowing
waters (lotic systems).
In flowing waters passive movements are predominantly unidirectional
(Bärlocher 1992a; Hynes 1970). Particles, including fungal propagules, suspended
in the water will be transported downstream for a certain distance before resettling.
This process is referred to as “spiraling” (Minshall et al. 1985; Mulholland et al.
1985) and has a great significance for the dispersal pattern of fungi and also the
availability of their substrates in such environments. What are the convincing mech-
anisms that permit aquatic organisms with little or no active movements to persist in
a given river reach? If water current were the only effective mechanism, it would be
inevitable for their gradual depletion and ultimate elimination from rivers. The
explanation to their sustained presence in rivers must, thus, recline in processes that
annul the effects of streamflow (Bärlocher 1992a).
Organic particles [litter, feces, precipitates of dissolved organic matter (Lock
1981; Suberkropp and Klug 1980)] transported by streamflow within the “spiraling”
process provide degradable energy sources for saprobic fungi and also for other
microorganisms attached to these particles. The major part of these particles derives
from leaves (Abelho and Graca 1996; Grigg and Mulligan 1999) which represent
the major energy source in streams (Kovács 2012). Before a leaf falls and blows into
the stream, the litter is already colonized by terrestrial fungi and yeasts (Bärlocher
1992a) which may contribute to decomposition, but they decline steadily and are
replaced by aquatic fungi, the main decomposer (Suberkropp 1992), aquatic hypho-
mycetes (Ingold 1942), Ingoldian fungi (Bärlocher 1992b), or amphibious hypho-
mycetes. Some studies concluded that the average distance that leaves travel from a
starting point in streams was 200 m, and within 1000 m all leaves were generally
entrained (Young et al. 1978). Shorter distances (19 and 91 m) were found by
Prochazka et al. (1991). Since fungal asexual spores (conidia) of aquatic hyphomy-
cetes are nonmotile, all of these pioneer studies clearly indicated the importance of
this transporting pathway (water movements) in dispersal of fungal propagules
attached to drifting matters, since conidia seem ill suited for longer-distance disper-
sal (Duarte et al. 2012). Independent conidia of aquatic hyphomycetes released into
flowing water may be carried from a few hundred meters to a few kilometers
(Thomas et al. 1990) and can maintain their ability to germinate for several days
(Iqbal and Webster 1973; Sridhar and Bärlocher 1994). The distances that the drift-
ing particles (with attached fungal propagules) travel are also relevant for microbial
movements, strongly determining the possible dispersal of fungi (Bärlocher 1992a)
at a local scale. However, leaves and their fragments quickly develop less suitable
for fungal growth and reproduction (Bärlocher 1982) and compel the fungal species
to passively and very rapidly move from substrate to substrate after leaves secede
and drop (Bärlocher 1992a). Against the risk of being drifted from their target (sub-
strate) and swept away from the favorable upper reaches of a stream, Bärlocher
suggested that the shapes and sizes of conidia of aquatic hyphomycetes (Bärlocher
1992a) and being carried by leaf-eating animals, shredders [i.e., Gammarus pulex
(Bärlocher 1981)], attempt to minimize this impact. Many species have branched
spores (e.g., tetraradiate, consisting of four arms diverging from a point) or sigmoid/
filiform spores (with a curvature lying in more than one plane), but ovoid or spheri-
cal shapes also occur in some species (Ingold 1942, 1971). In Webster’s (Webster
1959) experiment, a horizontal water tunnel apparatus was used to determine the
efficiency of impaction of some common spores of aquatic hyphomycetes. He found
that the tetraradiate spores (e.g., Articulospora tetracladia, Tetracladium marchal-
ianum) have higher trapping efficiency than those with longer, sigmoid
(Anguillospora longissima) or other types of spores (Heliscus lugdunensis). Results
of Dang et al. (2007) led to a similar conclusion in their microcosm experiment with
three aquatic hyphomycetes (Heliscus lugdunensis, Flagellospora curvula, and
Tetrachaetum elegans). In contrast, Cornut et al. (2014) found that leaf litter buried
by streambed sediments (hyporheic zone) is colonized more efficiently by filiform
spore species than by species with tetraradiate (branched) spores due to the filtering
effect of sedimentary matrix. Conidia are not likely to have long-distance aquatic
dispersal (Bärlocher 2009), since conidia are comparatively fragile and quickly lose
their germinability even under favored conditions (Sridhar and Bärlocher 1994).
Several studies focused on the exchanges with the surrounding coastal/terrestrial
habitats try to reveal more detailed image about the dispersal pathways of typical
aquatic fungi (Bärlocher 1992a). The exchange between groundwater and stream-
water (Hynes 1983) is well known and raises the question whether this hydrological
pathway occurs in both directions. Kuehn and Koehn (1988) proved subterranean
origin of terrestrial hyphomycetes and zygomycetous fungi, but not for aquatic
hyphomycetes in a study on artesian. Although Bärlocher and Murdoch (Bärlocher
and Murdoch 1989) demonstrated that multi-radiate and sigmoid conidia can pen-
etrate quite deeply into the sediment, but in that form they did not reach the ground-
water bases. Overall, it might be concluded that those aquatic fungal propagules
which cannot overcome the currents of flowing waters (drifting to the lower reaches
of the stream) also cannot be returned to the upper favorable reaches of a stream by
the exchanges with currents of the groundwater.
It is widely accepted that occurrence of aquatic fungi is not restricted to the lotic
systems itself. There must be some unexplored transport mechanism between flow-
ing waters and terrestrial habitats, thereby offering reservoirs for these aquatic bio-
tas where they may survive in terrestrial sexual state (Bärlocher 1992a). There are
many records evidencing that many aquatic hyphomycetes can occur and survive in
soils (Bandoni 1972; Park 1974; Waid 1954); in ephemeral aquatic micro-ecosys-
tems, like tree holes (Gönczöl 1976; Gönczöl and Révay 2003; Karamchand and
Sridhar 2008; Vass et al. unpublished data); on roofs of several buildings (Czeczuga
and Orlowska 1999) and gutters (Gönczöl and Révay 2004); and even in the tree
canopies (Czeczuga and Orlowska 1994; Sridhar 2009; Sridhar and Karamchand
2009). Thus, it seems that they survive in these habitats not as conidia rather as
mycelia or dormant structures associated with plant detritus (Bärlocher 2009).
Shearer (Shearer 1992) described the wood as a potential source of inoculum of
aquatic hyphomycetes, but their transport mechanisms into the previously listed
terrestrial habitats have not been explored yet. Even though, Selosse et al. (2008) in
their letter have raised the idea of “flying from water to plants” by wind or aerosols
as a potential process, but without aerobiological observations this hypothesis is not
supported firmly. For tetraradiate spores, one potential explanation might be the
aqueous film theory described by Bandoni (1974) and Bandoni and Koske (1974).
It may explain the movement of spores in the water film on leaves and also on tree
barks (Karamchand and Sridhar 2008). As Webster and Descals (1981) stated, one
of the most interesting aspects of the aquatic fungi, especially of the aquatic hypho-
mycetes, is their successful worldwide distribution, which does not show any con-
sistent inter- and intracontinental phylogeographic structure (Duarte et al. 2012).
The observation of identical morphospecies on geologically young islands a long
way from mainlands, such as the Hawaiian Islands (Ranzoni 1979), points to effi-
cient long-distance transportation of viable inocula. Existence of unexplored
pathway(s) has been the greatest challenge in the field of aquatic mycology, and to
go beyond speculations molecular investigations are essential in the future. One
possible explanation of the paradox of the worldwide distribution of freshwater
fungi is that such fungi are not dispersed in their staurosporous form for long dis-
tances but rather with their teleomorphs. Regarding many aquatic hyphomycetes
(anamorph) have a sexual state, teleomorph (Webster 1992). Many teleomorphs of
Ingoldian fungi do not need to be submerged in water in order to discharge their
spores, but do so freely in air. These airborne meiospores are penetrator types and
permit dispersal over large distances (Bärlocher 2009).
The main difference of lentic systems (standing waters) to those discussed above
is the lack of permanent, strong, unidirectional water currents. Regarding the spatial
heterogeneity of standing waters, we can distinguish three main zones: profundal,
littoral, and pelagic zones (Hutchinson 1967). Due to the high diversity of lakes, we
cannot ultimately characterize these zones by main types of their energy sources
(allochthonous or autochthonous), but there are habitats permanently or temporarily
under dominant influence of allochthonous or in situ produced (primary production
by algae) autochthonous material (Pieczynska 1990). In the former case, the main
part of the external input consists of leaf litter like flowing waters in general.
Therefore, leaves colonized by terrestrial fungi often enter lakes via wind and
inflowing streams. Moreover, the high loads of airborne propagules by dry or wet
deposition also occur (Smirnov 1964) (see subchapters above). Reaching a lake
does not mean unambiguously that all transported species from lotic systems are
able to continue their activities in standing waters but represent merely an additional
energy source for local decomposer biota.
Pollen grains as allochthonous sources for lentic systems could be already colo-
nized by pollen-degrading fungi in air or on bark tissues, but their entering the lakes
results in a fungal replacement by aquatic fungi (like chytrids) (Skvarla and
Anderegg 1972). Wurzbacher et al. (2014) provided a detailed analysis on pollen
degradation by aquatic fungi in which the major fungal part consists of
Chytridiomycota beside the three other phyla (Ascomycota, Basidiomycota, and
Cryptomycota) with a majority of lower zoosporic fungi. The zoosporic fungi
[Chytridiomycota, Oomycota, and plasmodiophorids; only Chytridiomycota being
true fungi (Deacon 2006)] seem to have an aquatic evolutionary origin, developing
motile spores with a posterior flagellum of the whiplash type which liberate them-
selves from the sporangium due to their own motility in water (Ingold 1971; Money
and Fischer 2009) and dispersal capacity in the water column (Sparrow 1968).
Zoospore-like motile form is also present in Myxogastria (myxomycetes).
Depending on the environmental conditions, either a myxoflagellate or myxamoe-
bae bud from the spore. Myxoflagellates develop two flagella. One is usually shorter
than the other and occasionally only remnant. Myxamoebae move like amoebae—
that is, crawling on the substrate. The flagella function for locomotion to assist to
move close to food sources (Ingold 1971). Certain features of zoospores are impor-
tant from the point of view of dispersal: their lifetime, swimming amplitude, and
ability in the selection of suitable substrates. In average, zoosporic fungi may swim
from several hours to several days (Ingold 1971; Deacon 2006) with a speed of
about 0.25 cm min−1 (Ingold 1971) to 1.0 cm min−1 (Royle and Hickman 1964).
However, Deacon (2006) mentioned lower rates (0.006 cm min−1) for zoospores of
Oomycota. These numbers allow estimating that these organisms may colonize a
minimum of 3–4 m new environments during their lifetimes. Therefore, biological
advantage of motility might focus on the mechanism of escape spores from the
zoosporangium, and also there are some major advantages in selecting a substra-
tum, but comparatively little benefit in actual dispersal. As Ingold thought, the zoo-
spores of water molds are chemotactic based on the observation of Saprolegnia spp.
(Fischer and Werner 1958), which are highly attracted to small quantities of amino
acids and salts released by dead animals as their potential nutrient sources.
In the littoral zone of lakes, benthic community dominated by extended algal
mats could be very beneficial for saprobic and parasitic fungi, and their detached,
floating parts could be heavily colonized by fungi (Zopf 1884). Such biotic surfaces
like algal mats are often used as microhabitats for predatory fungi to capture nema-
todes, rotifers, or tardigrades (Sommerstorff 1911).
According to available knowledge, we may conclude that dispersal of aquatic
fungi in lakes is, similarly to the fungal dispersal strategies in lotic systems,
restricted to habitats where decomposing materials are concentrated [in lakes it
means littoral region (Pieczynska 1990)]. We may interpret this as “edge effect”—
an increase of biota abundances in ecotonal habitats (di Castri et al. 1988).
Despite cell numbers or biomasses of aquatic fungi are much lower in the open
water (Wurzbacher et al. 2010) than in littoral habitats, their role in matter and
energy transport might be important and described as mycoloop (Kagami et al.
2007) of aquatic food web (Sigee 2005). The role of saprobic or parasitic fungi with
zoospores establishes an association between filter-feeding zooplankton and large,
“inedible” algae (e.g., Asterionella) (Kagami et al. 2004). In this case, the individual
motility of zoosporic fungi allows fulfilling this ecological function in aquatic eco-
systems along with the successful fungi distribution in the open water (pelagic
zone). Therefore, there are important pathways in fungal dispersal carried out by
other biotas, focusing on parasitic fungi.
Beyond freshwaters, in marine zones approximately 500 fungal species have
been observed. Most of these species belong to Ascomycota or Mastigomycotina
and species from Basidiomycota are represented with low numbers. Their classifi-
cation as true aquatic (marine) fungi is strongly questionable. In groups of fungi
occurring in marine zones, similar tetraradiate spores could be detected as in fresh-
waters (Jakucs and Vajna 2003), but in contrast, the ascospores often have flakes of
wall material or mucilaginous appendages, which must be of functional signifi-
cance influencing sinking properties as the shapes give high surface tension to stay
afloat (Padisák et al. 2003; Rees 1980) in marine conditions (Deacon 2006). Most
of them are saprobic, but parasitic and symbiotic species have also been found. For
example, the spores of Gloeosporidina cecidii can easily colonize brown algae
species of Sargassum (Kohlmeyer and Demoulin 1981), but the parasitic fungus
(Cytospora rhizophorae) of red mangrove (Kohlmeyer and Kohlmeyer 1971; Wier
et al. 2000) and the Rhabdospora avicenniae living on black mangrove (Kohlmeyer
and Kohlmeyer 1971) are documented, and the mycological observation (Ananda
et al. 1998) of fungi growing on different animal substrates (like shells, fish
“bones”) in coastal zones is also worth to mention. There are detected species in
deep sea as well: Allescheriella bathygena in 1722 m and Periconia abyssa in 3975
and 5315 m of depths (Kohlmeyer 1977). Moreover, the surprisingly high fungal
activities in deep-sea hydrothermal ecosystems and its composition (in which
members are considered key organisms of terrestrial habitats) (Le Calvez et al.
2009) further confuse the patterns that we gained when trying generalize dispersal
mechanisms of fungi. However, the ocean currents probably are the major trans-
porting pathways in the case of microfungi as well as in marine systems.
Freshwater environments include all those sites where freshwater serves as the
main external medium, either in the liquid or frozen state. Frozen aquatic environ-
ments have long been considered as microbiological deserts, but studies found that
this is contrary (Sigee 2005). Ekelöf (1907) reported fungi from the Antarctica for
the first time. The Antarctic subcontinent, for example, is now known to be rich in
microorganisms (Vincent 1988). More than 20 species of microfungi including
Stachybotrys chartarum had been isolated from freshwater on the King George
Island, Antarctica (Kong and Qi 1991; Yu and Wang 1995). Most Antarctic environ-
ments get microbial propagules (including fungi) via air as result of spore traps
showed and reports of the microflora in Antarctic snow and ice or at geothermal
sites, with a high occurrence of cosmopolitans in most habitats (Pearce and Galand
2008; Ruisi et al. 2007). A majority of fungi reported from Antarctica are asexual
microfungi including both endemic and indigenous ones (Ruisi et al. 2007). Besides
the other biota, fungi are often locally abundant and interacting within highly struc-
tured communities. However, their dispersal patterns and origins have remained
largely unexplored.
Dispersal of fungi in tap water via plumbing system is also an emerging issue.
A wide variety of fungi is known to be common in wet indoor environments, as well
as in the drinking water resources [i.e., (Hageskal et al. 2007)]. Formation of tena-
cious and massive black biofilms was occasionally observed at the water–air inter-
phase of water taps and in associated habitats at several locations in Germany.
Exophiala lecanii-corni is the dominant component of these biofilms. A retrograde
route of contamination in case of E. lecanii-corni can be assumed (Heinrichs et al.
2013). Meanwhile in a German survey (Göttlich et al. 2002), the fungal flora was
dominated by species of Acremonium, Exophiala, Penicillium, and particularly
Phialophora. Some of them occurred throughout the entire drinking water system
and constitute a resident fungal flora in water supplies. It has been also concluded
(Short et al. 2011) that the plumbing systems serve as a significant environmental
reservoir of human-pathogenic fungi, like Fusarium. Fusarium oxysporum f. sp.
cucumerinum was detected with biofilm-forming capacity in a recent study as well
(Peiqian et al. 2014). Picioreanu et al. (2001) considered the detachment process is
due to internal stress created by moving liquid past the biofilm. Two biofilm detach-
ment processes, sloughing (large-biomass-particle removal) and erosion (small-
particle loss), were modeled. Simulations showed that erosion resulted in smoother
biofilm surface, while sloughing made biofilm surface rougher. Under similar
hydrodynamic conditions and biofilm strength, faster-growing biofilms lead to a
quicker rate of detachment than slow-growing biofilms. The experimental data
showed that detachment depends on both shear and microbial growth rates.
Instability in biofilm accumulation and rapid biomass loss (sloughing) were trig-
gered by high growth rates. High liquid shear, combined with low biomass growth
rates, can avoid massive sloughing. Biofilm patches filled the entire cavity where
they commence their growth, but they are not able to spread out the carrier peaks
and to completely colonize the substrate. Although most of the knowledge of bio-
film dispersal regards to bacteria, similar processes may distribute biofilm-forming

fungi in indoor wet environments.
Apparently some pathogenic fungi, especially fusaria, are able to disperse more
efficiently in other fluids than water as well, like sap inside living plants or animal
blood. Some pathogenic fungi are introduced into plants through lesions in stems,
twigs (Sieber et al. 1995), or roots. Small hyphal parts or spores might then be trans-
ported with the transpiration stream from root lesions to the leaves. Fusarium oxys-
porum infects healthy plants penetrating the plant’s root tips, root wounds, or lateral
roots by means of mycelia or by germinating spores. The mycelium grows intracel-
lularly through the root cortex and into the xylem. Once in the xylem, the mycelium
remains exclusively in the xylem vessels and produces microconidia. The microco-
nidia enter into the sap stream and are transported upward where the flow of the sap
stops and the microconidia germinate (Agrios 2006).
Fusarium verticillioides is a dimorphic fungus (Szécsi and Magyar 2011). In the
nature or in axenic cultures at 25–30 °C, it has a mold-like appearance while chang-
ing to a yeastlike form in rich culture media at 37 °C. There are several reports that
this fungus has been isolated from human and animal bodies associated with dis-
eases (Nucci and Anaissie 2007). In many cases, fungemia—the presence of a fun-
gus in the blood—is the only manifestation of the infection. The yeastlike form
diffuses more readily in human and animal bodies through blood and lymph sys-
tems than mold-like cells (Wang et al. 1999).
Dispersal by Plants and Substrate Particles
Plant seeds are efficiently dispersed. Therefore fungal particles on or in the seeds
will be distributed with equal effectiveness. Seed-borne fungi are of particular inter-
est to plant pathologists, as contaminated seeds are responsible for some plant dis-
ease outbreaks. Humans also have a huge influence over seed dispersal through a
number of effective and largely generalist dispersal vectors (Auffret et al. 2014).
Smuts, e.g., Tilletia and Ustilago species, infect the developing caryopsis
(“seed”) of grasses (Poaceae). Sori (bunt balls) are formed in the ovary of the host
plants, where hyphae of the pathogen replace the tissue of the young ovary, which
is then converted into a powdery, brownish-black mass of teliospores. Each dis-
eased “seed” (bunted kernel) can produce thousands to millions of teliospores.
Teliospores are often released when bunt balls are ruptured during harvest. However,
some of the bunt balls remain intact and are found among the harvested grains.
Teliospores are released from these grains during maintenance or processing.
Interestingly, teliospores of Tilletia caries are combustible, so explosions and fires
result from their ignition by sparks from mechanical harvesters (Wiese 1998). Such
teliospores are easily dispersed by wind. If impacting on the host plant and reaching
the sterigma of a healthy flower, the teliospores germinate and infect the ovary.
Because large-scale seed treatments as well as resistant cultivars are widely applied,
some smut species are less damaging nowadays than 50 years ago (Wiese 1998).
Transmission of endophytes is through seeds, or other vegetative propagules are

also known (see subchapter below regarding man-made dispersal).
Orchids have been found to associate with saprobic basidiomycetes of several
lineages, collectively named Rhizoctonia (as anamorph). These fungi may strongly
impact seed germination patterns of orchids (Oros et al. 2014). Orchid seedlings are
fully dependent on nutrients supplied by a mycorrhizal fungus. Its recruitment suc-
cess will be strongly influenced by the availability of a suitable fungal strain.
Mycorrhizal fungi have an aggregated distribution within the habitats (Jersáková
and Malinová 2007). However, in certain circumstances the mycorrhizal fungi are
likely to be distributed independently of the orchids (Feuerherdt et al. 2005).
Co-distribution model of the mycorrhizal fungi (Rhizoctonia solani) and orchid spe-
cies (i.e., Pterostylis acuminata) was observed in a laboratory experiment by Perkins
and McGee (1995).
Spores and mycelia adhering on detached particles of their substrate can be
transported as “hitchhikers.” Host plants or its tissues, fruits, etc. usually contain a
wide variety of free spores of different species, including pathogens. Puccinia psidii
(one of the most important pathogens of Eucalyptus and other Myrtaceae) can be
spread mainly by windblown fungal spores, but infected rooted cuttings, seedlings,
pollen, and other host tissues increase the risk of its introduction to rust-free coun-
tries as well as of spreading new pathogen lineages (Lana et al. 2012). Strong dis-
turbance increments the drift of colonized substrate particulates as vectors (Pasanen
et al. 1991). Plant debris carrying F. verticillioides are abundantly aerosolized dur-
ing the operation of harvesters (Magyar et al. 2011). Once airborne, large particles
may be carried by the wind to a distance of 300–400 km from the source (Ooka and
Kommendahl 1977).
Joint dispersal is prevalent in diverse symbioses and a predominantly common repro-
ductive mode in lichens (Wornik and Grube 2010). Lichenized algae can be dispersed
together with their fungal partners in asexually produced organs, either produced directly
on the vegetative thallus or in specialized regions. It is an efficient approach to retain
successful associations and to circumvent low symbiont availability.
Dispersal by Animals
In most cases, the dispersal agents are animals, from arthropods and gastropods to
mammals and birds (Castellano et al. 2004). There are numerous fungus–vector
associations, ranging from almost incidental associations to highly evolved mutual-
ism (Deacon 2006). Associations between fungi and insects are among the most
species rich, diverse, and complex in terrestrial ecosystems. Spores dispersed by
insects are recognized in many groups of fungi, including Ascomycota,
Basidiomycota, anamorphic fungi, and zygomycetous fungi (Kendrick 1985; Ingold
1953), as well as in the myxomycetes (Stephenson and Stempen 1994). Nevertheless,
the role of fungivorous insects as spore vectors has been poorly documented
(Schigel 2012). The adaptations in various fungal groups as a result of selection for
arthropod dispersal are discussed in Abott’s article (Abbott 2002). The significance
of insects as vectors of fungal phytopathogens has frequently been undervalued

(Agrios 1980). Compared to the general postulation that wind transport is the prin-
cipal mode of dispersal of fungi, Kluth et al. (2002) showed that the occurrence of
spores dispersed by insects may also effectively spread the pathogen to isolated
weed stands (alternative host) (Morrison et al. 1998). The unique adaptations and
dispersal abilities of both fungi and insects result in a variety of interactions (Schigel
2012). Interactions between animals and fungi, such as attraction, mycophagy,
mutualism, and parasitism, are occasionally documented, but some of them have
high ecological or economic impact. Specialized entomophilic species are far less
common in the kingdom of Fungi than in plants. Spores with adhesive or echinate
surface can spontaneously attach onto or trapped into animal hairs.
Many lichens are dispersed by animals since soredia adhere electrostatically to
the cuticle of invertebrates [insects, spiders, mites; (Stubbs 1995)] or to the feathers,
fur, and extremities of vertebrates. Short- and long-distance transport is also pro-
vided by animals which use lichen fragments to camouflage either their own body
(insects) or their nests [birds, squirrels, (Gressitt 1965; Richardson 1975; Gerson
and Seaward 1977; Scharf 1978; Seyd and Seaward 1984; Brodo et al. 2001;
Allgaier 2007)].
Masses of spores and sticky sap droplet exuded form pycnidial and perithecial
cirrhi or often on the top of an elongated stalk are carried away by arthropods pass-
ing through colonies. Microfungi such as Gliocladium, Graphium, Leptographium,
Myrothecium, Pesotum, Stilbella, and Stachybotrys develop complex conidiophores
and utilize this dispersal mode (Ingold 1953; Abbott 2000; Seifert 1985; Upadhyay
1981; Wingfield et al. 1993). These relatively large, complex structures develop
vertically from the substrates and are tall enough to hitchhike onto large insects
moving over the surface.
Other microfungi, including Acremonium, Fusarium, Gliomastix, Trichoderma,
and Verticillium, produce large numbers of droplets at the apex of conidiophores
(Ingold 1953; Carmichael et al. 1980). The conidiophores are developed in different
orientations throughout the mycelium and are particularly efficient to touch small
insects traveling through a mycelial mass. The genus Cephalotrichum develops its
asexual spores in a dry head at the apex of a synnema. These synnemata are fre-
quently up to 1 mm high and are produced at a right angle to the substrate surface.
The fused hyphae of the synnemata offer resilience and spring back into the upright
position when gently manipulated in the laboratory (Abbott 2000). As the insects
are in motion through a sporulating colony resembling miniature forest and brush
against the synnemata, small clouds of conidia are released, effectively dusting the
insect with conidia. This differs from the “paintbrush” method used by the
slimy-spored synnemata of fungi, such as Graphium. Some myxomycetes (slime
molds) may develop spores in similar stalked structures (e.g., Stemonitis) and are
dispersed by tiny slime mold beetles (e.g., Anisotoma, Agathidium), which feed on
the fructifications of the slime molds (Stephenson and Stempen 1994). Ing (1967)
reports that woodlice distribute myxomycete spores. The sporocarps of Enteridium,
Tubifera, and Fuligo commonly provide habitat for beetles and several other biotas
(members of Anisotomidae, Leiodidae, or Agathidiidae). Their spores can be found
in the fecal pellets of these animals. Therefore, they play a major role in the disper-
sal mechanism of myxomycete fungi. A number of fungi have developed a strategy

to attract animals when spores are mature and lead the animals to consume the
spores. The attractants are volatile organic compounds, including pheromones
(Claus et al. 1981).
Komonen (2008) studied the abilities of Ciidae, the minute tree fungus bee-
tles to colonize Trametes, and the results show that the beetles are able to fly up
to 1.5 km toward the odors of fruit bodies of Trametes. The fungal spores are
often covered by sugary matter with attractive odors to attract insects (Webster
1980). For Phallus and Mutinus, a strong fetid odor is produced to attract flies
to feed on the glebal surface of the fungi. The basidiospores adhere to the legs
and bodies of the flies, and the insects may deplete the entire slimy basidiospore
layer in several hours (Abbott unpublished). The spores are transported to adja-
cent sites by the flies and are excreted, relatively intact (Ingold 1965). The
insects are vectors for some fungi as they carry spores in the wound site. Some
fungi from leaves eaten by invertebrates survive passage through the digestive
tract (Bärlocher 1981). Endophytic fungi colonize plant tissues without causing
symptoms of disease and live cryptically in all higher plants. Endophytes which
are once deposited on the leaf surface might easily infect the plant through
openings that are caused by herbivores. There are indications that endophytes
can be dispersed by herbivores. For instance, when the infected herbivore
migrates to new feeding places, endophytes such as the entomopathogenic
Beauveria bassiana could be transferred (Vega et al. 2008). Grasshoppers or
other phytophagous insects could serve as an agent for the dispersal of non-
grass fungal endophytes (e.g., Colletotrichum gloeosporioides) in plant com-
munities such as tropical forests. It was shown that endophytic fungi can pass
the gut of grasshoppers without being destroyed and can be dispersed in this
way (Devarajan and Suryanarayanan 2006).
In plant-pathogenic fungi, insect transmission of spores is extensively studied.
It is necessary to possess some knowledge of vector behavior for reliable epidemio-
logical predictions (Dye 1986). For instance, patchy distribution of hosts and
aggregation of vectors within patches cause an increase in parasite’s basic reproduc-
tive rate which is predicted by random host–vector contact models. Conditions for
steady coexistence between parasites and hosts, as well as the equilibrium frequency
of infection in host populations, are dependent on the degree of feeding preference
of vectors for either healthy or diseased hosts. Different vector species may exhibit
different efficacies of transmission due to differences in morphology or behavior
(Webber 1990). Therefore, in order to better understand the epidemiological conse-
quence of the dispersal of a vector-borne pathogenic fungus, it is important to inves-
tigate the mechanisms of transmission efficiency and the interactions among
vectors, pathogens, and hosts (Shykoff and Bucheli 1995).
Microbotryum violaceum (Pers.) G. Deml and Oberw. is a causative agent of the
anther smut disease common in members of the pink family (Caryophyllaceae).
Flower visitors that usually serve to pollinate flowers can transfer infectious spores
from diseased to healthy plants (Fig. 14.6a). The female flower of the infected dioe-
cious plant develops anther-like structures filled with spores instead of pollen grains.
Fig. 14.6 Silene acaulis infected by Microbotryum violaceum. (a) Purple spores are produced in
anthers (photo courtesy from Michael Hood). (b) Insect-attracting pseudoflowers on Arabis sp.,
caused by Puccinia monoica (photo courtesy from Michael Wood)
Flowers of infected plants last significantly longer than those of healthy plants,
probably because the infection strengthened floral organs, such as the flower base
and the anther filaments (Uchida et al. 2003). Pollinators prefer plants with large
floral displays and also prefer males to females and healthy to diseased plants
(Shykoff and Bucheli 1995). Male plants consistently produced nectar with higher
sugar concentration, thereby offering higher-quality floral rewards than either
females or diseased plants. It was suggested that more attractive plants may be pre-
disposed to infection since pollinating insects also serve as vectors for this fungal
disease. In a field experiment, those plants that became infected produce larger
numbers of flowers than those healthy individuals (Thrall and Jarosz 1994).
Pollinators visit more healthy plants after an “accidental” visit to a diseased plant
than they might if visiting plants at random (Real and Rathke 1991).
Puccinia monoica Arthur is a rust fungus, which inhibits flowering in its host
plant, Arabis. The fungus causes the plant to produce pseudoflowers, which attract
insects to spread the rust spores. These pseudoflowers attract insects not only by
visual means but also by producing a pungent fragrance and exuding a sugar-rich
solution (Raguso and Roy 1998).
The dispersal of plant pathogens by adult shore flies was found to be aerial vec-
tors by an experiment for three microfungal pathogens: Fusarium oxysporum f. sp.
basilica, Thielaviopsis basicola, and Verticillium dahliae. Adult shore flies are
attracted to sporulating cultures and infected plant tissues. Shore flies acquired fun-
gal conidia and other structures both by feeding and physical contact. The minimum
acquisition time for soilborne fungi was 10–20 min. Acquisition incremented with
time to reach 100 % frass deposits infestation following 2 h of acquisition. Pathogens
dispersed by adult shore flies were fast over time at 2.21 cm2/h/insect. The dispersal
area by adult shore flies incremented with the lengthening in exposure time
(El Hamalawi 2008).
Claviceps conidia were found on a range of insects, including moths, flies, leafhop-
pers, Orius spp., beetles, midge flies, head bugs, hymenopterous insects, and thrips.
Such conidia are carried by up to 100 % of moths and 75 % of flies collected from
some fields (Butler et al. 2001). However, this fungus uses an arsenal of strategies to
provide successful spore dispersal. The anamorph of these species (Sphacelia) pro-
duces three different types of single-celled, hyaline spores (micro-, macro-, secondary
conidia). An ergot kernel develops when a spore of Claviceps sp. infects a floret of
flowering grass or cereal. The infection process is similar to a pollen grain growing
into an ovary during fertilization. Infection requires that the spores of the fungal
pathogen have access to the stigma, and as a result plants infected by Claviceps are
primarily outcrossing species with open flowers. The growing mycelium subsequently
destroys the host ovary and connects with the vascular bundle originally developed for
seed nutrition. The first stage of ergot infection develops a white soft tissue (known as
sphacelia) exuding sugary honeydew, which contains millions of macro- and micro-
conidia (Fig. 14.7). Macroconidia are immersed in a nearly clear sugary liquid which
takes on an opaque, yellow-brown, or orange to pink color because of the conidia. The
syrup-like honeydew is exuded onto the surface of the sphacelia and continues to drip
down across the sorghum head and onto other plant parts and the soil surface. The
macroconidia have limited capacity to spread because of their occurrence in a sticky
liquid environment. When exposed to high relative humidity (>90 %), the hygro-
scopic honeydew droplets acquire an increased water content, which stimulates ger-
mination of the macroconidia from which germ tubes extend and penetrate the
honeydew surface that function as sterigmata, ending in apical secondary conidia that
are easily disseminated by wind (long-distance spread) and/or fall onto soil where
secondary conidia can also infect plants and act as primary inoculum for disease ini-
tiation. Moreover, honeydew containing conidia may adhere to farm personnel and
equipment leading to the dispersal of the fungi from field to field. Honeydew in con-
taminated seed lots is also easily transferred, causing rapid uncontrollable spread.
Although insects as vector of the conidia of C. purpurea is known, they may not play
a significant role as water splash or wind in spreading sorghum ergot. The teleo-
morphs of Claviceps species produce long, filiform ascospores, which utilize passive
dispersal pathways by wind and rain (Bandyopadhyay et al. 1998).
Thrips obscuratus is capable of carrying conidia of Botrytis cinerea on its body.
Adult thrips artificially contaminated with B. cinerea had most conidia on the head,
thorax, legs, and the abdominal distal segments; few were found on the wings. The
conidia were observed most frequently in sculptured areas and intersegmental regions
or trapped under setae, but very few on the smooth areas. This distribution pattern
suggests that adhesion is mechanical. Up to 17 % of adult thrips sampled were natu-
rally contaminated by the fungus, and up to 12 propagules per contaminated thrips
were found. Field infestation of kiwifruit by T. obscuratus was shown to increase the
susceptibility of kiwifruit petals to B. cinerea (Fermaud and Gaunt 1995). Fungal
pathogens of insects (e.g., Entomophthorales) have also adapted to dispersal from one
individual to others within an insect community (Pirozynski and Hawksowrth 1988).
Fig. 14.7 (a) High severity of ergot (Claviceps africana) with abundant production of honeydew
containing large amounts of conidia. Opacity of honeydew is due to the high content of macroco-
nidia. (b) Secondary conidia production as the whitish area at the upper ends of the ergot droplets.
(c) White secondary sporulation on the surface of honeydew that dripped onto the sorghum leaf
from sphacelia in the sorghum head above. (d) Sporulation of C. africana on soil surface where
honeydew dripped. Courtesy from G. Odvody. Reproduced with permission from APS Feature
(Odvody et al. 1998)
Some birds and mammals (mouse and shrew) are also proved to be vectors of
plant-pathogenic fungi, e.g., in transporting Cryphonectria parasitica (chestnut blight
pathogen) in mixed hardwood forests (Scharf and DePalma 1981). Birds are visually
attracted to some fungi. Mycophagy by birds is also observed many times and well
reviewed (Bailey 1904; Simpson 1998, 2000). However, Simpson (2000) marveled
the reason why the fungi, regarding to its wide availability and nutrient benefits
Fig. 14.8 (a) Douglas squirrel (Tamiasciurus douglasii) feeding on and transporting a truffle
(photo courtesy from Bernie Krausse). (b) Paurocotylis pila imitating fruits of Podocarpus spp.
that are consumed by birds (photo courtesy from Clive Shirley)
(Cork and Kenagy 1989), are not utilized by more birds than observed in total. In
New Zealand, which lacks native mammals, birds appear to be important vectors of
sequestrate fungi. Paurocotylis pila develops a scarlet peridium. As ascomata of this
fungus enlarge, the ascomata are elevated to the surface of the humus, occasionally
detaching completely and positing loose on the surface. Their size and color imitate
fruits of nearby Podocarpus spp. and other plants that are consumed by birds
(Fig. 14.8b). In addition, P. pila matures at the same time as the fruits of Podocarpus.
Paurocotylis ascomata on the forest floor among podocarp fruits are almost surely
ingested by birds (Castellano et al. 1989). Picoa lefebvrei in deserts of the Arabian
Peninsula and North Africa produces different visual signals. Several to greater than a
dozen small ascomata congregate to form a distinct hump on an otherwise flat desert
floor. Birds find these humps, scrape away the covering soil, and ingest the ascomata
(Alsheikh and Trappe 1983). All sterile fungal tissues are digested, but the ascospores
pass through the digestive systems undamaged. As feces containing ascospores dete-
riorate with age, the ascospores are released into the soil and potentially contact feeder
roots of receptive mycorrhizal hosts (Trappe and Maser 1977). According to the first
two present authors’ unpublished observations, the songbirds have high importance in
dispersal of tree bark-inhabiting and plant-pathogenic fungal spores and mycelia by
their movements (Vass and Magyar unpublished).
Hypogeous Fungi
Besides insects and other arthropods, mammals are also often utilized, especially by
hypogeous fungi to move their spores from the site of growth and production to new
substrata for colonization. Such fungi lack mechanisms of spore discharge to the air
(Fogel and Trappe 1978). Loss of forcible discharge of spores to air must be accom-
panied by mutations that adapt sequestrate fungi to other spore dispersal tactics.
Spore dispersal of hypogeous fungi including gourmet fungi, truffles, is dependent
exclusively on animals. The role of mycophagous small mammals to disperse hypo-

geous fungi is well established (Urban et al. 2012).
Such fungi (and other macrofungi) are part of the diet of small rodents (Rodentia),
such as mice (Murinae, Muridae, Myomorpha), dormice (Gliridae, Sciuromorpha),
voles (Arvicolinae, Cricetidae, Myomorpha), and squirrels (Sciuridae, Sciuromorpha)
(Maser et al. 2008) and insectivorous shrews (Sorex spp., Soricidae, Eulipotyphla)
frequently feed on hypogeous fungi (Kataržytė and Kutorga 2011; Schickmann et al.
2012). However, at present no information is available on whether spore dispersal by
small mammals contributing to productivity is analogous to the relevance of pollina-
tion in fruit orchards (Urban et al. 2012). Mycophagy can also affect fungal diversity
within their home ranges by making sure the unremitting and efficient dispersal of
spores from one site to others. Movement of bush rats (Rattus fuscipes) transports
fungal spores among different fungal communities. These animals have the potential
to affect the structures of vegetation communities via spore dispersal (Vernes and
Dunn 2009). The significance of small mammals as consumers and dispersal agents
of mycorrhizal fungal spores in tropical and temperate ecosystems had been reported
in several studies. A study strongly suggests that the subterranean rodent Ctenomys
cf. knighti may play an important role as a dispersal agent of arbuscular mycorrhi-
zae and pigmented septate endophytic fungi (Fracchia et al. 2011). Rodent dispersal
of fungal spores also promotes seedling establishment away from mycorrhizal net-
works in host plants (Frank et al. 2009).
Mycophagous mammals excavate and consume fruiting bodies of hypogeous
ectomycorrhizal fungi and discharge excrement which contains numerous spores.
However, the spores passing through vertebrates’ digestive systems can influence
the viability and activity of the spores consumed (Schickmann et al. 2012; Castillo-
Guevara et al. 2011, 2012). Passage through the digestive system of flying squirrels
(Glaucomys sabrinus) may improve germination and inoculation potential of
spores, while active mycelia in forest soils may be the most important and efficient
way for seedlings to develop mycorrhizae under natural environments (Caldwell
et al. 2005).
The hypogeous and epigeous fungi ingested by red squirrels (Sciurus vulgaris)
in subalpine conifer forests in the Alps were studied to determine the presence and
taxa of fungal spores in excrement samples. Almost all live-trapped squirrels had
fed on fruit bodies of hypogeous fungi in summer and fall, but only some red squir-
rels had ingested epigeous fungi, Boletus and/or Laccaria. It seems that fruit bodies
of hypogeous and, to a lesser extent, epigeous fungi provide an important seasonal
food resource for red squirrels in conifer forests in the Alps. Squirrels which have a
large home-range size and long dispersal distances perhaps play a key role to
disperse spores for hypogeous fungi (Bertolino et al. 2004; Teron and Hutchison
2013, Fig. 14.8a).
Piattoni et al. (2012) investigated the significant role of wild boars in dissemina-
tion of hypogeous fungal spores. They have found poor abundance of spores in
boars’ feces which suggested that the wild boar can be considered an opportunistic
mycophagist, but due to the movements of wild boar during seasonal migrations,
wild boar could have an important role though in truffle long-distance dispersal.
Mycophagists can indirectly affect vegetation succession by dispersing spores and

other reproductive structures of mycorrhizal fungi, thus helping the distribution and
regeneration of mycorrhizal plant species (Bruns 1995; Wiemken and Boller 2006).
Maser et al. (2008) postulate that growth, regeneration, and adaptation of the mycor-
rhizal fungi–tree network would be greatly impaired, if not impossible, without
dispersal of spores of hypogeous fungi by animals. The distributions of spores of
microfungi were studied in soil litter and on the fur of small mammals (Sorex ara-
neus, S. caecutiens, S. minutus, and Clethrionomys glareolus). There are 156 spe-
cies of microfungi reported on the animal fur. Fungal spores were observed to attach
to hairs with mucus or warty walls under electronic microscopy. The composition
of spores isolated from the fur of the mammals and the soil litter was significantly
different and had a seasonal variation. The microfungi peculiar to the fur were dif-
ferentiated. All the studied species of small mammals are different in the composi-
tion of microfungi on their fur. The spore composition can be used as a marker for
studying ecological niches of animals and fungal dispersal. The differences in the
spore compositions on the fur increment with the distance between habitats of the
animals (Shchipanov et al. 2006).
Transportation of an immense number of Gorgomyces by nematodes in soil litter
was observed using apparently specialized mucilaginous appendages on the spores
[(Gönczöl and Révay 1985), Fig. 14.13].
A number of Basidiomycota produce resupinate (crust-like) basidiomata buried
in the soil. The basidiospores of these fungi are actively discharged, but it seems that
they are often not well adapted for aerial dispersal. Tomenlella sublilacina, a wide-
spread ectomycorrhizal fungus, sporulates in the soil organic horizon and can estab-
lish from the spore bank in a short time following disturbance. Gut contents of
centipede and feces of centipede and salamander contained many apparently intact
spores of this fungus. These results showed the potential for T. sublilacina spore
dispersal by arthropods (mites, springtails, millipedes, beetles, fly larvae) and their
predators (centipedes, salamanders) in soil food webs and might assist to elucidate
the widespread distribution of this fungus. It is possible that this is a common mech-
anism of dispersal for fungi developing resupinate basidiomata in the soils, signify-
ing a necessity to better understanding of the linkages of soil food webs and spore
dispersal (Lilleskov and Bruns 2005) by arthropods and vertebrate predators.
According to their scanning electron microscopic (SEM) studies, the role of
some beetle species (Leiodes cinnamomea, Agaricophagus cephalotes, Colenis
immunda, Agaricophagus reitteri) in spreading of the fungus, burgundy truffles
(Tuber uncinatum), was documented. Large numbers of fungal spores were found
adhering to the underparts and legs of adult beetles feeding on truffles. Additionally,
SEM revealed no spores on flies hatched from the fruit bodies of truffles, thus
excluding their role in spreading the fungus (Bratek et al. 2010).
Each sequestrate (truffle-like) species produces its unique range of aromas, often
a mixture of a number of compounds (Marin et al. 1984). Immature fruiting bodies
have little or indistinctive odor. When spores start to mature, the attractant com-
pounds are developed, and as more spores mature, the aroma increments in pun-
gency and intensity (Trappe and Maser 1977).
The results of Varga and Naár (2002) supported the assumption that Collembola
species have an important role in the distribution of fungi living in bryophytes as well
as in the distribution of the ones living in the soil. According to the results of Dromph
(2001) who had observed the dispersal mechanism of insect-pathogenic fungi by
three different Collembola species (Folsomia fimetaria, Hypogastrura assimilis, and
Proisotoma minuta), the fungal propagules in the feces of their carrier species could
preserve their viability. Thus, their dissemination is supported by the consumer
(Collembola) biota, establishing these fungi in new habitats (Williets et al. 1989).
Mutualism
Mutualistic relations between fungi and insects also exist. Leaf-cutter ants (e.g.,
Atta and Acromyrmex) cultivate fungus gardens in subtropical and tropical Americas.
The ants carry and maintain a selected fungal species (e.g., Leucoagaricus) to inoc-
ulate the piles of harvested leaf pieces to provide a food source for their larvae
(Fisher et al. 1994; Wheeler 1907). Other fungi (e.g., Termitomyces) are associated
with termites (e.g., Termes) in Africa and Asia (Wheeler 1907). Ophiocordyceps
species known as “zombie ant fungi” control the ant hosts by inducing a behavior of
anchoring biting (Fig. 14.9; Hughes et al. 2011). The fungi emit a mixture of
behavior-controlling chemicals when encountering the brain of its natural target
host, but not when infecting other ant species (de Bekker et al. 2014). These fungi
infect many insects, and the species that infect ants induce hosts to die attached by
their mandibles to plant material to provide a platform from which the fungus can
Fig. 14.9 The “zombie ant” dispersing pathogenic Ophiocordyceps spores. Photo: David Hughes,
with permission
develop and actively discharge spores to infect other ants. The jet ant, Lasius fuligi-
nosus, cultivates Cladosporium myrmecophilum, which functions to provide stabil-
ity to the walls of its carton nest (Fig. 14.10). The spores of this fungus (and those
of many other species) are carried inside the mouth of the ant and ingestion is pre-
vented by a filter-like organ (Magyar and Babinszkyné Nagy unpublished).
Fig. 14.10 a: Jet ant (Lasius fuliginosus, picture courtesy from Kenneth Ervik), b: infrabuccal
pocket of the ant where fungal spores are transported [redrawn after Gotwald (1969)], c–b’: spores
found in the infrabuccal pocket (Magyar and Babinszkyné Nagy unpublished). c–f: Cladosporium
myrmecophilum (the ant’s carton nest fungus), g: Cladosporium sp., h–j: Oncopodiella guamensis,
k–m: Oncopodiella trigonella, n: unknown basidiospore, o–r: unknown species, s: Alternaria sp., t:
Ellisembia sp., u–b’: Corynespora sp.
Xyleborus spp. (ambrosia beetles), bark borers, cultivate Ambrosiella spp., the
ambrosia fungi to feed their larvae and adults. The ambrosia fungi are transported
in mycangia (specialized pockets) and are an essential part of beetle brood galleries
(Wheeler 1907; Cassar and Blackwell 1996). Most blue-stain fungi or lumber molds
(Ophiostomatales) are well documented to be dispersed by insects, and such disper-
sals associate with bark or ambrosia beetles. Ophiostomatoid fungi frequently colo-
nize the wood in the galleries of bark beetles. Ascospores are developed in wet
spore masses at the apices of the perithecial necks in Ophiostoma, Ceratocystis, and
Sphaeronaemella. The long-necked ascomata and long-stalked conidiophores stick
out into the insect passing routes and efficiently force the insects to touch the spore
masses as they pass through the restricted spaces and to pick up spores (Upadhyay
1981; Wingfield et al. 1993).
Bark beetles, Dendroctonus and Ips, are well documented for their roles in dis-
persal of the ascospores and the conidia of blue-stain fungi (e.g., Leptographium
and Pesotum) (Upadhyay 1981; Wingfield et al. 1993). One classic example is the
dispersal of Dutch elm disease caused by Ophiostoma ulmi and O. novo-ulmi by
specialized bark beetle vectors (Scolytus and Hylurgopinus). These pathogens
enter the plant via wounds chewed by bark beetles and then translocated in the
xylem vessels by developing in a yeastlike budding phase. It results in reactions in
the xylem vessels to lead to obstruction and death of part or all of the xylem. The
disease cycle commences when pathogen-carrying beetles emerge from the bark of
dead or dying elms in early spring, fly to adjacent healthy trees, and eat the bark of
the young shoots. The bark beetles damage the xylem during feeding, so to infect
the elms with the pathogens. Then the fungal pathogen spreads in the xylem, result-
ing in the death of the whole tree or some of its major branches. The bark of the
newly died elms is then targeted by the female beetles to oviposit. The female
beetle bores into the inner bark and chews out a channel, oviposits along its length
of the bark beetle gallery. The eggs hatch and the young larvae make a series of
radiating channels by feeding prior to pupating for overwintering. At the same
time, the pathogen develops from the xylem into the bark and produces spores in
the tunnels. In the following spring, the adult beetles emerge from the pupae and
are covered by spores. The adult beetles fly away from the bark and search for new
elms. The disease cycle repeats.
The southern pine beetle (Dendroctonus frontalis) has two mycangia, separated
by a sclerotized mycangial bridge, suggesting that each mycangium functions inde-
pendently. Mycangia are surrounded by abundant tracheoles connecting the struc-
tures to outside via openings within the prothorax. It was hypothesized that these
openings may play roles in determining the species of fungi entering to and growing
in the mycangium (Fig. 14.11) (Yuceer et al. 2011).
It has been reported that myxomycete spores are stored in the pits in the mandi-
bles of Sphindidae, an entirely myxomycophagous family, and the venter of slime
mold-feeding latridiid species.
There is a hypothesis that a loose mutualistic association is present between dust
mites and fungi. It is beneficial to the mites for feeding on animal (or human) skin
scales colonized by fungi, and in return it benefits the fungi since spores are dis-
Fig. 14.11 (a) The southern pine beetle (Dendroctonus frontalis, picture courtesy from the USDA
Forest Service Archive, USDA Forest Service, Bugwood.org). (b) Locations of the mycangia. (c)
Spore-like fungal cells (arrows) in the mycangium (b, c courtesy from Cetin Yuceer)
persed by the mites. Conidia are moved on the mite cuticle, but the spores are con-
centrated within the feces of the mites as well (Fig. 14.12). Spores survive passage
through the digestive tracts and able to germinate in the fecal pellets, which serve as
a sufficient substrate to the fungi to develop hyphae and conidiophores without any
other food sources (Colloff 2009). A mutualistic relationship between Acarus siro
and the molds and yeasts that colonize the wheat endosperm is hypothesized by
Levinson et al. (Levinson et al. 1991). The mite is apt not to feed on the endosperm
unless it becomes moldy. The fungi produce ammonia from broken and sprouted
wheat as a metabolite. Ammonia is a rather efficient attractant for colonizing mites
(kairomone effect). Also, the guanine in the mite feces is solubilized by ammonia to
Fig. 14.12 The dispersal of mold spores by feeding dust mites [redrawn by Magyar after Colloff
with permission (Colloff 2009)]
attract opposite sex for copulation (pheromone effect). The mites mate and dis-
perse, distributing the fungal spores via their digestive tracts. Shed human dander
is colonized by Aspergillus glaucus group and their lipids are broken down to form
free fatty acids. One species of Fungitarsonemus (Prostigmata, Eleutherengonides),
common foliar mites in tropical areas, releases an adherent layer to allow spores and
pollen grains to adhere as an additional layer (perhaps tactile or visual camouflage).
However, their role in fungal dispersal remains poorly understood. Mites are also
involved in dispersal of a broad range of coprophilous fungi (Malloch and Blackwell
1992) and several myxomycetes (Keller and Smith 1978). Fungal mites (e.g.,
Tyrophagus) ingest the mycelia and spores. Fecal materials of insects and mites
often contain fungal spores, and these spores often appear intact and undamaged by
passing through the digestive systems of arthropods (Abbott, unpublished). The
effects of panphytophagous oribatid mites on the recovery of the microbial com-
munity in partially decomposed litter layer from forests after strong disturbance
(freezing and heating) were investigated in a laboratory microcosm experiment.
Generally, oribatid mites enhanced the recovery of the disturbed systems by accel-
erating the recolonization of litter materials by fungal species and the recovery of
the microbial metabolism by dispersal of spores and by grazing on microbial popu-
lations (Maraun et al. 1998).
Mutualism between snails and microfungi has also been observed. Snails
(Littoraria irrorata) graze grass primarily not to feed but to prepare substrate for
growth of Phaeosphaeria and Mycosphaerella spp. and consume these invasive
fungi. However, unlike ants and termites, snails do not seem to carry spores to inoc-
ulate prepared substrate to initiate fungal growth (Silliman and Newel 2003).
Coprophilous Fungi
Coprophilous fungi use the dung of herbivores as substrates. The mechanisms

of spore dispersal of these fungi guarantee that the spores are propelled from
the dung onto the nearby vegetation (mostly by ballistic mechanisms), where
the fungi will be fed on along with plant materials and pass through an animal
digestive system to repeat the cycle. The coprophilous ascospores are dispersed
by a number of dispersal mechanisms from the dung to the nearby vegetation.
The ascospores are often covered by mucilage or have gelatinous appendages
for attaching to the plant parts on which they land without difficulties (Wicklow
1981). Herbivores eat the ascospores that frequently are darkly pigmented and
well protected against both gastric juices and the UV light from the sun. Spore
germination can even be triggered by gastric juices of the herbivores (Webster
1970). Basidiobolus ranarum (Entomophthorales) growing on the feces of liz-
ards and frogs employs a different discharge mechanism. The sporangium of this
species is developed on a subsporangial vesicle, when the vesicle ruptures at its
base, jetting the sap backwards to propel the sporangium forwards, like a rocket.
Pilobolus, the hat-thrower fungus, is known for their explosive spore dispersal.
Pilobolus has to pass through the digestive systems of grazing animals in their
life cycle. Since the animals circumvent foraging near their dungs, Pilobolus
uses a phototropic (light-following) squirt gun mechanism to propel their spo-
rangia up to 3 m away onto uninfested vegetation. The ballistic discharge of
Pilobolus may reach speeds of up to 90 km/h due to the release of highly pressur-
ized fluids from the sporangiophore stalk after the rupture of the subsporangial
vesicle. The sporangia have a sticky mucilaginous ring for adhering to vegetation
when wetted by the propelling fluids. Once ingested, the spores pass through the
digestive systems undamaged and are deposited into a fresh pile of dung, thus
Fig. 14.13 Nematode carrying Gorgomyces conidia. Photo by Ágnes Révay, with permission
continuing the asexual life cycle. Sphaerobolus stellatus, the cannonball fungus
(Basidiomycota), develops basidiospores in a large ball-like structure within a
cup-shaped basidioma. At maturity, the inner layer of the cup separates from the
outer layer and suddenly inverts, like a trampoline, forcibly ejecting the spores
containing the ball into the air (Ingold 1971).
There are more than 40 species of bioluminescent fungi (Weitz 2004). This phe-
nomenon may raise the role of luminescence for attracting invertebrates to assist
fungal spore dispersal (Sivinski 1981, 1998).
Aquatic animals also represent main keys in dispersal of aquatic fungi (amphib-
ians—chytridiomycosis caused by Batrachochytrium dendrobatidis; fish—some
species of the fungus-like organism called Oomycetes; zooplankton—Microsporidia
species on copepods) in lakes.
Man-Made Spore Dispersal
Human activity also plays an important role in spore dispersal. High amount of fungi
are aerosolized worldwide by man-made kinetic energy, e.g., by harvesting (Magyar
et al. 2012; Skjøth et al. 2012), or demolition of moldy buildings (Bouza et al. 2002).
Spore dispersal by man-made thermal energy, especially in heat production by indus-
trial composting, is also remarkable (Sebők et al. unpublished). The effect of these
human activities can be intensive and drastic and often higher than energy require-
ment for the natural fungal spore liberation, either passive or active.
An example for the unintentional dispersal of a pathogen by humans is the global

emergence and spread of the pathogenic, virulent, and highly transmissible fungus
Batrachochytrium dendrobatidis, belonging to zoosporic aquatic fungi. It has
caused the decline or extinction of up to about 200 species of frogs, resulting in the
disease chytridiomycosis (Fisher et al. 2009; Skerratt et al. 2007). In the distribution
of this fungus, human activities like visiting waterbodies one by one (regional scale)
or human transport (continent scale) also have huge relevance and impact (Skerratt
et al. 2007).
Spores of microfungi are also intentionally dispersed by human activity, e.g., in
fermentation and food industry, research laboratories, and some fields of mycotech-
nology (Magyar 2007; Samson 2010). Open-field cultivation of microfungi is rarely
in practice, since inoculation may pose a risk in agriculture. Some exceptions are
corn smut, ergot, and Jiaobai smut [Ustilago esculenta infecting Zizania latifolia as
a vegetable (Chan and Thrower 1980; Terrell and Batra 1982)] cultivation.
Corn smut is a delicacy in Mexico and is known as huitlacoche there. It is pre-
served and sold for a considerably higher price than uninfected corn. Corn smut
consumption originates from Aztec cuisine. For culinary use, the immature galls are
harvested. As fully mature galls are dry and almost entirely full of spores. In the
mid-1990s, due to demand from high-end restaurants, farms in Florida and
Pennsylvania were permitted by the United States Department of Agriculture
(USDA) to purposely infect corn with huitlacoche. The initiative is still under way
(Pataky and Chandler 2003).
Jiaobai (or gau sun) has been widely cultivated as a vegetable for several centu-
ries in China (Terrell and Batra 1982). When Zizania latifolia is infected by Ustilago
esculenta, its cum apex becomes swollen forming a juicy stem gall and contains
smut mycelia (Chan and Thrower 1980). The galled stems are harvested as vegeta-
ble at the stage when no smut spores are developed (Chung and Tzeng 2004). This
vegetable is produced by vegetatively segregating infected plants.
Endophytic fungi are common in leaves and stems of grasses (Szécsi et al.
2013). Their systemic infection also produces infected seeds. These fungi do not
cause any disease in the grasses, but under most circumstances they are beneficial
to the growth and survival of infected plants, enhancing drought tolerance, summer
survival, and insect resistance. In recognition of the beneficial effects, turf grass
breeders now offer seeds of a variety of “endophyte-enhanced” cultivars (Grewal
and Richmond 2004). This man-made seed-borne dispersal of microfungi has an
emerging agricultural and commercial potential. Fungal spores could also be inten-
tionally dispersed as biological weapons. Karnal bunt (Tilletia indica) spores were
produced in Iraq as an anticrop agent. It must have been intended for use in the war
against Iran, whose main crop is wheat (Whitby 2002; Suffert et al. 2009). The
M115 anticrop bomb also referred as the feather bomb or the E73 bomb was a US
biological cluster bomb aimed to deliver Puccinia graminis f. sp. tritici, wheat
stem rust (Wheelis et al. 2006). Wheat stem rust bomb production includes a dry
particulate matter (rust spores) which was adhered to a lightweight vector, usually
feathers.
Applications of Dispersal Strategies of Microfungi:

Future Trends
Research of dispersal strategies of microfungi lead for new applications and inven-
tions in surprisingly different fields of science. Analysis of the spore discharge pro-
cess using high-speed video is proved to be a useful modern tool to understand
micromechanical processes. The ballistospory biomechanics were analogous to the
recent development of a surface tension motor (Pringle et al. 2005). The production
of this nanoscale machine driven by the coalescence of liquid droplets demonstrates
that processes powered by surface tension may have significant applications in
engineering (Regan et al. 2005). Investigations of other forcible spore discharge
mechanisms and the discharge of basidiospore may offer a natural model for this
kind of devices, and the findings have had major significance for future develop-
ment of machines that operate on the micrometer and nanometer scales.
The practical use of insect dispersal is in the biological control. Western honey-
bee, Apis mellifera L., was used to deliver conidia of Clonostachys rosea to control
Botrytis cinerea on strawberry (Fragaria × ananassa) (Peng et al. 1992). Bumblebee,
Bombus impatiens, was studied as a delivery vehicle of C. rosea (reported as
G. roseum and G. catenulatum) to control Botrytis cinerea on raspberry (Rubus
idaeus) and rabbiteye blueberry (Vaccinium virgatum) (Yu and Sutton 1997; Smith
et al. 2012). Promising research results showed that the western honeybee (Apis
mellifera) could be used to disperse Trichoderma spp. to reduce disease incidence
of sunflower head rot caused by Sclerotinia sclerotiorum (Escande et al. 2002).
Free fungal spores can be studied concerning their biodiversity in a sample. Spore
composition is characteristic, and their analysis is a useful tool to identify the origin
of samples, indicating vegetation (air samples from a known trajectory, stemflow and
honeydew samples, forensic, archeological, or geological sediments). It was sug-
gested that the possibility of identification of origin of honeys could obviously be
tested with the multivariate analysis of fungal diversity data (Magyar et al. 2005).
Plasmodium movements of slime molds are driven by the grainy cytoplasm,
which flows by unidirectional pulsation in a cell. The cell attains a speed of up to
1 mm/s (Nowotny 2000). Such movements of slime molds are used to imitate the
formation of the motorway transport network (Adamatzky et al. 2013) or the migra-
tions of human population (Adamatzky and Martinez 2013) in laboratory condi-
tions. Plasmodium movement will be used in future designs of self-growing wetware
circuits and devices and integration of slime mold electronics into unconventional
biohybrid systems (Adamatzky 2013).
Acknowledgments We thank Ágnes Révay, Zoltán Bratek, and Gyula Oros for their excellent
suggestions and providing literature.
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Chapter 15
Microfungi in Indoor Environments: What
Is Known and What Is Not
Chin Yang, Sepideh Pakpour, John Klironomos, and De-Wei Li
Introduction
Indoor molds refer to the microfungi occurring in indoor environments. A majority

of indoor fungi belong to the microfungi. A few of them are members of
Basidiomycota, such as wood-decaying fungi and species of Coprinus s.l. A mem-
ber of Ascomycota, Peziza domiciliana is occasionally found in indoor environ-
ments. The World Health Organization (WHO) recognized that “[h]ealthy indoor
air is recognized as a basic right” (WHO 2009). The indoor bio-deterioration and
health effects associated with fungal infestation in indoor environments have
become a research priority (Green et al. 2011). More and more studies in the last
decade demonstrated the association between indoor fungi and their detrimental
effects on human health. “WHO guidelines for indoor air quality: dampness and
mould” concluded that “the most important effects are increased prevalence of
C. Yang, Ph.D.
Prestige EnviroMicrobiology, Inc, 242 Terrace Boulevard Suite B-1, Voorhees, NJ 08043, USA
e-mail: chins.yang@prestige-em.com; csyang1000@gmail.com
S. Pakpour • J. Klironomos, Ph.D., F.R.S.C.
Department of Biology, University of British Columbia,
Okanagan campus, Kelowna, BC, Canada V1V 1V7
Okanagan campus, ASC 402, 3333 University Way, Kelowna, BC, Canada V1Y 4S8
e-mail: sepideh.pakpour@ubc.ca; john.klironomos@ubc.ca
D.-W. Li, Ph.D. (*)

DOI 10.1007/978-3-319-29137-6_15
374 C. Yang et al.
respiratory symptoms, allergies and asthma as well as perturbation of the immuno-

logical system” (WHO 2009). Molds associated with poor indoor air quality are
important risk factors for human health in both low-income and middle- and high-
income countries (WHO 2009).
Part of the difficulty is that several hundred indoor microfungi have been reported
in indoor environments. This number of microfungi is significantly underestimated
based on authors’ observations. It is common to find more than two dozen fungi
coexisting in a residence from a single investigation of indoor molds. Frequently,
the indoor fungi are only identified to genus level due to the costs and a lack of well-
trained mycologists. It poses a huge challenge to segregate multiple factors that
determine which fungus or fungi are responsible for the adverse effects on health,
since the occupants are exposed to multiple agents simultaneously. Also, other bio-
logical agents, such as dust mites, pollen, bacteria, viruses, and pets, are present in
conjunction with indoor molds. These confounding agents/factors make the deter-
mination of causal agents even more difficult. The other challenge is to accurately
estimate exposure level and to link it to relevant symptoms and health outcomes due
to the exposure (WHO 2009). It is understandable why at present the causal rela-
tionship between indoor microfungi and some associated health problems, such as
sick building syndrome, is still subject to debate.
It is indisputable that significant development has been made in indoor molds
research. It has led to the publication of “WHO guidelines for indoor air quality:
dampness and mould” in 2009. In 2012, the American Society of Heating,
Refrigerating and Air-Conditioning Engineers (ASHRAE) changed their position
and recognized “the association between damp buildings and the potential for
adverse health effects” and advised its membership that the association of molds
with health risks is real (Harriman 2012).
Since several books and review papers on indoor molds have been published
(Straus 2004; Prezant et al. 2008; Yang and Heinsohn 2007; Jaakkola et al. 2013;
Quansah et al. 2012; Nevalainen et al. 2015), this chapter mainly focuses on the
latest developments and new directions of research in the last decade.
Fungal Systematics and Indoor Microfungi Research
Systematic studies on a number of indoor microfungi-related genera, such as

Acremonium, Aspergillus, Cladosporium, Penicillium, and Stachybotrys, using
DNA sequence data had led to the redelineation of a number of these genera in the
last decade (Bensch et al. 2010; Zalar et al. 2007; Summerbell et al. 2011).
Scott et al. (2004) studied indoor Penicillium chrysogenum in Ontario, Canada.
The results showed that P. chrysogenum is the most common fungus indoors (Scott
et al. 2004). It is also the most common fungus in water-damaged buildings in
Denmark and Greenland (Andersen et al. 2011). The isolates collected during the
study by Scott et al. (2004) formed four clades. The first clade contained more than
5.6 % of house isolates clustered with the ex type strains of P. chrysogenum and
15 Microfungi in Indoor Environments: What Is Known and What Is Not 375
P. notatum. The 4th clade clustered with Fleming’s strain and was the dominant one
which included more than 90 % of house isolates (Scott et al. 2004). A further study
P. chrysogenum was conducted by Houbraken et al. (2011). Their results showed
that Fleming’s strain is not Penicillium chrysogenum, but P. rubens which is the
most common fungi (clade 4 in Scott et al. study) in indoor environments.
Cladosporium cladosporioides s.l. was studied by Bensch et al. (2010) and 22
species were segregated and described as new species according to phylogenetic
analysis using sequencing data and cryptic morphological differences (Bensch et al.
2010). Cladosporium sphaerospermum s.l. was also redelineated. Cladosporium
dominicanum, C. halotolerans, C. psychrotolerans, C. spinulosum, and C. velox
were described, all with globoid conidia (Zalar et al. 2007). The subtle morphologi-
cal differences among these newly described species result in a major challenge to
identify them morphologically.
According to the study by Summerbell et al. (2011), Acremonium strictum, a
very common indoor microfungus, was combined as Sarocladium strictum and
Acremonium kiliense, another indoor microfungus, was changed to Sarocladium
kiliense.
A number of new species and records have been described or reported from
indoor environments, such as the new species, Balaniopsis triangularis D. W. Li
and W. B. Kendrick, and a new record in Canada and the USA, Triadelphia aus-
traliensis B. Sutton (Li et al. 2008, 2013). Several noteworthy fungi isolated from
indoor environments in the USA were described and reported for the first time:
Ascotricha erinacea Zambett., Sporoschisma saccardoi Mason & Hughes apud
Huges, Stachybotrys microspora (Mathur & Sankhla) Jong & Davis, S. nephrospora
Hansford, and Zygosporium masonii Hughes (Li and Yang 2004b). (Li and Yang
2004b). In fact, this was the first report of Ascotricha erinacea since it was described
by Zambettakis in 1955 (Zambettakis 1955). Stachybotrys elegans (Pidopl.)
W. Gams and Parascedosporium putredinis (Corda) Lackner & de Hoog were
reported from indoor environments for the first time (Li et al. 2013). Recently,
Stachybotrys elegans was reported from intensive care units in Assiut University
Hospitals in Egypt (Aboul-Nasr et al. 2014). Stachybotrys chartarum and S. elegans
are species complexes and further studies are necessary to delineate the two species
complexes in the future (Wang et al. 2015).
Wallemia sebi, a xerophilic and mycotoxigenic microfungus, was sometimes
reported from indoor environments (Samson et al. 2004). Wallemia was previously
considered to be Ascomycetes (Kirk et al. 2001). Its dolipore/parenthesome septa
were reported over four decades ago (Terracina 1974). Moore (1986) opined that W.
sebi may be a basidiomycete and a teleomorph. Moore’s former opinion was con-
firmed by a phylogenetic study not long ago. The genus Wallemia indeed belongs to
Basidiomycota and a new class Wallemiomycetes and a new order Wallemiales
were erected to accommodate it (Zalar et al. 2005). A genomic study by Padamsee
et al. (2012) suggested that W. sebi has cryptic sexual reproduction. Moore’s latter
opinion may not be far off.
Tritirachium spp., a hyphomycete genus occasionally found indoor environ-
ments, were formerly placed in Pezizomycotina, Ascomycota (Kirk et al. 2008).
376 C. Yang et al.
However, a recent study by Schell et al. (2011) found that this genus is a new lin-
eage in Basidiomycota. Subsequently, subphylum Pucciniomycotina, class
Tritirachiomycetes, order Tritirachiales, and family Tritirachiaceae were proposed
to accommodate this genus (Schell et al. 2011).
Since the passage of the International Code of Nomenclature for algae, fungi,
and plants (Melbourne Code) or (ICN) adopted by the 18th International Botanical
Congress Melbourne, Australia, July 2011 (McNeill et al. 2012), the principle one
fungus = one name has been in effect. Dual name systems (asexual and sexual
names) for fungi were terminated. Only one name is used for each species. It has led
to name changes for many fungi. Name changes for some taxa are pending, such as
Aspergillus, Penicillium, Fusarium, and their teleomorphs (Geiser et al. 2013; Pitt
and Taylor 2014). This change of the Melbourne Code has significant implications
for indoor microfungi research and investigation. Both states of Aspergillus and its
teleomorphs, Eurotium and Emericella, as well as Penicillium and its teleomorphs,
Eupenicillium and Talaromyces, are common in indoor environments or frequently
found on culture media. The availability of the genus name of Paecilomyces remains
to be determined. Bipolaris and Curvularia are linked to teleomorphic Cochliobolus.
A recent study placed Bipolaris and Curvularia into two separate clades (Manamgoda
et al. 2012) and may solve the problem and allow us to continue using these two
names. The name Tetraploa, the conidia of this genus sometimes found in the air, is
linked to its sexual stage, Massarina, which is polyphyletic including two other
genera, Acrocalymma and Ceratophoma. Unfortunately, many more names related
to indoor microfungi can be listed. Uncertainty of name changes for these common
indoor fungi is leading to an increasing concern to indoor mold researchers and
investigators. To better understand the changes to article 59 of the Melbourne Code
and its implication to indoor mold research, please refer to Chap. 2 by Walter Gams
for a detailed discussion.
Fungal Fragments
Airborne fungal fragments as a component of bioaerosols have been overlooked in

aeromycological studies in the past. Samir et al. (2014) indicated that hyphal frag-
ments and hyphae as aeroallergens are underestimated. A significant number of
studies did not include fungal fragments as one of the fungal categories/groups/
taxa. Fungal fragments include hyphal and conidial fragments and partial conidio-
phores, ascomata and basidiomata, and other partial fungal structures. Hyphal frag-
ments were reported in various aeromycological studies and laboratory reports in
the past (Li and Kendrick 1995a, b; Li and Yang 2004a), but not other fungal frag-
ments. The difficulties in identifying and enumerating these fungal fragments are
implicating scientific understanding of the significance of fungal exposure in indoor
environments and associated health effects. Exposure to fungal fragments has
attracted attention in the last decade. There is an increasing interest in aerosolized
fungal fragments and their association with asthma severity (Green et al. 2006),
especially the submicron fungal fragments. The health effects of exposure to respi-
rable sized fungal fragments in indoor and occupational environments infested by
fungi remain less clear and need further studies in the future.
Green et al. (2011) coined two new terms for fungal particulates: gonomorphic
and non-gonomorphic. Gonomorphic refers to the fungal structures which are dif-
ferentiated as separable, dispersive reproductive structures (sexual and asexual
spores). Non-gonomorphic particles are defined to be mechanically severed from
the parent mycelium but were not programmatically differentiated as separable
(Green et al. 2011). Non-gonomorphic particles include hyphal fragments (<100
μm), chlamydospores, partial multicellular conidia, and subcellular fragments of
hyphae and conidia (Eduard et al. 2012). A number of researches have focused on
it. Immunodiagnostic methods demonstrated that non-gonomorphic particles con-
tain antigens as well as allergens (Eduard et al. 2012). These preliminary studies
have initiated collaborative studies into the occurrence and possible health effects
associated with personal exposure to non-gonomorphic particles.
Particle fragmentation can be facilitated by several biotic (fungal autolysis,
hyphal vacuolation, shizolytic/rhexolytic separation, as well as prokaryote, proto-
zoan, and microarthropod comminution) or abiotic processes (wind, vibration,
anthropogenic, and mechanical disturbances). In some environments, larger non-
gonomorphic particles (>2.5 mm) may represent a significant proportion of the fun-
gal bioaerosol load (ca. 56 %) and are derived from species within the orders
Capnodiales, Eurotiales, and Pleosporales (Green et al. 2011).
Fungal fragments (< spores) were found to be released from fungal colonies in
air chamber studies (Eduard et al. 2012). Hyphal fragments are non-gonomorphic
particles that tend to be primarily derived from outdoor sources during the summer
and from indoor sources during the winter (Li and Kendrick 1996). Hyphal frag-
ments in the air were often within 7–100 μm in length and viable (Pady and Kramer
1960; Pady and Gregory 1963). Meirer and Lindbergh (1935) reported airborne
hyphal fragments near the Arctic Circle.
Green et al. (2005) demonstrated that airborne fungal fragments (hyphal frag-
ments and fragmented conidia) and conidia of a number of previously undocu-
mented genera are new aeroallergen sources. Green et al. (2006) indicated that
respiratory deposition models suggest that submicron fragments of Stachybotrys
chartarum may be deposited in 230–250-fold higher numbers than spores. Their
studies showed that the concentrations of total airborne hyphae were frequently
significantly higher than those of conidia of individual allergenic genera.
Approximately, 25 % of all hyphal fragments expressed detectable allergens and
conidia of previously uncharacterized genera were sources of allergens (Green et al.
2006).
Samir et al. (2014) demonstrated that immunostaining of fungal hyphae was het-
erogeneous, and ∼ 27 % of all fungal fragments and hyphae expressed detectable
allergens compared with nonstained hyphae. Their results showed that fungal
hyphae and fragments are underestimated sources of airborne allergens since posi-
tively immunostained hyphal fragments were detected in all samples and the quan-
tity of the detected fungal hyphae in any of the individual protein-binding membranes
378 C. Yang et al.
was significantly higher than the spore counts of Alternaria spp., Aspergillus spp.,
and Cladosporium spp. (Samir et al. 2014).
Madsen (2012) found that aerosolization of conidia and fungal fragments of
Botrytis cinerea occurred under an airflow of 1.5 m s−1 or 0.5 m s−1. She determined
that the size of the respirable fraction of the aerosolized particles was dependent on
the RH. At high RH, about 30 % of the aerosolized particles were of respirable size,
while at low RH, about 70 % were of respirable size. Under low RH, more fungal
(1 → 3)-β-D-glucan and chitinase became airborne than under high RH.
Size-selective studies on exposure to fungal spores and fragments were not well
studied until a decade ago. Reponen et al. (2007) stated that fungal fragments with
smaller sizes (<1 μm) may contribute to mold-related health effects. A number of
laboratory studies showed that large numbers of submicrometer-sized fungal frag-
ments (30 nm–1 μm) were liberated along with unbroken spores from infested sur-
faces. The number of released fungal fragments was constantly higher, up to 500
times, than the number of unfragmented spores, and the number of fragments is not
correlated with spores (Górny et al. 2002; Cho et al. 2005). Reponen et al. (2007)
studied airborne fungal particles in three sized groups: (a) >2.25 μm (spores), (b)
1.05–2.25 μm (mixture), and (c) <1.0 μm (submicrometer-sized fragments). The
authors concluded that the real contribution of fungal fragments to the overall expo-
sure may be very high, even much higher than those estimates from previous labo-
ratory studies.
A recent size-selective study on personal exposure of agricultural workers to
airborne fungi and fungal fragments showed that Alternaria and Botrytis were
highly correlated with (1 → 3)-β-D-glucan at the aerodynamic size < 1 μm, which was
much smaller than the expected spore sizes (the average aerodynamic sizes were
18.5 μm for Alternaria and 6.1 μm for Botrytis). Thus, the assumption is that
Alternaria and Botrytis might release small fragments into the air and these aerosol-
ized submicron fungal particulates could enter the deep lung and cause respiratory
diseases (Lee and Liao 2014).
Cho et al. (2005) found that the released fungal fragments of Stachybotrys char-
tarum were 380 particles cm-3, which was approximately 514 times higher than
those of conidia, and Aspergillus versicolor released comparable numbers of spores
and fragments. Their modeling showed that S. chartarum fragments had 230–250-
fold higher respiratory deposition than its conidia and A. versicolor had a compa-
rable result.
Adhikari et al. (2013) studied particulates of fungal fragments in three aerody-
namic size fractions: <1.0, 1.0–1.8, and >1.8 μm and used N-acetylhexosaminidase
(NAHA) as a marker of fungal cell biomass. Significant relationships were found
between the amounts of NAHA in the total amount and in the size fraction >1.8 μm
but not in the smaller fractions.
Seo et al. (2014) found that the concentration of (1,3)-β-D-glucan in submicron
fungal fragments in indoor air was 2 times higher in homes with asthmatic children
(50.9 pg/m3) compared to homes with non-asthmatic children (26.7 pg/m3) in South
Korea and that relative humidity was negatively correlated with the concentration
of (1,3)-β-D-glucan in submicron fungal fragments. At present, a number of studies
have concluded that fungal hyphae and fragments are underestimated sources of
aeroallergens (Green et al. 2006; Samir et al. 2014). A number of studies are starting
to focus on using nanotechnology in indoor molds (Gong et al. 2014; Filip 2009;
Rácová et al. 2013).
Mold, Dampness, and Water Damage
Water damage/intrusion and dampness in buildings without immediate corrective

actions are the determining factor which allows indoor molds to grow, eventually
leading to fungal infestations indoors. Dampness/high humidity and long-term
water damage lead to different fungal compositions and fungal succession. The dif-
ference is determined by water activity (aw) in the substrates/building materials.
Exposure to molds associated with dampness and water damage in indoor environ-
ments has received significant attention in recent research (Kennedy and Grimes
2013). For an assessment of dampness in indoor environments, the review paper by
Kennedy and Grimes (2013) can be consulted.
Dampness due to high relative humidity (RH) in the air is an often overlooked
problem which results in fungal infestation. High RH allows fungi, especially xero-
philic taxa, to develop in indoor environments. A number of fungi (Alternaria spp.,
Cladosporium spp., Acremonium sp., and Ulocladium sp., etc.) and visible fungal
colonies developed on ceiling tiles in a school with inadequate operation of the A/C
system during the summer in a coastal area of Connecticut (Li unpublished
observation).
Karvala et al. (2014) studied patients with occupational asthma (OA) or work-
exacerbated asthma (WEA) and concluded that adverse work ability outcomes are
associated with asthma in relation to workplace dampness. They opined that there
was a need to put effective preventive measures in place to assist workers with
indoor air symptoms to sustain their work ability. Haverinen-Shaughnessy et al.
(2012) found that moisture problems (water damage, dampness, and molds) are
rather common in schools in the Netherlands, Spain, and Finland. The occurrence
and severity may show a discrepancy across geographical areas, which can be partly
explained by building characteristics.
Black Aspergilli (Aspergillus section Nigri) were studied in indoor environments
in six countries: Algeria, Croatia, Hungary, the Netherlands, Thailand, and Turkey
recently (Varga et al. 2014). The highest species diversity (seven species including
a new species) was observed in indoor samples from Thailand, while the lowest
(two species) was found in Algeria. A. niger, A. tubingensis, A. luchuensis, and A.
welwitschiae were identified in all three temperate European countries, while A.
tubingensis, A. luchuensis, and A. welwitschiae were also detected in Turkey, but
not A. niger (Varga et al. 2014).
A recent study with 13,335 participants on associations between home dampness
and asthma and related symptoms in 4- to 6-year-old children in Shanghai found
that home dampness was strongly and significantly associated with dry cough,
wheeze, and rhinitis symptoms. Children exposed to visible mold spots had a 32 %
higher risk of asthma (adjusted OR, 95 % CI: 1.32, 1.07–1.64) (Hu et al. 2014).
380 C. Yang et al.
Mites
House dust mites and other indoor mites and their association with indoor molds are
little studied, although they have been observed and reported on a few occasions
(Yang 2007; Samson and Houbraken 2011). Mites can be serious contaminants in
laboratories by feeding on fungal colonies and carrying fungal spores around.
House dust mites (HDM) are major allergens indoors and most studies were focused
on allergens of HDM and associated allergies in the past. However, HDM are often
present on building materials, especially on drywall infested by indoor molds. The
relationship between HDM and indoor molds has been overlooked in the past. Part
of the reason is lack of expertise in mites. Very few people working on indoor
molds have the acarological background to identify mites to its taxon. Very few
acarologists are available to assist identifying mites collected from indoor environ-
ments. The ecological roles played by house dust mites associated with fungi in
indoor environments are unclear, such as feeding behaviors on fungal colonies and
spreading fungal infestations by transporting fungal spores on their bodies. In addi-
tion, little is known concerning the viability and allergenicity of fungal spores and
fragments in mite fecal matter.
It is common knowledge that dust mites are the predominant producers of inhala-
tion allergens in many regions in the world (Cui 2014). The most common mite
species that produce allergens are Dermatophagoides pteronyssinus, the European
house dust mite, and Dermatophagoides farinae, the American house dust mite
(WHO 2009). These two species are not confined to Europe or North America as
their common names imply. Another species, Euroglyphus maynei, also has a wide
distribution. Eleven other dust mite species have been reported indoors (Cui 2014).
Most research has focused on D. pteronyssinus and D. farina and their allergenici-
ties and health effects. The major allergens produced by D. pteronyssinus are prote-
ases (Der p I and Der p II), which are reported in large amounts in fecal materials,
while D. farinae produced the major allergen Der f I (Institute of Medicine 2000).
Increased levels of these allergens have been reported in house dust, mattress dust
bedding, and upholstery (Van Strien et al. 2004; Cui 2014).
Recently, Naegele et al. (2013) studied feeding preferences of Dermatophagoides
farinae to 16 indoor fungi. They found that D. farinae preferred to feed on Alternaria
alternata, Cladosporium sphaerospermum, and Wallemia sebi. Penicillium chrys-
ogenum, Aspergillus versicolor, and Stachybotrys chartarum were at the bottom of
its preferential list. Naegele et al. (2013) also suggested that the food preferences of
D. farinae may play dual roles in indoor mold infestation: a decrease in spore num-
bers by feeding on fungal structures and spreading fungal spores its bodies carry.
However, it is necessary to study the factors which determine the feeding prefer-
ences of mites associated with indoor molds. MVOC emitted by fungi, mycotoxins/
secondary metabolites in fungal structures, or water activities in building materials
should be further studied.
Indoor Substrates and Indoor Molds
There are a number of materials present in indoor environments that can serve as
substrates for fungal growth such as drywall, wooden structures, furniture, carpet,
paints, books, paper products, leather products (shoes, jackets, couches), uphol-
stery, and wallpaper, etc.
Wood
Wood is highly susceptible to fungal colonization by fungi such as Cladosporium,

Penicillium, and Aspergillus (Bjurman 1994). Wood drying, specifically kiln drying,
results in a higher amount of nitrogen and low molecular carbohydrates on the wood
surface which makes it more susceptible to mold growth (Thelander et al. 1993;
Viitanen 1997). Other studies illustrated that some engineered wood products, such
as oriented strand boards (OSB), plywood, and medium-density fiberboard (MDF),
are more susceptible to growth of Aspergillus, Trichoderma, and Penicillium than
solid wood, particleboard (Chung et al. 1999), acylated wood (Suttie et al. 1998),
and wood polyethylene composites (Mankowski and Morrell 2000).
Wallpaper and Plasters
Both paper and glue are very good media for many indoor fungi and thus wallpapers
are very susceptible to mold growth (Bissett 1987; Grant et al. 1989). Synthetic
polymers such as synthetic rubber and plastic can also be degraded by different spe-
cies of fungi such as Aspergillus niger, Aspergillus flavus, Aureobasidium pullulans,
Chaetomium spp., Penicillium funiculosum, Penicillium luteum, and Trichoderma
spp. (Flannigan and Miller 2001).
Recently, prefabricated gypsum plaster paper boards (drywall) are commonly
used in new buildings. The boards are highly susceptible to mold growth, mainly by
the cellulolytic S. chartarum because of the paper used to reinforce the material. In
addition, the gypsum itself can support fungal growth due to its nutrient contents
and additives that make it more hygroscopic at lower humidity levels (Nielsen et al.
1998; Eppley and Bailey 1973; Andersen et al. 2002).
Plastic and Glass
Fungi can also grow on polyethylene and polyvinyl chloride (PVC) as they can
degrade most plasticizers including common organic acid esters such as dioctyl
phthalate (DOP) and dioctyl adipate (DOA) (Webb et al. 2000). Glass-reinforced
382 C. Yang et al.
plastic (GRP), popularly known as fiberglass, and fiberglass ceiling tiles which con-
tain 10 % ureaphenol-formaldehyde resin are other susceptible materials that can
support fungal growth, especially A. versicolor and Penicillium spp. (Steyn and
Vleggaar 1976).
Paint
Fungi can also grow on water-based or solvent-based paints; however, it is not clear
whether molds found on the surface are using the paint components or taking nutri-
ents from dirt also found on the surface (Allsopp et al. 2004). In general, paints can
either enhance or decrease the susceptibility of a given base material to fungal
growth. For example, paints prevent the growth of Aureobasidium pullulans, while
Penicillium and Aspergillus species can grow rapidly on the same paints (Nielsen
and Thrane 2002).
Cheng et al. (2014) showed that the surface water ratio and moisture content of
mortars, brick, and tiles were affected by the pore size and distribution of these
materials and different environments also showed significant effects on the surface
water ratio of the building materials. The surface water ratio was a major factor
affecting mold growth on these building materials (Cheng et al. 2014).
Chunduri (2014) reported Aspergillus niger, Ascotricha chartarum, Fusarium
solani, Aspergillus spp., Penicillium spp., Cladosporium spp., Phoma spp.,
Stachybotrys spp. Ascospora spp., Curvularia spp., and Alternaria spp. from indoor
cement walls of residential/commercial construction with water damage/intrusion.
However, these results need to be verified, since cement itself may not support the
growth of some of these fungi, such as Stachybotrys spp. and Ascotricha chartarum
without organic dust or wooden furniture against the cement walls.
Other Substrates
Fungal infestation in buildings is rather complex and facilitated by a number of fac-

tors, such as water damage, dampness, indoor environmental conditions, substrates,
exposure time, building maintenance, building design, etc. The physical properties
of building materials, e.g., surface structure, can be a key factor for fungal spore
germination and fungal growth (Ryu and Moon 2014). Ryu and Moon (2014) inoc-
ulated Aspergillus versicolor, Penicillium chrysogenum, and Stachybotrys charta-
rum (reported as S. atra) on four kinds of wallpapers and their results showed that
the wallpapers with irregular patterns or a flat surface structure delayed fungal
growth. Wallpapers with higher moisture absorption capability prevented the devel-
opment of fungi. A clear correlation between growth rates of the fungi and hygro-
scopic properties of the wallpapers was determined in the study also. Aspergillus
versicolor grew faster than P. chrysogenum and S. chartarum on the wallpapers.
Mattresses are recognized as reservoirs for dust mites, but are also important reser-
voirs for molds (Verhoeff et al. 1994). The concentrations of molds in mattresses
were reported to be 103–107 spores/g of dust (Verhoeff et al. 1994).
Natural Disasters and Indoor Molds
One of the effects of global warming is the increase in numbers of severe tropical
storms (hurricanes and typhoons) and floods throughout the world (Mousavi et al.
2011). Hurricanes, superstorms, and floods cause tremendous damage to residences
and other properties. In addition to physical damage, mold infestation and elevated
mold and other particulate exposure of home owners, residents, first responders,
and restoration workers in the damaged buildings lead to an increasing health con-
cern for healthcare providers and disaster medicine throughout the world (Johanning
et al. 2014). In the last 10 years, two major hurricanes, Katrina in 2005 and Sandy
in 2012, made landfall on the east coast and caused catastrophic damage. A number
of studies on indoor molds associated with these two major hurricanes have been
published.
Rao et al. (2007) found that culturable fungi were significantly higher in the
moderately/heavily water-damaged houses (67,000 CFU/m3) than in the mildly
water-damaged houses (3700 CFU/m3) (p = 0.02) 1 month after hurricanes Katrina
and Rita and the predominant fungi in those houses were Aspergillus niger,
Penicillium spp., Trichoderma, and Paecilomyces. At the same time, the fungal taxa
and their concentrations were different from those previously reported from build-
ings without water damage in the southeastern USA. Fungi, fungal glucans, and
endotoxins in the environment after Hurricanes Katrina and Rita in New Orleans
were detected at concentrations that have been associated with health effects (Rao
et al. 2007).
A retrospective, cross-sectional study of flooding, mold exposure, remediation,
and respiratory symptom prevalence was conducted in Bound Brook, New Jersey,
after Hurricane Floyd in September 1999 (Jones et al. 2013). The results showed
that flood damage was a strong predictor of mold growth (p < 0.001), and flooding
was strongly associated with physician-confirmed respiratory symptoms in the
aftermath of the flood (28 of 29 cases vs. 10 of 18 referents; p < 0.001). Individuals
involved in cleanup work without adequate personal protection were inclined to
report five or more symptoms (p < 0.002). The study concluded that exposure to
molds during cleanup of moldy materials was a significant contributor to symptoms
(Jones et al. 2013).
A study on total and respirable dust exposure for restoration work activities
(demolition, mold remediation, trash and debris management, landscape restora-
tion, and sewer/waterline repair) was conducted from 2005 to 2012 after Hurricane
Katrina devastated the city of New Orleans in 2005 (Rando et al. 2013). The results
showed that the most significant exposures were for demolition work, with average
respirable dust exposures in 2005 above the action level of 2.5 mg/m3 and 17.6 %
384 C. Yang et al.
of exposures exceeding the permissible exposure limit (PEL) (5 mg/m3). Average

exposures to endotoxin and microbial glucan in 2005 were as high as 256 EU/m3
and 118 μg/m3, respectively. The results of this study support the conclusion that
there is an association between respiratory illness and exposure during post-Katrina
restoration work in the years immediately after the hurricane (Rando et al. 2013).
Studies on indoor molds after major natural disasters do not always support each
other. The results showed some discrepancies. Grimsley et al. (2012) reported
indoor and outdoor airborne mold levels were 501 and 3958 spores/m3, respec-
tively, in the homes of HEAL children that had been damaged by rain, flooding, or
both. Alternaria antigen was reported in dust from 98 % of the homes, with 58 %
having concentrations > 10 μg/g, and Mus m 1, Der p 1, and Bla g 1 were found in
60 %, 35 %, and 20 % of homes, respectively, at low concentrations. This study
concluded that except for Alternaria antigen in dust, concentrations of airborne
mold (ratio of indoor to outdoor mold) and dust allergens in the homes of HEAL
children were lower than measurements found in other studies. They speculated that
extensive post-Katrina mold remediation and renovations, or because children
moved into cleaner homes upon returning to New Orleans, might be the reason
(Grimsley et al. 2012).
Barbeau et al. (2010) evaluated the levels of indoor and outdoor molds in the
months following hurricanes Katrina and Rita and found the homes with greater
flood damage, especially those with >3 ft of indoor flooding, had higher levels of
mold growth in comparison with homes with little or no flooding. Water intrusion
originating from roof damage was also associated with mold growth. However, they
did not find an increase in the occurrence of adverse health outcomes from pub-
lished reports to date (Barbeau et al. 2010).
Sato et al. (2014) conducted a study on fungi from paper-based cultural objects
damaged by seawater due to the tsunami in March 2011. Stachybotrys chartarum
was found to be one of the key species causing the black alterations on tsunami-
damaged, paper-based cultural items that remained wet for several months after the
tsunami. Chaetomium and Cladosporium were observed on blackened documents
also. Myxotrichum deflexum and Streptomyces sp. were isolated from the red-altered
paper samples (Sato et al. 2014). The results showed that NaCl tolerance and
cellulose-utilizing capacity were important for the microfungi that outcompeted the
fungal taxa intolerant to NaCl on paper and paper products following the tsunami.
A number of species of Aspergillus and Penicillium are very tolerant to NaCl, up to
20 %, while S. chartarum was tolerant up to 10 %. This may explain why recent
studies reported S. chartarum was isolated from sponge in marine environments
(Ma et al. 2013).
At present, there are several projects supported by NIOSH to train professionals
who are involved in restoration or demolition work following Hurricane Sandy to
avoid or reduce their exposure to indoor molds and other particulates when han-
dling fungal infested/hazardous materials. Clearly, more studies are necessary in
this field in the future.
There is an increasing concern by medical professionals worldwide over ill-
nesses and health issues experienced by unprotected first responders, workers,
home owners, and volunteers in recovery, restoration, and demolition of moldy

indoor environments after hurricanes, typhoons, tropical storms, and floods.
Avoiding and minimizing unnecessary fungal exposure are recommended and
appropriate personal protective equipment (PPE) in disaster response and recovery
work should be used (Johanning et al. 2014).
Dust
House dust is a repository, concentrator, and a long-term reservoir of indoor fungi.

Continuous elutriation of organic and inorganic airborne particles, originating from
various indoor and outdoor sources, forms dust (Scott 2001). Dust is a complex
mixture of organic and inorganic material whose composition varies depending on
a given building type and use and major particle sources (Macher 2001; Chew et al.
2003). Dust samples are frequently collected during indoor fungi inspections and
research to evaluate indoor environments and cumulative exposure of occupants to
fungi as well as fungal composition.
The first global study on indoor fungi using dust samples collected from indoor
environments on different continents found that indoor fungal composition is geo-
graphically patterned and more diverse in temperate zones than in the tropics
(Amend et al. 2010). This result deviates from the previous understanding that
indoor mold diversity in tropical areas is higher. Amend et al. (2010) reported that
among 4473 operational taxonomic units (OTUs) defined at 97 % ITS sequence
identity, only 31 OTUs were reported in more than half of the samples. The same
authors also found that all but 3 of these 31 indoor fungi belonged to the phylum
Ascomycota, and among these common indoor fungi over 80 % (25 of 31) were in
the class Dothideomycetes. A recent study using polymerase chain reaction (PCR)–
denaturing gradient gel electrophoresis (DGGE) and culture methods was con-
ducted on household dust. It found that fungal diversity varied between 9 and 56
operational taxonomic units. Culture methods demonstrated that the most abundant
genus was Aspergillus, followed by Penicillium, Mucor, and Rhizomucor.
Trichoderma, Chrysosporium, Fusarium, Rhizopus, and Stachybotrys, which were
present in a limited number of houses in central Portugal (Sousa et al. 2014).
There are some conflicting results about the health effects of indoor fungi. A
recent study showed that culturable fungi, (1–3, 1–6)-β-D-glucan, and ergosterol
concentrations in dust collected from residences in Sweden were not associated
with asthma, rhinitis, or eczema diagnoses in children (Choi et al. 2014). However,
the validity of this conclusion was questioned by Rylander (2014). No doubt, the
disagreement will continue.
Adams et al. (2013b) adopted a simple, passive, innovative sampling device
modified from the dustfall collector published by Würtz et al. (2005) for their recent
study in student housing in Berkeley, CA. With this method, a Petri-dish sampler
suspends a sterile, empty, 9-cm disposable Petri dish, 0.3 m from the ceiling to col-
lect settling airborne dust for a defined period of time at a sampling site. The main
386 C. Yang et al.
advantage of this method is to collect dust samples with clearly defined starting and
finishing points. When a dust sample is collected from an indoor surface with tradi-
tional methods, it is very difficult to know the time frame the dust sample repre-
sents. The study of Adams et al. (2013b) produced some very interesting results
using pyrosequencing (ITS region 1). The most abundant fungus is Cryptococcus
victoriae. Does this result suggest that yeasts and yeast-like fungi are significantly
under-evaluated or its dominancy represent a geographic difference in fungal diver-
sity? Among the 25 most abundant microfungi, two fungi could not be identified by
BLASTing against the fungal taxa in GenBank. It indicated that some indoor fungi
are overlooked and even not described previously. They found that richness of indi-
vidual indoor samples ranged from 17 to 271 OTUs and 643 OTUs were reported in
both winter and summer across seasons and 619 OTUs were reported in both indoor
and outdoor samples across localities (Adams et al. (2013b).
Health Effects
Exposures to indoor fungi may lead to a range of diseases and symptoms, both
respiratory and non-respiratory. Respiratory diseases and symptoms that originated
from exposure to indoor fungi include asthma, hypersensitivity pneumonitis, cough,
wheeze, dyspnea (shortness of breath), nasal and throat symptoms, respiratory
infections, rhinosinusitis, and sarcoidosis (Park and Cox-Ganser 2011).
Stachybotrys chartarum is notorious as an indoor microfungus and mycotoxin
producer that can cause mycotoxicosis (stachybotrytoxicosis) in animals and
humans. This fungus has been suggested to cause various medical conditions in
humans, such as acute infant pulmonary hemorrhage, asthma, adult nasal and tra-
cheal bleeding, allergies, asthma-like symptoms, inflammation, and lung injury
(Etzel et al. 1998; Vesper et al. 2001; Vesper and Vesper 2002; Al-Ahmad et al.
2010; Bhan et al. 2011; Yike and Dearborn 2011; Pieckova et al. 2009). Infant pul-
monary hemorrhage incidents in Cleveland caught the attention of the media, the
public, and the medical community to this toxigenic fungus and other indoor molds
in the last 15 years. The causal relationship between S. chartarum and pulmonary
hemorrhage and the risk of exposure to this species to public health is still subject
to debate (Pestka et al. 2008; Yike and Dearborn 2011). Cases of infant pulmonary
hemorrhage associated with S. chartarum in patients’ residences in Cleveland keep
increasing from nine cases in 1990s to 52 cases in 2011. Of the cases investigated,
91 % of patients were living in residences infested by S. chartarum (Yike and
Dearborn 2011). Pulmonary hemorrhage in acute animal models of instillation of
S. chartarum conidia into rodent airways had been reported in more than 10 papers
(Yike and Dearborn 2011). One of the early studies isolated Stachybotrys charta-
rum (reported as S. atra) from bronchoalveolar lavage fluid of a child with pulmo-
nary hemorrhage (Elidemir et al. 1999). Another one found S. chartarum exposure
in an infant that developed laryngeal spasm and hemorrhage during general anesthe-
sia (Tripi et al. 2000). Nagayoshi et al. (2011) demonstrated for the first time that
inhalation exposure to the conidia of S. chartarum led to the remodeling of pulmo-

nary arteries in mice. Yike and Dearborn (2011) opined that this study made a sig-
nificant contribution to our understanding of the pathologic effects of S. chartarum.
Recent studies showed that pulmonary hypertension and evoked pulmonary arterial
remodeling in mice were caused by repeated inhalation of S. chartarum conidia
(Nagayoshi et al. 2011).
Rakkestad et al. (2010) indicated that cell death (apoptosis) was induced by heat-
treated conidia of S. chartarum within 3–6 h due to DNA damage. Pollard et al.
(2013) demonstrated that the conidial extracts of S. chartarum altered surfactant
protein expression. Exposure to the conidial extracts of S. chartarum resulted in
significantly reduced viability of fetal rat lung epithelial cell and human A549 cells
and their ability to produce pulmonary surfactant. This study has demonstrated that
inhaling mycotoxins produced by S. chartarum may induce potential damage to
surfactant production and function (Pollard et al. 2013).
Bhan et al. (2011) demonstrated that hypersensitivity pneumonitis was induced
by S. chartarum. Although a low rate of IgE mediated allergic responses to the
toxigenic fungus Stachybotrys chartarum has been reported in some studies, it is
unlikely a strong allergen in the clinical setting and the toxic-irritant effects appear
to be more important (Barnes et al. 2002). After comparing the allergenicity of S.
chartarum to house dust mite extracts in a mouse model, Chung et al. (2010) devel-
oped a suggested threshold dose (10 μg) for S. chartarum allergy induction. For a
susceptible population in damp water-damaged environments, the authors’ conclu-
sion is that exposure to S. chartarum might be easily over the sensitization thresh-
old. A high prevalence of pulmonary diseases was reported among office workers
of Florida court buildings following prolonged indoor exposure to S. chartarum and
Aspergillus versicolor (Hodgson et al. 1998).
Gareis and Gottschalk (2014) studied guttation droplets of Stachybotrys spp. and
found that all 15 strains of S. chartarum and S. chlorohalonata but one developed
various amounts of guttation droplets. Among the isolates, five are toxigenic iso-
lates and produced highly toxic guttation droplets. These toxin containing guttation
droplets were confirmed by all methods: ELISA, effect-based bioassay (MTT cell
culture test), and tandem mass spectrometry (LC-MS/MS). The concentrations of
macrocyclic trichothecenes, satratoxin G and H, are reported to be between the
Limit of detection (LOD) and 7160 ng/mL exudate and 280 and 4610 ng/mL,
respectively. Roridins and verrucarins were also reported from guttation droplets.
The study demonstrated that S. chartarum were able to produce toxic exudates. It
suggests that the toxic exudates may be significant in understanding its toxic poten-
tial in indoor environments (Gareis and Gottschalk 2014), exposure, and its health
effects. The genome of S. chartarum has been sequenced (Semeiks et al. 2014). The
genomic information of this species will assist us to better understand the mecha-
nisms of its medical implication to human beings and the production of secondary
metabolites including mycotoxins and more.
Satratoxin-G (SG) is a trichothecene mycotoxin produced by Stachybotrys char-
tarum. Islam et al. (2006) showed that intranasal exposure to SG induced apoptosis
of olfactory sensory neurons and acute inflammation in the nose and brain of mice.
388 C. Yang et al.
Carey et al. (2012) demonstrated that SG induced acute rhinitis, atrophy of the
olfactory epithelium, and apoptosis of olfactory sensory neurons occurred in both
groups in monkeys. These studies shed some light on the potential risk of nasal
airway injury and neurotoxicity caused by exposure to mycotoxigenic molds in
water-damaged buildings.
Dannemiller et al. (2014) showed that early-life exposure to low fungal diversity
in dust collected from residences was associated with increased risk for later child-
hood asthma development using DNA sequencing data. Tischer et al. (2011) stated
that exposure to visible mold and/or dampness was associated with an increased
risk of developing asthma during the first 2 years of life. The authors found that
meta-analyses showed a significant association with early asthma symptoms in four
cohorts and with asthma later in childhood in six cohorts and 3–10 years. There was
a significant association in six cohorts with symptoms of allergic rhinitis at school
age 6–8 years and at any ages between 3 and 10 years (Tischer et al. 2011).
Wallemia sebi, a basidiomycetous hyphomycete and xerophilic fungus, is also
very common in house dust (Desroches et al. 2014; Zalar et al. 2005). Desroches
et al. (2014) demonstrated that isolates of Wallemia sebi from indoor environments
produced several secondary metabolites including the known compound wallemi-
none and a new compound 1-benzylhexahydroimidazo [1,5-α] pyridine-3,5-dione
(wallimidione). Desroches et al. (2014) stated that wallimidione is likely the most
toxic mycotoxin produced by W. sebi.
Exposure to mycotoxigenic molds has been recognized as a significant health
risk for the last 20 years. Research has shown that mycotoxins are possible causes
of human disease in water-damaged buildings (Brewer et al. 2013). However, the
health effects from mycotoxins carried in airborne fungal structures remain hotly
debated at present.
Brewer et al. (2013) tested urine of patients with a prior diagnosis of chronic
fatigue syndrome for aflatoxins, ochratoxin A, and macrocyclic trichothecenes
using Enzyme-Linked Immunosorbent Assays (ELISA). Urine specimens from 104
of 112 patients (93 %) were positive for at least one mycotoxin and nearly 30 % of
the cases were positive for more than one mycotoxin. The results from a healthy
control population without exposure to indoor molds were negative.
Sercombe et al. (2014) found that the predominant fungal conidia that bound IgE
were derived from common environmental genera including Cladosporium and
other fungi that produce 1-celled spores and inhalable fungal aerosols are the pre-
dominant aeroallergen sources in the homes in Sydney, Australia, in comparison
with Der p 1, Fel d 1, and Bla g 1 allergen particles. Simoni et al. (2011) indicated
that school children in Italy, Denmark, Sweden, Norway, and France exposed to
viable molds ≥300 CFU/m3 showed a higher risk than those exposed to lower levels
for dry cough at night in the past year (odds ratio, OR: 3.10, 95 % confidence inter-
val, CI: 1.61–5.98) and rhinitis (OR: 2.86, 95 % CI: 1.65–4.95), as well as for per-
sistent cough (OR: 3.79, 95 % CI: 2.40–5.60). Aspergillus/Penicillium DNA showed
significant and positive association with wheeze, and Aspergillus versicolor DNA
with wheeze, rhinitis, and cough. Significant inverse associations of Aspergillus
versicolor DNA were found with forced vitality capacity (Simoni et al. 2011).
Alternaria population was associated with wheezing symptoms for children with
maternal mold sensitization [OR = 9.16; (1.37–61.22)], but not for those without
maternal mold sensitization [OR = 1.32; (0.79–2.20)] (Behbod et al. 2013). The evi-
dence indicated that dampness and molds in the home were determining factors for
the development of asthma. The association of visible molds, and especially mold
odor (MVOCs) to the risk of asthma development, implicated mold-related causal
factors (Quansah et al. 2012).
Hsu et al. (2010) indicated a statistically significant dose-dependent relationship
between total serum IgE levels and severity of indoor visible mold growth. However,
further analysis of specific IgE to commonly examined fungal allergens failed to
substantiate the correlation (Hsu et al. 2010). Holme et al. (2010) failed to find an
association between the spore concentration indoors and moldy odor as well as
signs of visible dampness in the homes. They did not determine an association
between the airborne spore concentration indoors and asthma/allergy in the children
(Holme et al. 2010). A recent study found that exposure to mold odor was related to
reduced lung function among non-asthmatic individuals, particularly among women
(Hernberg et al. 2014). Airborne fungi were found to be the second most frequent
allergen after mites (Ceylan et al. 2013). The number of household residents was
found to positively correlate with the populations of airborne fungi. However, the
authors did not find an association between the airborne fungi and dosage of inhaler
corticosteroids used or symptom levels in asthmatics (Ceylan et al. 2013).
Racial/ethnic, neighborhood, and socioeconomic factors are found to link to
asthma, but few studies have been conducted on the relationship between these fac-
tors and indoor allergens (Camacho-Rivera et al. 2014). Home owners were less
likely to report the presence of mice, cockroaches, and mold within their house-
holds in comparison with renters in Los Angeles. At the neighborhood level, their
results showed that neighborhood-level racial/ethnic and socioeconomic influences
on indoor allergen exposure exist (Camacho-Rivera et al. 2014).
A recent study focused on the interaction of genes with environment. The result
showed that there are obvious combined effects between IL-4 promoter (CT, CI,
TT) and mold exposure on both additive and multiplicative scales (Hwang et al.
2012). The risk of asthma was found to be significantly associated with children
carrying the CT genotype and visible mold exposure in comparison with those car-
rying the TT genotype without any exposure indicator. There is a similar tendency
for children who were exposed to mold odor and carried CT genotype. The authors
opined that gene–environment interactions between the IL-4 promoter and an
indoor mold infestation may play a significant role in childhood asthma (Hwang
et al. 2012).
Slime molds (Myxomycetes) are polyphyletic. Common slime molds can be
encountered indoors and outdoors, such as Physarum, Fuligo, and Stemonitis
(Fiore-Donno et al. 2012; Stephenson 2011). Stemonitis species have been observed
growing on water-damaged wooden structures in indoor environments (Li and
Yang, pers. obser.). Airborne and indoor slime molds and their potential health
effects are often overlooked and inadequately studied due to the difficulties in iden-
tifying their spores from the air and inability to isolate these slime molds with regu-
lar culture media.
390 C. Yang et al.
Lierl (2013) recently extracted allergens from nine species of slime molds. Her
results of allergy prick testing in 69 subjects with symptoms typical of seasonal
allergic rhinitis found that 42 % of the subjects were positive for at least 1 slime
mold extract, with 9 % to 22 % reacting to each extract. Lierl (2013) further
opined that the spores of these slime molds in the air might be significant
aeroallergens.
For the association between indoor mold exposure and exacerbation of asthma,
the conclusion is still subject to debate. In the latest review by Kanchongkittiphon
et al. (2014) on literature published since 2000, the authors concluded that there is
sufficient evidence of a causal association between outdoor culturable fungal expo-
sure and exacerbation in asthmatics sensitized to fungi. However, they opined that
evidence of an association between indoor culturable Penicillium as well as total
culturable fungal exposure and exacerbation in asthmatic children is limited or sug-
gestive (Kanchongkittiphon et al. 2014). They attributed their conclusion to the
sampling methods using 1-min air samples. In their opinion, the short sampling
time led to highly unreliable assessments of fungal concentrations in the air and
temporal variability of fungal spores in the air is high.
MVOC
Musty odor is often the first indication that catches our attention when we walk into
a moldy property. The odors we smell are volatile organic compounds (MVOCs)
emitted from microfungi growing in the moldy property. Some fungi, though not
all, produce noticeable MVOCs. In the last decade, the health effects of MVOCs
started to attract scientists’ attention and their importance has been gradually recog-
nized (Piechulla and Degenhardt 2014). A recent study concluded that some
MVOCs may be a risk factor for sick building syndrome and certain MVOCs were
slightly higher in homes with reported dampness and mold (Sahlberg et al. 2013).
Exposure to mold odor (MVOCs) was related to lower lung function levels among
non-asthmatic individuals, especially among female adults (Hernberg et al. 2014).
S. chartarum was found to emit MVOCs on gypsum wallboard and ceiling tiles
(Betancourt et al. 2013). Most of the MVOCs emitted by S. chartarum were alco-
hols, ketones, ethers, and esters. Anisole (methoxybenzene) was emitted from all
strains of S. chartarum with a maximal concentration observed when the strains
were incubated for 7 days. Betancourt et al. (2013) suggested that MVOCs are suit-
able markers for fungal identification and could be used for early detection of
hidden molds because MVOCs easily diffuse through weak barriers, such as wall-
paper (Betancourt et al. 2013).
Polizzi et al. (2012) studied MVOCs collected in water-damaged buildings to
evaluate their use as possible indicators of indoor fungal growth using two sampling
methods. Their results showed that dynamic headspace absorption using the Tenax
method is more sensitive than Solid-Phase Microextraction. The detected MVOC

profiles allowed them to identify Aspergillus versicolor group, Aspergillus ustus,
and Eurotium amstelodami based on species-specific MVOCs or MVOC patterns.
MVOCs produced by Chaetomium spp. and Epicoccum spp. were different from 76
fungal strains of various genera. This study indicated that chemotaxonomic dis-
crimination of fungal species may be used as a supplementary method to the classi-
cal morphological and molecular identification techniques for research on indoor
microfungi (Polizzi et al. 2012).
An animal study found that two fungal volatiles (E)-2-octenal and oct-1-en-3-ol
had cytotoxic effects on murine bone marrow stromal cells and exposure to both
(E)-2-octenal and oct-1-en-3-ol led to a shift to unsaturated fatty acids and lower
cholesterol levels in the cell membrane (Hokeness et al. 2013). The results indicated
that the MVOCs increased membrane fluidity. These changes to the cell membrane
contribute to the failure of normal cell function. Considering the importance of bone
marrow stromal cells to the appropriate development and activation of immune
cells, the results of this study provide crucial information to understand the mecha-
nism at a cellular level for how exposure to MVOCs may lead to immune-related
disease conditions (Hokeness et al. 2013).
Matysik et al. (2008) studied MVOCs emitted from six microfungal species
Aspergillus versicolor, A. fumigatus, A. niter, Penicillium expansum, P. chrysoge-
num, and Cladosporium cladosporioides on DG 18 and wet wallpaper. There are
differences in MVOCs emitted by the fungi growing on wet wallpaper and DG18.
Fungi growing on wet wallpaper had changed MVOC patterns with less signals and
significantly reduced emission rates. The authors also demonstrated that some
MVOCs are species specific on wallpaper, such as 2,4-pentandione for A. fumigatus
and 1,3-dimethoxybenzene for A. versicolor (Matysik et al. 2008).
Bingley et al. (2012) detected more than 150 volatile compounds from 16 fungal
strains (mainly Aspergillus spp. and Penicillium spp., also including Stachybotrys
chartarum, Trichoderma, Alternaria sp., and Cladosporium sp.) isolated from cin-
ematographic film using headspace solid-phase micro-extraction coupled with Gas
Chromatography–Mass Spectrometry. Three MVOCs, 1-octen-3ol from 13 of the
isolates, 3-octanone from 10 of the isolates, and 3-octanol from 4 isolates, are indic-
ative of viable fungal growth on the cinematographic film. The study is significant
in preventing unnecessary discarding of valuable historical film due to health and
safety concerns regarding spore inhalation and would allow safe handling (Bingley
et al. 2012).
A new online MVOC database (http://bioinformatics.charite.de/mvoc/) was
designed for microbial volatiles and their emitting organisms (Lemfack et al. 2014).
Lemfack et al. (2014) reported 846 compounds (5431 synonyms), which are pro-
duced by 349 bacterial and 69 fungi species. Many fungi compiled in the MVOC
database are species of Aspergillus, Penicillium, and Trichoderma (mVOC 2015).
This database is a significant start for MVOC research in the future, despite the fact
the newly published MVOCs studies may not be found in the MVOC database.
392 C. Yang et al.
Infections Caused by Indoor Fungi
A wide variety of fungi have been observed by the authors or reported in the litera-
ture growing in the indoor environment (Samson 1999; Li and Yang 2004a; Li et al.
2013; Samson et al. 2011). They include members of the Myxogastria (formerly
known as Myxomycota) (e.g., Stemonitis spp.), the Zygomycetous fungi (e.g.,
Rhizopus stolonifer, Mucor spp., Syncephalastrum racemosum), the Ascomycota
(e.g., Ascotricha erinacea, Ascotricha chartarum, Chaetomium spp., Emericella
spp., Eurotium spp., and Peziza domiciliana), the Basidiomycota (e.g., Asterostroma
cervicolor, Coprinus spp., Gloeophyllum spp., Sistotrema brinkmanii, Schizophyllum
commune, and Serpula lacrymans), and many species belonging to asexual states of
Ascomycota (Li and Yang 2004b; Li et al. 2013; Samson et al. 2011; Schmidt
2007). Many of these indoor fungi have been associated with allergic diseases,
asthma, hypersensitivities, occupational respiratory diseases, and other syndromes
(Li and Yang 2004a; Yang and Johanning 2007; Ellis and Day 2011; Hodgson and
Flannigan 2011). More importantly, many indoor fungi are also known human
pathogens. A table of pathogenic fungi compiled from several different sources was
provided (Li and Yang 2004a). A table of infectious fungi is available in the article
titled “Respiratory tract infections caused by indoor fungi” (Summerbell 2011). The
focus of this section is on the importance of infections by these fungi in the indoor
environment.
Although the health impacts of damp indoor environments and resulting fungal
growth have been extensively reviewed (Favata et al. 2000; Li and Yang 2004a;
Yang and Johanning 2007; Ellis and Day 2011; Hodgson and Flannigan 2011;
Storey et al. 2004; WHO 2009; IOM and Health 2004) and guidelines for control of
infectious agents, including fungal pathogens, in healthcare facilities are available
(CDC 2003), there has been little concern expressed regarding the home and work
environments where sensitive individuals or immune-deficient patients are likely to
spend significant amounts of their time. With the sensitive and immune-deficient
populations at almost 20 % of the US population in 1996, these populations were
expected to increase significantly because of increases in life span and the number
of immunecompromised individuals (Gerba et al. 1996).
Summerbell (2011) described three categories of fungal respiratory tract infec-
tions caused by virulent fungal pathogens, Pneumocystis (a genus of yeast-like,
atypical fungi), and opportunistic fungal pathogens. The first category includes
Blastomyces dermatitidis, Histoplasma capsulatum, Paracoccidioides brasiliensis,
Coccidioides immitis and C. posadasii, Cryptococcus neoformans and C. gattii,
Sporothrix schenckii, and Penicillium marneffei. The second category includes
Pneumocystis species that are contagious and cause respiratory infections, usually
in immunocompromised individuals. The third category includes many opportunis-
tic species, primarily a few species in the genus Aspergillus.
Among the first category, nine fungi are considered virulent and capable of
infecting healthy individuals. Their number is relatively small and they are usually
limited in their geographical area of distribution or grow in some unique environ-
ments, such as avian excreta or bat guano. However, there have been few reports
confirming these fungi in fact growing in typical indoor environments. Because
their distributions are usually endemic to certain geographical areas, this indicates
that they may have certain growth requirements that are unique to their environment
for their presence. Their detection and isolation may be limited or restricted by such
requirements.
Among the first category, the only species that is considered to have a wide dis-
tribution is Sporothrix schenckii, which is often associated with vegetative debris
(including dried straw, dried reeds, or dried Sphagnum peat moss). The infections
by S. schenckii are mostly subcutaneous abscesses but rarely respiratory or through
airborne spores (Summerbell 2011). The other eight species are either endemic
within certain geographic regions, associated with avian excreta (Tille 2014) or
with certain animals (e.g., Penicillium marneffei). Because of their saprobic nature,
all nine species are potentially capable of growing indoors when their environmen-
tal needs are present. However, a few cases of infections caused by these fungi due
to their growth indoors have been reported. Cases of infections due to the introduc-
tion of spores from outdoors or other sources were discussed by Summerbell (2011).
An unusual case of blastomycosis was reported in Ontario, Canada. Blastomyces
dermatitidis was isolated from a petroleum filtering shed laden with diatomaceous
earth (Bakerspigel et al. 1986). The petroleum filtering shed is not a typical indoor
living or work environment. However, this does suggest that buildings within the
geographical distribution areas of the fungus may harbor it if the indoor environ-
ment allows the presence or even the proliferation of this fungus. Histoplasma cap-
sulatum, Cryptococcus neoformans, and C. gattii are the three species that have
been confirmed capable of growing indoors, particularly in association with roost-
ing birds and bats (Summerbell 2011).
In the second category, Pneumocystis is a genus currently including at least two
species, P. jirovecii [reported as P. jiroveci prior to 2009 (Stringer et al. 2009)] and
P. carinii. P. jirovecii is reserved for the isolates causing human Pneumocystis cari-
nii pneumonia (PCP), while P. carinii is applied to those causing PCP in rodents
(Stringer et al. 2002; Tille 2014). Pneumocystis is difficult to grow in culture outside
the lung (Stringer et al. 2002; Tille 2014). There has been no report of Pneumocystis
species growing outside of a host or in water-damaged environments.
In the third category, there are many species that are known or documented to
grow indoors. A list of fungi, other than endomycetous yeasts which have been
confirmed as causing respiratory tract infection, sinusitis, or disseminated infec-
tions potentially invading the lung in humans, includes species in the Zygomycetous
fungi, the Ascomycota and its anamorphs, and the Basidiomycota and its anamorphs
(Summerbell 2011). The list includes many species that are well documented as
capable of growing indoors. The most important taxon in this group is the genus
Aspergillus.
Although the genus Aspergillus has been extensively studied by mycologists
(Thom and Church 1926; Thom and Raper 1945; Raper and Fennell 1965; Klich
2002; Samson and Pitt 1990; Pitt and Hocking 2009; Samson and Varga 2007;
Bennett 2010), many taxa within the genus were still not well delineated until the
394 C. Yang et al.
last 10 years. The most medically important species in the genus, Aspergillus fumig-
atus, was generally regarded as a very variable species in morphology (Pringle et al.
2005; Hong et al. 2005). In fact, many common species in the genus, such as A.
niger and A. terreus, are variable and often treated as a complex (Geiser et al. 2007).
Significant efforts have been made to better define the species taxa in the genus
Aspergillus using molecular techniques in an integrated taxonomic approach (Hong
et al. 2005; Geiser et al. 2007; Samson and Varga 2007).
The genus Aspergillus comprises approximately 285 species (Seifert et al. 2011),
of which 34 have been associated with human disease. Historically, A. fumigatus
caused 90 % of aspergillosis cases (Barnes and Marr 2006). Aspergillus fumigatus
is considered both a primary and opportunistic pathogen (Nierman et al. 2005). In
immunocompromised individuals, the incidence of invasive infection can be as high
as 50 % and the mortality rate is often about 50 % (Denning 1998). Increasingly,
aspergillosis is caused by non-fumigatus species (Barnes and Marr 2006).
Aspergillus spp. were reportedly associated with infections occurring after hemato-
poietic stem cell transplantation (HSCT) included A. fumigatus (56 % of cases), A.
flavus (18.7 %), A. terreus (16 %), A. niger (8 %), and A. versicolor (1.3 %) (Barnes
and Marr 2006).
Within the genus Aspergillus, A. fumigatus, A. flavus, A. terreus, A. niger, and A.
nidulans are the primary causative agents of human infections (Dagenais and Keller
2009). Four species, A. fumigatus, A. flavus, A. terreus, and A. nidulans, accounted
for the majority of Aspergillus infections (Summerbell 2011). One of the common
traits among these species is that they are rapid growers at a body temperature of 37
°C (Pitt and Hocking 2009). Such a trait has been shown to correlate with pathoge-
nicity (Dagenais and Keller 2009).
Of particular concern is that some aspergilli have been found to develop resis-
tance to antifungal treatments. A. terreus is resistant to amphotericin in vitro. In
addition, other species with variable susceptibilities to antifungal agents are being
described (Barnes and Marr 2006; 2006). Denning and Perlin (2011) reported
increasing resistance of Aspergillus fumigatus to an azole drug, itraconazole.
Snelders et al. (2012) reported that resistance of Aspergillus fumigatus to medical
triazoles was due to cross-resistance from triazole fungicides.
Aspergillus fumigatus is found in composting vegetation, wood chips, garbage,
and other materials at above ambient temperatures. Its spores are common in out-
door air and in carpet and mattress dusts. It can grow on warm, wet building, and
finishing materials such as wallpaper (Samson et al. 2011). It has been reported
from food products, such as oilseeds, soybeans, vegetables, nuts, coffee beans, cere-
als, meat products, and cheeses (Pitt and Hocking 2009). However, its confirmed
growth in the indoor environment has been very limited, probably because there is
usually very little decaying vegetation indoors.
Although A. fumigatus is one of the most important opportunistic microfungi to
infect humans, little is known about its ecology, its dissemination and dispersal, and
its periodicity. It was indicated that all humans will inhale at least several hundred
airborne spores of Aspergillus fumigatus per day (Latgé 1999). However, the sug-
gestion was based on three references of limited studies. In review of the three refer-
ences cited in the report, there are major discrepancies of the claim. Chazalet et al.
(1998) reported 0 to 3 conidia/m3 of A. fumigatus using impactors on Sabouraud
medium. Goodley et al. (1994) conducted a 1-year study to monitor the frequency
of spores of Aspergillus spp. in a hospital ground in the London area. The study
involved once a week air sampling using a Surface Air Sampler for 2–3 min between
2 and 4 pm. On a monthly basis, airborne spores of A. fumigatus varied from 0 to
273 CFU/m3, with the highest concentration in March. Hospenthal et al. (1998)
used an Andersen single-stage impactor and Czapek-Dox agar to monitor airborne
spores of Aspergillus species in the Washington DC area. The sampling was carried
out at an average of three times per month over a 54-week period. Results of A.
fumigatus and A. flavus were grouped together. Weekly concentrations varied from
0 to 12 CFU/m3. An average of 2 CFU/m3 were recovered for A. fumigatus and A.
flavus combined. In addition, there was no seasonal variation observed; variations
in concentrations from different locations were in the graphic presentations. Because
all three studies were based on limited scopes and intermittent sampling, it is very
difficult to suggest or to predict an average airborne concentration of A. fumigatus.
This raises the important issue of how much do we know about the ecology of this
important opportunistic human pathogen.
Mullins et al. (1984) conducted intermittent sampling of airborne Aspergillus
fumigatus spores using Andersen samplers on a 3 days/week schedule over a
12-month period at Cardiff, UK, and St Louis, USA. On average, A. fumigatus
spore concentrations were 15 CFU/m3 in St Louis and 11 CFU/m3 in Cardiff.
Seasonal variations were observed with highest concentrations in October for St
Louis and in November for Cardiff.
Aspergillus flavus is most likely from an agricultural setting and rarely growing
on building materials (Samson et al. 2011). It has only been detected growing in the
indoor environment on a couple of occasions by the authors. Aspergillus terreus is
common in tropical and subtropical regions and uncommon on building materials
(Klich 2002; Samson et al. 2011). There is little indication that it grows indoors but
has been isolated from indoor dust (Samson et al. 2011). Aspergillus niger is report-
edly not generally associated with contaminated building materials (Samson et al.
2011) but has been observed on occasions to grow on water-damaged books and
sheetrock wallboards. Emericella (Aspergillus) nidulans is common in tropical and
subtropical regions (Klich 2002; Samson et al. 2011). It has been observed on occa-
sion growing on water-damaged building materials, e.g., sheetrock wallboard, by its
cleistothecia, globose to subglobose Hulle cells, and red and bi-flanged
ascospores.
In addition to causing respiratory infections and diseases, fungal contaminations
in the indoor environment can impact human health in other ways. Reports of fun-
gal infections caused by contaminated injectable medicines, surgical wounds, or
contact lens solutions have been increasing. Faulk and Lesher (1995) reported that
a patient receiving injections of prednisone corticosteroids developed draining
lesions. Biopsies and isolations were made. An atypical mycobacterium,
Mycobacterium fortuitum, and a dematiaceous fungus, Phialophora verrucosa,
were recovered from the lesions and identified (Faulk and Lesher 1995). Two cases
396 C. Yang et al.
of Exophiala infections of the subcutaneous tissues in organ transplant patients

were reported by Gold et al. (1994). Outbreaks of fungal infections were reported in
the USA due to fungal contaminations in contact lens solutions and injectable drugs.
These outbreaks have caused many cases of fungal infections, including fungal
meningitis and fungal keratitis.
In 2002, five cases of systemic fungal infection caused by Exophiala (Wangiella)
dermatitidis occurred in patients receiving injectable steroids were reported
(Casadevall and Pirofski 2013). In one case, the infection was not evident until 152
days after injection of the contaminated solution. Four cases developed into menin-
gitis, while the fifth case was sacroiliitis. The outbreak was traced back to a single
compounding pharmacy, which was later found by a governmental agency to have
inadequate quality control for sterility (CDC 2002).
Exophiala (Wangiella) dermatitidis is cosmopolitan and commonly known as a
black yeast which is an inhabitant of cool moist soils but has been found in sinks,
drains, and steam baths. It has also been isolated from steam baths and hot tubs
(Matos et al. 2002; Summerbell 2011). It can cause fatal infections of the central
nervous system in otherwise healthy, mainly adolescent individuals in East Asia
(Samson et al. 2011). The source of E. dermatitidis contamination was not reported.
In 2006, the US CDC reported an outbreak of fungal keratitis among 164 con-
firmed patients in 33 states and 1 US territory. Microbial corneal infection is a rare
but is a serious complication that can lead to permanent vision loss or the need for
corneal transplantation. In this outbreak, corneal transplantation was required or
planned in 55 cases (34 %). One hundred fifty-four (94 %) of the confirmed patients
wore soft contact lenses. Use of a specific brand of contact lens solution was impli-
cated because case patients were found to be significantly more likely than controls
to use the brand solution. Fusarium was not recovered from the factory, warehouse,
solution filtrate, or unopened solution bottles. Implicated solution was not clustered
in lots or in time. Among 39 isolates tested, at least 10 different Fusarium species
were identified, comprising 19 genotypes (Chang et al. 2006). Fusarium spores are
not uncommon but usually found at low densities in outdoor air during growing and
Fall seasons. In a Canadian study, using a high-throughput jet sampler Fusarium
avenaceum, F. graminearum, and F. sporotrichioides were found to account for 93.9
% of the total Fusarium airborne spores populations during the sampling period.
Nine other Fusarium species at low frequencies were also detected in the study
(Martin 1988). In another Canadian study, the researchers detected spores of several
Fusarium species, including F. graminearum, F. crookwellense, F. sporotrichioides,
F. moniliforme, F. equiseti, F. subglutinans, and F. culmorum within a 20-day
sampling period in July, 1994 (Fernando et al. 2000). These findings plus the iden-
tifications of 10 different Fusarium species and 19 genotypes strongly suggest that
the source of contaminations was more likely from airborne spores than systematic
contamination.
Another outbreak caused by fungi was reported in 2012 from patients receiving
contaminated steroid methylprednisolone acetate (MPA) injections. The outbreak
was first reported in Tennessee where sixty-six cases were reported with a total of
22 patients that had laboratory confirmation of Exserohilum rostratum infection (21

patients) or Aspergillus fumigatus infection (1 patient) (Kainer et al. 2012). The
CDC and Food and Drug Administration (FDA) announced that E. rostratum had
been identified in two lots of unopened vials of methylprednisolone.
The CDC expanded the investigation to other affected States and reported the
outbreak of fungal meningitis and other infections among patients who received the
contaminated preservative-free MPA injections from the New England
Compounding Center in Framingham, Massachusetts. Chiller et al. (2013) reported
clinical findings of 328 patients without peripheral-joint infection. It found that 265
(81 %) had central nervous system (CNS) infection and 63 (19 %) had non-CNS
infections. Laboratory evidence of E. rostratum was detected in samples from 96 of
268 patients (36 %). For those with CNS infections, strokes were associated with an
increased severity of abnormalities in cerebrospinal fluid (P < 0.001). Non-CNS
infections were more frequent later in the course of the outbreak (median interval
from last injection to diagnosis, 39 days for epidural abscess and 21 days for stroke;
P < 0.001), and such infections developed in patients with and without meningitis.
The authors warned that fungal infections caused by epidural and paraspinal injec-
tion of a contaminated glucocorticoid product can result in a broad spectrum of
clinical disease (Chiller et al. 2013).
The final report of the CDC’s investigation included not only fungal meningitis,
but also localized spinal or paraspinal infections, and infections associated with
injections in a peripheral joint space, such as a knee, shoulder, or ankle. The index
case had Aspergillus fumigatus meningitis. Subsequent reports showed that the pri-
mary fungus identified in patients is Exserohilum rostratum (Lockhart et al. 2013).
Exserohilum rostratum is considered an extremely rare cause of human fungal dis-
ease (Summerbell 2011). The final statistics shows that the outbreak infected 751
people in 20 states and caused 64 deaths. The injections were found to be contami-
nated with Exserohilum rostratum (CDC 2013).
Although ubiquitous, members of the genus Exserohilum are rarely pathogenic
to humans. Only three species have been shown to parasitize humans: E. rostratum,
E. longirostratum, and E. macginnisii. The most common type of infections are
sinusitis and skin infections, though also a few cases of cerebral abscesses, keratitis,
osteomyelitis, prosthetic valve endocarditis, and disseminated infection have been
described. They described a child undergoing treatment for acute lymphoblastic
leukemia (ALL) that was infected by Exserohilum rostratum, causing cutaneous
phaeohyphomycosis. The infection was traced to contaminated intravenous dress-
ings. Treatment of the infection is primarily based on aggressive surgical removal
combined with antifungal therapy (Saint-Jean et al. 2007).
In the case of the outbreak caused by Exserohilum rostratum, presumably from
an environmental source, the fungus was not on the list provided by Li and Yang
(2004a, b). It was listed in Summerbell’s table as “opportunistic” and “rare” in caus-
ing respiratory tract infection. The fungus was reported as common on grass and
many other plants, substrates, and soil (Ellis 1971; Sivanesan 1987). In fact, it is one
of the three known pathogenic species in the genus (Saint-Jean et al. 2007). It
398 C. Yang et al.
appears that a rare, opportunistic fungus can cause a major outbreak if given the
opportunity and under an unfortunate situation.
New Technologies
New technologies often provide better methods or tools to assist us to better study
indoor microfungi and associated health effects. No doubt, the proper application of
newly emerged technologies is beneficial to indoor fungi and aeromycological
studies.
Raman Microspectroscopy-Based Identification of Individual Fungal Spores has
been used to develop a reference library of Raman spectra from a number of micro-
fungi typically associated with damp indoor environments. The acquired reference
spectral library has subsequently been utilized to identify individual spores of
microfungi via direct comparison of the spore Raman spectra with the reference
spectral signatures in the library. In addition, the distinct peak structures of Raman
spectra provide detailed understanding of the overall chemical composition of
spores. Potential application of this novel methodology is anticipated in the fields of
public health, forensic sciences, and environmental microbiology (Ghosal et al.
2012).
Nanotechnology–Effectiveness of antimicrobial nanometals has been exten-
sively studied recently (Yu et al. 2013). Some of the studies explored the potential
of using nanotechnology for management of indoor molds. Yu et al. (2013) showed
that Ag concentration to inhibit the germination and growth of Aspergillus niger
conidia of 5 wt% nano Ag catalyst was 65 mg/mL and ozone has a synergetic effect
on nanometals’ antifungal efficacy.
High-throughput sequencing technology has been applied to study compositions
of indoor molds. It is a very good supplementary method to traditional culturable
methods for indoor mold studies, since many fungi cannot be cultured or grow
poorly on artificial media. A number of studies have used this technology to study
indoor fungi (Adams et al. 2013a; Amend et al. 2010).
Every method has its pros and cons. There is no exception for high-throughput
sequencing. Adams et al. (2013a) found that the method may lead to bias in com-
munity richness and composition when high abundance of a few fungal taxa of the
dust samples differs to a large degree. To address this shortcoming, Adams et al.
(2013c) used UPARSE, a new method aimed at clustering globally trimmed
sequences into operational taxonomic units (OTUs) with a focus on reducing OTU
inflation. With this method the number of OTUs was dropped from 1305 taxa in
QIIME (Protocol S1) to 966 in UPARSE for indoor fungi.
Their results showed the composition detected on residents’ foreheads had a
surprising richness of nonresident fungi, including plant pathogens such as Claviceps
purpurea, ergot. However, they opined that it is unlikely the majority of the fungi
would grow on the indoor surfaces which seems to be more or less passive collec-
tors of airborne fungi of putative outdoor origin, while some fungi did grow on typi-
cal household surfaces, particularly on drains and skin (Adams et al. (2013c).
Future Perspectives
The research, especially the report by Amend et al. (2010), in the last decade raises
some important questions. Is it possible that some indoor fungal species evolve
independently from their outdoor populations of the same taxon? If so, can we
determine the evolutionary rate at which the indoor species evolve? How do indoor
microfungi evolve? Is there any difference between the evolutionary directions of
fungi indoors and the ones on natural substrates outdoors? Should we still empha-
size that all indoor fungi have origins of outdoor sources?
Morphology-based methods and gene-based methods are supplementary. Each
has their advantages and disadvantages. One should not exclude the other. After all,
morphology-based or classic fungal taxonomy is the basis of gene-based fungal
systematics.
The exact number of microfungi in indoor environments is severely underesti-
mated and remains unclear. Systematic studies on indoor microfungi should be car-
ried on in local areas and worldwide.
House Dust Mites and Indoor Molds
Do we really know the number of species of indoor mites, except for the three
known species which have been studied for their allergenicity in the past and 11
other species reported from indoor environments? Our personal observation has
suggested that some soil mite species may occur on building materials with water
damage. Soil ecological studies showed that mites feed on organic matter which
was partially decomposed by fungi. Is this relationship present on building materi-
als indoors also? What are the ecological roles of the mites in indoor environments?
What are the exact relationship and interaction between domestic mites and micro-
fungi in indoor environments? It seems that mites are attracted to fungal colonies.
Are the mites indoors chemotaxic? Are MVOCs, mycotoxin, or chitin involved in
chemotaxis?
Nanoparticles are particles between 1 and 100 nm in size. They are also able to
pass through cell membranes in organisms, and their interactions with biological
systems are relatively unknown. Their sizes are similar to protein (SCENIHR 2015).
Nano-sized fungal fragments should be studied to understand whether they still
carry allergens and mycotoxins and what kinds of health effects result from expo-
sure to them.
400 C. Yang et al.
Nematodes, Termites, and Carpenter Ants
Nematodes are very common in soils. Nematodes were observed on fungal colonies
on wet dry walls damaged by a flood. Some nematodes can feed on fungi. Colonies
of Botrytis cinerea have been used as a food source to cultivate pine wood nema-
todes for studying pine wood nematode wilt (Futai 2013; Wu et al. 2013). A number
of fungi, such as Arthrobotrys oligospora, Dactylaria candida, Monacrosporium
cionopagum, and Nematoctonus geogenius, can trap or parasitize nematodes
(Barron 1977; Xie et al. 2010). Many residences have a crawl space with a bare soil
surface. It provides nematodes access to indoor building materials with fungal
infestation. It is not clear whether nematodes play any roles in indoor mold infesta-
tion and their interaction with indoor molds.
Slime Molds
Quantitative real-time polymerase chain reaction (QPCR) for detecting indoor fungi
and Environmental Relative Moldiness Index (ERMI) to screen indoor environ-
ments for molds were DNA-based methods developed in the late 1990s and 2000s,
respectively (Haugland et al. 1999; Vesper et al. 2007). Although QPCR primers
and probes have been developed for detecting 120 fungal species, commercial ser-
vice of QPCR is normally limited to a panel of 35 microfungal species. This method
is accepted in indoor mold research and investigations. ERMI was developed using
ca. 1000 dust samples collected from US houses to screen indoor environments for
molds (Vesper et al. 2007). However, ERMI is still subject to debate and further
evaluation and validation are necessary. It is questionable whether ERMI should be
applied to nonresidential environments or not. These two methods provided alterna-
tives to morphology-based methods for indoor microfungi research and investiga-
tion. Fungal systematics is developing and some indoor fungi-related genera may
be redelineated, such as Cladosporium cladosporioides sensu lato (species com-
plex) and Penicillium chrysogenum sensu lato. Thus, molecular methods should be
fine-tuned or updated following the advancement of fungal systematics. It is neces-
sary to verify the specificities of the primers and probes of qPCR for detecting
indoor fungi based on the latest development of fungal systematics. It will advance
our understanding of indoor molds and their effects on public health.
No method is perfect. The same principle is also applicable to DNA-based meth-
ods. DNA-based methods, comparable to morphology-based methods and other
methods, have demonstrated great advantages but also showed their limitations.
Morphology- and DNA-based methods are supplementary. Pitkaranta et al. (2008)
conducted a study comparing DNA sequence, cultivation, and quantitative PCR
methods. The results indicated that the three methods were complementary to each
other and combined results from the three methods depicted a more comprehensive
picture of diversity and population of indoor fungi than the one of any individual
method produced. Will and Rubinoff (2004) pointed out that “DNA sequence data
are an important and powerful part of taxonomy and systematics. Molecular data
have an indisputable role in the analysis of biodiversity. However, DNA-based data
should not be seen as a substitute for understanding and studying whole organisms
when determining identities or systematic relationships.” It is important to follow
the latest development of fungal taxonomy and understand the advantages and inad-
equacies of all the methods available for indoor fungi investigation and research.
Up-to-date knowledge will help us to choose proper methods suitable to our objec-
tives and to develop new hypotheses for future research.
Fungal Infections
With the increase of sensitive populations, fungal infections, whether primary or

opportunistic, are much frequently reported. Although many cases of infections are
respiratory, more are reported from non-respiratory routes of infections such as
wound or injections. The fungal spores that can germinate and grow at 37 °C can
also grow in the human body. Monitoring and testing for indoor fungal pathogens
must take into consideration such a characteristic.
Aspergillus fumigatus is considered the most important opportunistic fungal
pathogen. It has major health impacts on the health of immunecomprimised indi-
viduals, such as cystic fibrosis patients (Latgé 1999; LiPuma 2010). However, there
has been no continuous, systematic, long-term study of its airborne spores: their
geographical distributions, seasonality and periodicity, yearly variations, and tem-
poral and spatial fluctuations. Without such information, hospitals and other health-
care facilities will find it very difficult to control infiltration of A. fumigatus spores
from outdoors.
With frequent outbreaks of fungal infections associated with injectable medi-
cines and eye care solutions, environmental control and hygiene of manufacturing
facilities as well as users’ education are critical to avoid future outbreaks.
Acknowledgment We are very grateful to Dr. James LaMondia, The Connecticut Agricultural
Experiment Station, USA, for reviewing the manuscript.
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Chapter 16
Biology of the Whiskey Fungus
James A. Scott and Richard C. Summerbell
Introduction
Since the industrial revolution, sooty discoloration has become an ever-apparent

feature of the exterior surfaces of buildings and monuments worldwide. Discoloration
arises both from the deposition of airborne, carbonaceous combustion products and
from the growth of melanized microbes on surfaces. The development of dark dis-
coloration on outdoor surfaces such as concrete, brick, and wood as well as many
other construction materials exposed to low levels of ethyl alcohol (ethanol) vapour
has been associated with the aging of spirits, particularly in areas of damp climate,
for over a century. Known as “warehouse staining”, this phenomenon differs little
from disfigurement caused by the soot that billowed copiously from factories during
the industrial revolution. But unlike the sooty accumulations arising from coal com-
bustion, warehouse staining is caused by a microbial biofilm consisting of thick,
black, confluent, crust-like colonies that extend over large, exposed outdoor sur-
faces. The substrates for warehouse staining encompass a wide range of materials,
including man-made items such as construction materials, fences, road signs, out-
door furniture, and vehicles. Natural materials such as vegetation and rock are also
affected. Although this biofilm probably exists in nature sporadically as isolated
J.A. Scott, Ph.D. (*)

Division of Occupational and Environmental Health, Dalla Lana School of Public Health,
e-mail: james.scott@utoronto.ca
R.C. Summerbell, Ph.D.
Dalla Lana School of Public Health, Gage Occupational and Environmental Health Unit,
e-mail: rsummerbell@sporometrics.com

DOI 10.1007/978-3-319-29137-6_16
414 J.A. Scott and R.C. Summerbell
microcolonies where its growth may be engendered by natural ethanolic vapours, it

is only in the unnatural flux of ethanol emissions from industry that the stains flour-
ish prominently.
Even though warehouse staining has been recognized for many years, the phe-
nomenon received sparse study until recently. We trace the history of study on
warehouse staining, provide a summary of the current state of knowledge on this
interesting phenomenon, and identify areas for future research.
Nineteenth Century
In 1872, Antonin Baudoin, director of the French Distillers’ Association, noticed

the accumulation of a soot-like blackening of walls around distilleries along the
Charente River in Cognac, France. This “plague of soot”, he observed, obliged the
distillery proprietors to engage in regular cleaning. As he described it, the town of
Cognac seemed as though it were covered in mourning crape—a reference to the
crinkled black silk fabric of the Victorian era costumes worn by grief-stricken wid-
ows (Roumeguère 1881). Curiously, distilleries downstream in the historical prov-
ince of Aunis to the northwest as well as those in the southern countryside were
unaffected. Knowledgeable in biology through his training as a pharmacist,
Baudoin judged the growth to represent a species of the fungus Xenodochus; he
provided specimens to Durrieu de Maisonneuve (Richon and Petit 1881). The lat-
ter, in consultation with Roumeguère, concurred in his diagnosis and considered
naming the fungus “X. baudoini” to honour Baudoin (Roumeguère 1881). A formal
description was never published. However, Roumeguère circulated specimens from
the collection in his Fungi Gallici exsiccati, Centurie XVI, No. 1695. As outlined
by Scott et al. (2007), Roumeguère’s description referred the specimen to Torula
conglutinata var. compniacensis. His collection labels, however, erroneously listed
the specimens as “T. conglomerata * compniacensis” (DAOM 66898, Fig. 16.1).
Fig. 16.1 Packet containing Centurie XVI, No. 1695 of Roumeguère’s Fungi Gallici exsiccati
(DAOM 66898). The name written on the packet is “Torula conglomerata * compniacensis”
despite that the name published by Roumeguère (1881) was “Torula conglutinata Corda var.
Compniacensis Richon”
16 Biology of the Whiskey Fungus 415
The transcription error on the packet was carried forward by Saccardo (1886) and
Crane (2001).
In 1878, following a detailed study of specimens observed under higher power
magnification, Baudoin reconsidered his characterization of the causal agent and
decided the organism was cyanobacterial rather than fungal, a species of the genus
Nostoc (Richon and Petit 1881). Nearly a decade after his initial discovery, Baudoin
published a pamphlet outlining his theories on the blackening phenomenon, attrib-
uting the growth to Nostoc (Baudoin 1893).
Baudoin was not alone in his early observation of warehouse staining. While
presiding over the June 14, 1878, meeting of the French Botanical Society,
physician-mycologist Gaspard Adolphe Chatin presented to the society examples of
tile and stone collected near spirit maturation warehouses in Cognac. The fragments
were blackened, he believed, by an unknown cryptogam. No further information
was provided in the meeting transcript (Chatin 1878). Richon and Petit (1881) later
reasoned that Chatin’s discovery could only have been the fungus they formally
described as Torula compniacensis.
Twentieth Century
In the century following the work of Richon and Petit (1881) and Roumeguère
(1881), there were few sporadic reports in the scientific literature relating to ware-
house staining. In discussing anthropophilic fungi, Moreau (1954) referred to the
warehouse staining fungus (“Torula conglutinata var. compniacensis”) as an example
of a saprotroph—in this case, one responsible for blackening tiles of houses in the
village of Cognac that were exposed to the alcoholic vapours emitted from its famous
distilleries. Although he was clearly familiar with the fungus and earlier work done
by Baudoin, Richon, and Petit, Moreau did not contribute any original observations.
An elegant study done by Annelisa Kjøller (1961) at the University of Copenhagen
reported the presence of a dense, confluent, black fungal colonization on asbestos-
cement roofing at the Heering Distillery in Dalby, Sjælland, Denmark. Direct
microscopic observations of mounts from these materials revealed a predominant
form that Kjøller considered to be a single entity, although she noted the absence of
typical hyphae or differentiated conidiophores. Kjøller (1961) identified the fungus
as Torula compniacensis, and the identification was verified at the time by Dr.
S.J. Hughes in Canada, after he had compared the collections to authentic material
representing this taxon from Roumeguère’s Fungi Gallici exsiccati, Cent. XVI, No.
1695 “Torula conglomerata * compniacensis” (DAOM 66898, Fig. 16.1). Later
examinations of two specimens from Kjøller’s study by Scott et al. (2007), one a
stone chip from the factory roof (DAOM 6687) and the other a fragment of asbestos
tile (DAOM 109435), confirmed Kjøller’s conclusions and supported the placement
of her collections in the genus Baudoinia J.A. Scott and Unter. as Baudoinia comp-
niacensis (Richon) J.A. Scott and Unter.
In addition to her microscopic observations, Kjøller (1961) prepared cultures by
spreading cells scraped from dried specimens on malt agar, incubating the cultures
at 14 °C, and examining the agar plates directly under the microscope after 1 week.
Using this method she successfully germinated cells that had been stored dry for up
to a year. Although Kjöller is undoubtedly the first scientist to have successfully
cultured the warehouse staining fungus, lamentably no cultures remain from this
work for verification by modern methods.
In her studies of germinating “chlamydospores”, Kjøller (1961) observed consid-
erable variation and distinguished three basic patterns of germination and matura-
tion. She found that adulteration of the isolation medium with alcohol, sherry, or
prune juice failed to alter the germination or growth pattern. Kjøller’s first pattern
was characterized by initial swelling of the chlamydospore and bursting of the orna-
mented wall followed by the production of elongating germ tubes associated with
greenish-yellow mucilage (e.g. Fig. 16.2). The structures matured after 3 weeks to
become roughened, monilioid chlamydospores similar in appearance to those she
observed on the roofing material. Her illustrations of germinating chlamydospores
(Kjøller 1961, Fig. 16.1) bear close resemblance to the description and illustrations
of Scott et al. (2007, Figs. 16.4 and 16.5). These collections likely correspond to
Baudoinia. The second pattern involved the direct germination of chlamydospores to
a budding, yeast-like phase that ultimately matured into dark, smooth, muriform
chlamydospores. These structures connote the genus Aureobasidium, which con-
tains ubiquitous, unspecialized yeast-like fungi encountered commonly in the mature
warehouse staining mycobiota (Ewaze et al. 2008b; Scott et al. 2007; Watson et al.
1984). In her third pattern, chlamydospores germinated to produce hyaline germ
tubes that became moniliform, and dark, smooth-walled muriform chlamydospores
at maturity. Kjøller interpreted these to be arthroconidial chains. While the identity
of this fungus is unknown, it is unlikely to be affiliated with the genus Baudoinia.
Fig. 16.2 Germ tube of

Baudoinia photographed in
transmitted light
microscopy demonstrating
the greenish coloration of
the nascent cell wall
(SCCM 2488428) (2000 ×)
Several years after the publication of Kjøller’s work, Auger-Barreau (1966) pub-
lished preliminary findings on the identity of the agent of warehouse staining from
Cognac, France, as a basis for later investigations of allergy-related health effects in
communities affected by the fungus. Auger-Barreau was initially sent a specimen
from a Cognac cellar which she examined microscopically and attempted to culture.
She described the thick, brown, felt-like growth as macroscopically resembling the
velvety interior context of the “amadou” fungus, Fomes fomentarius (L.) Fr.
However, her microscopic and culture examinations suggested that it consisted of
multiple species. When Auger-Barreau visited the warehouse to collect more speci-
mens, she observed two distinctive morphological patterns of growth. One pattern,
seen on the interior walls of cellars, conformed to her observations of the specimen
she was initially sent. The other, a black and soot-like growth, was restricted to
building exteriors (Auger-Barreau 1966). Auger-Barreau (1966) reasonably con-
cluded that the outdoor fungus was probably Richon’s T. compniacensis, whereas
the indoor fungus more closely resembled a species of Cladosporium. In all likeli-
hood, the latter collection represented the cellar fungus, Zasmidium cellare (Pers.)
Fr. (Chlebicki and Majewska 2010; Tribe et al. 2006).
Two decades after the work of Kjøller and Auger-Barreau, Watson et al. 1984
reported on the warehouse staining phenomenon from bond warehouses in Scotland.
They subjected scraping samples to culture and reported a suite of non-specialist fungi
that included Alternaria tenuissima (Kunze: Fr.) Wiltshire, Ascochyta sp., Aspergillus
terreus Thom, Aureobasidium pullulans (deBary) Arnaud, Cladosporium tenuissi-
mum Booke, Curvularia sp., Epicoccum nigrum Link, Phoma capitulatum Pawar
Mathur and Thirum., P. nebulosa (Pers.) Berk., P. tropica Schneider & Boerema, and
Pseudodiplodia sp. They likened the black staining to a polymicrobial biofilm named
“Fumago vagans” that has been known to develop on aphid honeydews.
Watson et al. (1984) provided a series of calculations on ethanol losses from the
bond warehouses they examined, projecting a 2 % loss in the first year of aging and
a 1 % loss in the second and subsequent years. Based on approximately 2.25 million
litres of whiskey in storage, they calculated the loss to correspond to about 90,000 l
over the first few years of maturation. They proposed a relationship between the
ethanol emission from bond warehouses and the development of black fungal
growth on surfaces, observing that the “growth is denser and extends further from
the warehouses following the direction of the prevailing wind”. They further noted
that growth was absent near unused warehouses and only initiated once the ware-
houses were filled with whiskey barrels. Although they were clearly aware of the
association of the cellar fungus with fugitive emissions of ethanol, Watson et al.
(1984) were unfamiliar with prior work on the whiskey fungus.
Twenty-First Century
In 2007, the warehouse staining fungus was again rediscovered by Scott et al. (2007)
during a survey commissioned by a Canadian distiller, of fungal colonists of out-
door surfaces near spirit maturing warehouses. They compared their environmental
samples against those of Roumeguère (1881) and Kjøller (1961), concluding that
all specimens consisted predominantly of what appeared to be a single taxon con-
forming to the description of Torula compniacensis. Using a technique similar to
that of Kjøller (1961), Scott et al (2007) successfully germinated single cells, isolat-
ing them in pure culture. Scott et al. incorporated 5 ppm ethanol in their primary
isolation medium to enhance recovery of the whiskey fungus by reducing the pro-
liferation of contaminant moulds. Microscopic examination of the resulting isolates
showed darkly pigmented, extremely slow-growing colonies that resembled the first
predominant pattern described by Kjøller (1961).
Scott et al. (2007) examined 13 specimens and 8 cultures collected from a range
of geographic localities. Phylogenetic analysis of several isolates based on nucSSU
rRNA gene sequences supported the erection of a new genus in the order
Capnodiales. They named the genus Baudoinia in recognition of Antonin Baudoin’s
early contributions to the study of the organism (Scott et al. 2007). They provided a
synonymy partly based on Crane (2001), although as Illana-Esteban (2013) later
noted that Crane (2001) and Scott et al. (2007) had incorrectly cited the journal
reference for T. compniacensis Richon. Firstly, both authors listed the journal title
Rev. Mycol. (Paris). The journal abbreviation Rev. Mycol. (Paris) refers to Revue de
Mycologie, published from 1936 to 1979; by contrast, Rev. Mycol. (Toulouse)
denotes Revue Mycologique whose publication ran from 1878 to 1906 and included
the article in question. Illana-Esteban (2013) also observed that T. compniacensis
Richon was actually initially published by Richon and Petit (1881) in the third vol-
ume of another journal, Brebissonia, in February of 1881. Roumeguère then
reprinted Richon’s diagnosis in the Revue Mycologique which appeared in July of
the same year. Thus, the basionym of B. compniacensis is T. compniacensis Richon
in Richon and Petit, Brebissonia 3(8): 115 (1881).
Illana-Esteban (2013) incorrectly attributed authorship of Richon’s reprinted
diagnosis to Baudoin. As published, the last sentence of Roumeguère’s article
appears to read: “Je distribue la nouvelle variété dans ma centurie XVI. M. Baudoin
et M.”, implying that Baudoin and an unnamed individual, “M.”, authored the arti-
cle (“centurie XVI” here referred to the 16th set of 100 specimens distributed in
Roumeguère’s Fungi Gallici exsiccati in which the specimen of interest was circu-
lated as No. 1695 and incorrectly labelled as T. conglomerata * compniacensis).
However, the actual concluding sentence of the article was truncated, with the
remainder erroneously transposed to the head of the following page. The final sen-
tence of the article thus correctly reads: “M. Baudoin et M. Paul Brunaud après lui,
ont bien voulu m’en approvisionner, C.R.” (“Mr. Baudoin, and Mr. Paul Brunaud
after him have generously supplied it to me, C.R.”), confirming Roumeguère as the
article’s author. The synonym T. conglutinata Corda var. compniacensis (Richon)
Sacc. in Roum., Rev. Mycol. (Toulouse) 3(11):17 (1881), was created in a footnote.
Scott et al. (2007) designated a representative of Roumeguère’s exsiccata, centu-
rie XVI, No. 1695 (DAOM 238773) as the lectotype of B. compniacensis and
selected a living culture obtained during their studies near the type locality, Cognac,
France, as epitype (UAMH 10808) from which DNA sequence and cultural charac-
ters were derived. The genus Baudoinia was shown by Scott et al. (2007) to be a
member of the Capnodiales on the basis of nucSSU sequence analysis. Although no

sexual state has been demonstrated, Crous (2009) recognized Baudoinia as an ana-
morph linked to the genus Mycosphaerella. Using nucLSU sequence analysis,
Crous et al. (2009) treated the genus in the Teratosphaeriaceae, and this determina-
tion was later accepted by Wijayawardene et al. (2012).
Based on sequencing of multiple gene regions in 16 collections of Baudoinia
originating from a range of geographic locations, Scott et al. (2016) described four
additional species in the genus: B. antilliensis, B. caladoniensis, B. orientalis and B.
panamericana. They indicated that the global ranges of these taxa appeared disjunct
at a continental scale but proposed that international trade in used, colonized aging
barrels may promote global redistribution of whiskey fungi.
Physiology
As its unique habitat purports, B. compniacensis is able to metabolize a range of

carbon sources including acetate and ethanol in addition to the simple sugar, glu-
cose. Glucose is used optimally in the range of 1–10 mM, and acetate (in the form
of ammonium acetate) is used optimally at a concentration of 1 mM (Ewaze et al.
2007). Ethanol is used optimally at a continuous concentration in the range of 40–50
mM (0.25 % v/v) in the presence of 5–10 mM of a variety of nitrogen sources, both
inorganic (e.g. ammonium chloride, ammonium nitrate, potassium nitrate, sodium
nitrate) and organic (alanine, asparagine, glutamate, glutamine, casamino acids).
Urea, however, was not utilized (Ewaze et al. 2007). Other simple alcohols were
used poorly or not at all (Ewaze et al. 2007).
Higher concentrations of ethanol were associated with sharply attenuated growth.
For example, the biomass yield of cultures was substantially diminished at a con-
tinuous ethanol concentration of 172 mM (1 % v/v) and was completely arrested at
860 mM (5 % v/v) (Ewaze et al. 2008a). Concentrations up to 2410 mM (14 % v/v),
while not permitting growth, were tolerated for a brief period (Ewaze et al. 2008a).
Concentrations of 3440 mM (20 % v/v) produced complete cell death for all expo-
sure time periods investigated (Ewaze et al. 2008a).
Brief exposure of dormant cells to a very low concentration of ethanol in liquid
form (5 mM; 290 ppm v/v), while suboptimal for vegetative growth, resulted in
optimal germination and colony formation (Ewaze et al. 2008a). A similar pattern
of optimized germination activation was noted for dormant cells exposed briefly to
ethanol vapour at a concentration of 10 ppm (19 mg m−3 at 25 °C) (Ewaze et al.
2008a). At this ethanol concentration, germination exceeded the non-exposed con-
trol by 200 %. Similarly, an airborne concentration of 1 ppm ethanol increased
germination by 140 %, and 100 ppb resulted in an increase in germination of 60 %.
Enhancement of germination of the fungus in the presence of alcohol, while sus-
pected, was not observed by Kjøller (1961).
Protein electrophoresis studies confirmed that both alcohol dehydrogenase II and
acetaldehyde dehydrogenase were produced when isolates of the fungus were
grown on ethanol as a sole carbon source, but were absent when the isolates were
grown on glucose (Ewaze et al. 2008a). Both the phosphorylated (active) and non-
phosphorylated (inactive) forms of the TCA cycle enzyme isocitrate dehydrogenase
were found when isolates of the fungus were grown on glucose. This enzyme is
responsible for the oxidative decarboxylation of isocitrate to oxalosuccinate, and its
presence in glucose-grown cultures confirms the normal functionality of the TCA
cycle. When cultures were grown on acetate, however, both forms were absent. In
their place, Ewaze et al. (2008a) confirmed the presence of isocitrate lyase and
malate synthase, two glyoxylate pathway enzymes that support the TCA cycle-
mediated conversion of acetyl-CoA to oxaloacetate, when the fungus is grown on
ethanol or acetate in the absence of glucose. Greene et al. (2014) identified a gene
sequence of one isocitrate lyase isoform closely linked with a gene cluster support-
ing L-tyrosine degradation.
Stress Responses
Ewaze et al. (2007) reported that hydrated cells of B. compniacensis were readily
killed at 52 °C. Preconditioning of hydrated cells at 37 °C prior to transfer to 52 °C
resulted in significantly improved survival (Scott et al. 2007) and coincided with the
production of several putative heat shock proteins (Ewaze et al. 2007). A compa-
rable pattern of altered protein expression was stimulated following brief exposure
to 7 % ethanol followed by high temperature incubation (Ewaze et al. 2007), sug-
gesting a role for ethanol in the induction of stress response systems. Production of
the stress-protective disaccharide trehalose was similarly stimulated by both sub-
lethal heat shock and ethanol exposure (Al-Naama et al. 2009). As expected, dried
cells demonstrated much greater tolerance of high temperature than hydrated cells,
retaining viability after 21 d at 70 °C but exhibiting complete cell death at 75 °C
(Ewaze et al. 2007).
Nuclear Genome
The nuclear genome of Baudoinia compniacensis is the smallest that has been docu-
mented for a member of the class, Dothideomycetes, consisting of only 21.88 mB
(Ohm et al. 2012). The genome is almost entirely non-redundant (0.4 % repetitive
content) and contains roughly 10,500 predicted proteins. It also features fewer puta-
tive coding genes, fewer genes with introns, and shorter distances between coding
genes than are found in other Dothidiomycetes (Ohm et al. 2012). Only 428 putative
pathogenesis-associated genes were found in B. compniacensis, including small
secreted proteins (67), proteins involved in secondary metabolism (12), carbohy-
drate active enzymes (283), secreted peptidases (28), and lipases (38) (Ohm et al.
2012). Two polyketide synthases, two non-ribosomal peptide synthetases, and eight
terpene synthase genes were predicted (Ohm et al. 2012). Several putative
mycotoxin-like genes were also identified including orthologues of Ds-Nor-1,
Ds-AvnA, Ds-AdhA (dothistromin), and Af-verA, Af-omtB (sterigmatocystin) (Ohm
et al. 2012). Leducq (2014) interpreted the relatively small genome and paucity of
pathogenicity-related genes in Baudoinia as signifying a saprotrophic ecology.
Degradation of the amino acid L-tyrosine by fungi is accomplished by two alter-
nate pathways that diverge from the intermediate, homogenisate. The first of these
parallels the AKU pathway found in mammals and yields fumarate and acetoacetate
following two additional enzymatic steps. The second yields pyruvate and fumarate
after four additional enzymatic steps. Considerable variability is present across the
fungal phylogeny with respect to the presence of one or both tyrosine degradation
pathways. When present, each is typically supported by a tightly linked gene cluster
whose pattern of inheritance closely reflects phylogeny. Using comparative genomic
analysis, Greene et al. (2014) demonstrated the presence of only the former path-
way in B. compniacensis. It comprised a cluster of eight putative genes, four of
which shared high homology with genes found in the phylogenetically distant
Exophiala dermatitidis, although gene arrangement and orientation were not con-
served. Two additional putative homologous genes encoding the glyoxylate path-
way enzyme isocitrate lyase and a membrane transport protein were likewise
common to the L-tyrosine degradation linkage clusters in both taxa but absent from
other taxa surveyed. These findings were interpreted as evidence of horizontal gene
transfer from an ancestor of the extremophilic, melanized fungus Exophiala derma-
titidis (Chaetothyriales) to the Baudoinia clade (Wisecaver and Rokas 2015).
Mitochondrial Genome
The mitochondrial genome of B. compniacensis is similarly comparatively small in

size (26.0 kB) and encodes the 14 coding genes normally found in mycelial fungi
(ATP synthase subunits atp6, atp8, and atp9, cytochrome b, cytochrome oxidase
subunits cox1–cox3, and the NADH dehydrogenase subunits nad1–nad4, nad4L,
nad5–nad6) (Goodwin et al. 2014). A single additional unique open reading frame
(ORF) was identified that coded for an unknown protein. All 20 amino acids were
represented by 26 putative tRNA genes variously oriented in the forward and
reverse direction (Goodwin et al. 2014). The compact nature of the mitochondrial
genome reflects the absence of introns and superfluous ORFs.
Mating System
Riley et al. (2014) examined the nuclear genome sequence of B. compniacensis in

an effort to evaluate the mating system of the species. They identified a putative
sequence corresponding to the MAT1-2-1 gene encoding a high mobility group
(HMG) domain. The solitary presence of the MAT1-2-1 idiomorph implies a hetero-
thallic mating system, although a sexual state has so far not been recorded.
Ecology
Baudoinia forms a thick, soot-like crust on ethanol-exposed outdoor surfaces near

distillery aging warehouses and commercial bakeries (Scott et al. 2007). The sur-
faces most affected tend to be highly sun exposed and periodically subject to con-
densing conditions. Although the growth appears indiscriminately on a wide range
of surfaces ranging from stainless steel and paint to masonry and vegetation, cer-
tain materials remain predictably devoid of growth. For example, Watson et al.
(1984) remarked on the absence of fungal disfigurement in the drainage plane of
copper or zinc-clad surfaces, indicating the inhibition of growth by low concentra-
tions of ions of these metals (Fig. 16.3). Likewise, anecdotal reports have sug-
gested a low tolerance to salt based on observations that oceanfront elevations of
bond warehouses manifest less growth than landward-facing walls. Ewaze et al.
(2007), however, showed isolates of Baudoinia to withstand sodium chloride con-
centrations up to 2 M.
The genus is widely distributed geographically and is known so far from the
Americas, Europe, and Asia (Scott et al. 2007, 2016). The range of localities where
the fungus has been collected indicates a proclivity for damp riparian and coastal
climates. Although the fungus remains unknown from non-industrial habitats, the
Fig. 16.3 Exterior wall panel finished in white vinyl siding. The portion of panel to the left is situ-
ated beneath a cap of metallic copper flashing, whereas copper flashing is absent at the top of the
panel section at right. The area of siding located in the drainage plan beneath the copper flashing
shows substantially less colonization of Baudoinia compniacensis than the unflashed portion. This
is consistent with the observation of Watson et al. (1984) and their suggestion that copper and zinc
may have antimicrobial potential in the management of warehouse staining (Bar = 10 cm)
existence of a natural, non-industrial habitat is highly probable given that the

Baudoinia clade diverged within the Capnodiales between 55 and 75 million years
ago (Ohm et al. 2012). Spirit production and maturation, by contrast, has only been
practised for the past few centuries (Rogers 2011, 2014). Scott et al. (2007) specu-
lated that the clade may have arisen in non-anthropogenic, ethanol-exposed envi-
ronments such as fruit drops and other naturally occurring composts.
Ethanol Vapour as a Habitat Determinant
Environmental ethanol emissions arise naturally in terrestrial environments from

the aerial parts of plants that are subject to drought and other stresses. Typical air-
borne ethanol vapour levels in North America have been reported around 5 ppb
(roughly 9 μg m−3) (Farmer and Dawson 1982; Snider and Dawson 1985; Millet
et al. 2005). The failure of molecular-based environmental surveys to recover
Baudoinia sequences from environments lacking industrial ethanol emissions sug-
gests that the naturally occurring background levels of ethanol vapour common in
most terrestrial environments alone may be insufficient to engender the organism’s
ecological success on a scale sufficient to be noticeable. Clarification of this theory
awaits systematic study.
While Baudoinia colonizes surfaces exposed to a wide range of airborne ethanol
concentrations, laboratory studies have shown that it can grow entirely in the
absence of ethanol. Relatively high levels of ethanol vapour, such as those found
inside spirit aging warehouses coupled with the low levels of available moisture,
probably inhibit the growth of the fungus indoors or to halt its growth altogether. It
is noteworthy that the interiors of maturation warehouses typically lack any sign of
the fungus, yet the fungus will be found growing immediately outside the ware-
houses. Moderate ethanol levels, in the range of 5–10 ppm (9.5–19 mg m−3 at 25
°C), are optimally promotive of growth. The stimulation diminishes at successively
lower ethanol concentrations but remains perceptible at least as low as 100 ppb (190
μg m−3 at 25 °C).
The relationship between B. compniacensis and ethanol is complex. The fungus
can use ethanol as a nutrient. In addition, ethanol serves as a germination activator
and an inducer of cellular stress responses. Unlike the indoor-dwelling cellar fungus
Zasmidium cellare (Tribe et al. 2006), B. compniacensis grows outdoors, colonizing
ethanol-exposed substrates that experience extreme daily fluctuations in tempera-
ture and moisture (Scott et al. 2007). In Fig. 16.4, we propose a provisional model
for the role of ethanol in the diurnal cycle of Baudoinia. In this model, desiccated
cells are subject to moist air and cool substrates during the evening into the early
morning. The formation of dew on substrates serves to absorb airborne ethanolic
vapour concentrating the alcohol up to 10-fold. Hydrated, ethanol-exposed cells are
stimulated to germinate, and ethanol is then metabolized as a nutrient. At the same
time, the ethanol induces the production of cellular stress response mechanisms
including the synthesis of trehalose heat shock proteins. As sunlight warms the
Fig. 16.4 (a–d) Diagrammatic representation of the proposed generalized growth cycle of
Baudoinia, highlighting the role of ethanol vapour, moisture, and heat (left). The outer circle indi-
cates time of day divided into 24 hourly increments. Thickened hatch lines denote 0:00, 6:00,
12:00, and 18:00 h (clockwise from the left). The inner circle is sectioned according to the graph
(inset at lower right), depicting a prototypical temperature profile of an exposed outdoor substrate
during the growing season in a typical northern temperate region. (a) During the evening and early
morning, substrates bearing cells of the fungus become cool (blue, < 18 °C), leading to the forma-
tion of condensation near midnight (moon symbol). Ethanol, having a greater affinity for liquid
phase over vapour phase, readily dissolves in dew droplets, achieving an ethanol concentration up
to tenfold greater than that of the surrounding air (Ewaze et al. 2008a). Ethanol stimulates the
germination of dormant cells, serves as a nutrient for growth, and initiates cellular stress protective
physiology. (b) Sunlight exposure warms the substrate to average ambient temperature (green,
18–37 °C), evaporating the dew, dehydrating cells, and reinstating dormancy. (c) By midday (sun
symbol), the temperature of the sun-exposed outdoor substrate climbs towards its peaks (red, > 37
°C) (Scott et al. 2007). (d) Dry dormant cells may remain adherent to the substrate or be subject to
fragmentation (dashed arrow). The substrate cools (green) as the sun sets and the cycle repeats
substrate, the ethanol-rich dew evaporates and the fungal cells desiccate. As the
substrate warms further and the sun reaches its peak zenith angle, the dried, precon-
ditioned cells are subjected to peak midday temperatures. Then, as the sun sets, the
substrate temperature cools and the cycle repeats.
Dispersal
Despite the friable nature of dried colonies of Baudoinia, Scott et al. (2007) remarked
on the relative rarity of cells resembling B. compniacensis in air samples retrieved
around distillery aging facilities. This observation implies that mechanisms other
Fig. 16.5 (a–c) Molluscan grazing trails on colonies of Baudoinia on the surface of (a) discarded
stainless steel tank in an outdoor storage area (southwestern Ontario) and (b) precast concrete lamp
standard on a riverside boardwalk (Cognac, France). (c) Trails left by gastropods observed grazing
B. compniacensis mycelium on the surface of a truck of Betula sp. (Cognac, France) (Bars: a = 30
mm, b = 25 mm, c = 10 mm)
than aerosol propagation may contribute to dispersal. Some degree of vector-medi-

ated dispersal cannot be discounted. For example, during their study, Scott et al.
(2007) noted colonies in several geographic locations that bore signs of terrestrial
gastropod grazing where fungal material had been scraped from the substrate in pat-
terns resembling Brownian trails (Fig. 16.5a–c).
Taxonomic Composition
Early anecdotal reports attempted to characterize warehouse staining by culture

methods, recovering a number of common phyloplane fungi (e.g. Kjøller 1961;
Watson et al. 1984). Watson et al. (1984) did not connect their observations on
warehouse staining with earlier work on T. compniacensis. These authors misattrib-
uted the black growth to a suite of common environmental fungi. However, they
correctly recognized the characteristic ecological features of the whiskey fungus,
including its close association with fugitive ethanol vapour emissions and its pro-
clivity to colonize plant surfaces such as twigs, branches, and leaves to an extent
causing severe damage and death.
Scott et al. (2007) thought that the environmentally common fungi reported by
Kjøller (1961) and Watson et al. (1984) were unlikely to be responsible for the
highly characteristic, sooty appearance of warehouse staining. Direct microscopic
examination of freshly collected and archival samples of the growth from multiple
geographic sites revealed the strong predominance of what they interpreted to be a
single organism conforming to T. compniacensis (Scott et al. 2007). Despite lacking
the differentiated conidial structures typical of many ascomycetous asexual states,
Baudoinia has micromorphology that is distinctive and that cannot be confused with
the ubiquitous genera that Watson et al. (1984) reported from their culture studies.
Using a combination of a semiselective primary cultivation medium and careful
isolation techniques, Scott et al. (2007) obtained pure cultures that manifested the
same morphological characteristics as those represented in the descriptions and
illustrations of T. compniacensis by Richon and Petit (1881) and Kjøller (1961).
The presence of the taxa reported by Kjøller (1961) and Watson et al. (1984)
remains interesting. Because the taxa are all extremely common in outdoor air, Scott
et al. (2007) reasoned that passive deposition on colonized surfaces over time gave
rise to these allochthonous taxa as opposed to Baudoinia which they demonstrated
to be a true autochthonous colonist chiefly responsible for warehouse staining.
Studies carried out in recent years have further supported Baudoinia to be the
principal agent in the warehouse staining community (e.g. Scott et al. 2007; Ewaze
et al. 2007, 2008a, b; Al-Naama et al. 2009). Ewaze et al (2008b) found that the
incorporation of ethanol into a defined primary isolation at a concentration of 3 %
(520 mM) strongly inhibited rapidly growing mould contaminants and still allowed
the outgrowth of Baudoinia colonies. Al-Naama et al. (2009) acknowledged the
multiorganismal nature of warehouse staining biofilms but referred to species of
Baudoinia as “founding colonists” of the growth.
Conclusions and Future Directions
As public awareness of the whiskey fungus grows and consumer demand for aged
spirits increases, the need for information on the whiskey fungus by both the public
and the spirits industry is likely to intensify. There remain a number of unanswered
questions in relation to the lavish proliferation of the whiskey fungus in human
communities, and several of these address fundamental aspects of its biology that
remain unresolved. For example, what is the broader compositional nature and
complexity of the warehouse staining biofilm? In addition to Baudoinia, are there
other regularly occurring, autochthonous microbial participants? Despite intensive
study over the past few years, the genus Baudoinia remains mysterious. Still
unknown is the mechanism of ethanol sensing in the fungus remains uncertain.
Other outstanding questions are more of a practical nature. People living in areas
affected with heavy colonization often express concern about the potential for
negative health effects. Although plausible, this aspect of warehouse staining
broadly, and of the whiskey fungus specifically, has not been evaluated. Likewise
there is strong interest both from communities and the spirits industry to determine
safe and sustainable remedial measures and preventive strategies in relation to the
whiskey fungus.
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Chapter 17
Allergenic Microfungi and Human Health:
A Review on Exposure, Sensitization,
and Sequencing Allergenic Proteins
Mercedes Amado and Charles Barnes
Historical Association of Allergy and Mold
Charles Harrison Blackley (1820–1900) is generally credited with the initial under-
standing that pollen causes symptoms related to hay fever or allergic rhinitis. And, from
the very beginning he included fungi in the list of organisms related to allergy and
asthma (Blackly 1873). The Dutch pharmacologist and researcher Willem Storm van
Leeuwen (1882–1933) described the high frequency (50 %) of Dutch asthmatics whose
skin tested positive to molds (mostly Mucor, Penicillium, and Aspergillus spp.). He also
identified housing conditions related to mold sensitivity and asthma. He also developed
an “asthma-free room” as a form of environmental control (Van Leeuwen et al. 1925;
Denning et al. 2006). In 1935, Mott and Kesten reported ophthalmic hypersensitivity
reactions in rabbits using extracts of a yeast fungus (Monilia psilosis, current name:
Candida albicans) (Mott and Kesten 1931). Other pioneering fungal investigations by
F.T. Cadham (1924), Jiminez-Diaz (1932), and HA Hyde (1949) expanded the under-
standing of fungal spores as allergens. And SM Feinberg and OC Durham solidified
fungi as part of the allergen repertoire in the 1946 “Allergy in Practice Yearbook.” Yet,
they probably missed 90 % of the relevant taxa because they only identified easily
visually differentiated organisms or those that grew readily on agar plates.
M. Amado, M.D.
Pediatric Allergy and Immunology, Allergy Asthma and Immunology, Children’s Mercy
Hospitals and Clinics, 2401 Gillham Road, Kansas City, MO 64108, USA
e-mail: mamado@cmh.edu
C. Barnes, Ph.D. (*)
Division of Allergy/Immunology Laboratory, Children’s Mercy Hospitals and Clinics,
2401 Gillham Road, Kansas City, MO 64108, USA
e-mail: cbarnes@cmh.edu

DOI 10.1007/978-3-319-29137-6_17
430 M. Amado and C. Barnes
Fungal Extracts and Problems
A recent clinical comentary review on environmental fungi describes fungi as:

“True fungi have cell walls that contain beta-(1–3) and beta-(1–6) glucans and
chitin as structural components (Flannigan and Miller 2011) Fungi may be unicel-
lular when they are yeast forms, but typically have a thread-like or tube-like body
composed of hyphae, which range from 2 to 10 μm in diameter. Hyphae grow at
their tips and frequently branch resulting in an interconnected network of hyphae
called a mycelium. Fungi reproduce by producing spores, the majority of which are
adapted for airborne dispersal. Spores may be produced by asexual processes or
may be the products of sexual reproduction.” In a more practical vein, the American
Society for Testing and Materials International references The Fifth Kingdom by
Bryce Kendrick and defines fungi as: “eukaryotic, heterotrophic, absorptive organ-
isms that usually develop a rather diffuse, branched, tubular body (i.e., network of
hyphae) and usually reproduce by means of spores” (Levetin et al. 2015). The terms
“mold” and “mildew” though not scientific terms are frequently used by laypersons
when referring to various fungal growths (Kendrick 2008). One specific factor con-
cerning fungi that has come to light in recent years is the realization that fungal
surfaces contain a wide array of molecular patterns that can be important targets for
recognition by the immune system. In addition to beta glucans, they contain chitin,
mannans, and mannoproteins and galactomannans.
Medical use of fungal extracts is plagued by problems related to extracts for
immunotherapy and fungal sensitivity testing. These problems include the absence
of validated, standardized extracts recognized by the US Food and Drug
Administration, the number of different fungal species that can induce allergic reac-
tions, the extraction time- and culture condition-dependent production of allergenic
molecules (Portnoy et al. 1993), the complex number of different allergen structures
produced by fungi, and the presence of highly cross-reactive allergens in multiple
fungal taxa (Crameri et al. 2009).
Extracts Available
The fungal derived injectable preparations for humans are essential for the diagnosis
and treatment of allergic disease only. Fungi are not typically adapted for growing
at the warm temperatures found in the human body, and fungal infections of healthy
humans are found mostly in the peripheral cooler parts such as the scalp and feet
(Flint and Cain 2014). Failure to mount adequate immune response to invasive ther-
mophilic fungi is devastating (Fleming et al. 2014). Table 17.1 lists the major fungal
preparations available for diagnostic purposes either as extracts for skin testing or
as immobilized material for in vitro specific IgE testing. Since many of these prepa-
rations are approved for use, at least in the USA, through their historic or sometimes
obsolete names, the table sometime lists more than one name for an organism. And,
17
Table 17.1 Microfungi extracts available for diagnostic purposes (Greer Laboratories 2015)
Traditional scientific name Other name Current nomenclature
Genus Species In vivo (skin test) In vitro (immunocap)
Acremonium strictum Cephalosporium acremonium Acremonium strictum Yes
Acremonium kiliense Cephalosporium acremonium Acremonium kiliense Yes
Aureobasidium pullulans Pullularia pullulans Aureobasidium pullulans Yes Yes
Alternaria alternata Alternaria tenuis Alternaria alternata Yes Yes
Aspergillus flavus Aspergillus flavus Yes Yes
Aspergillus fumigatus Neosartorya fumigata Aspergillus fumigatus Yes Yes
Aspergillus niger Aspergillus brasiliensis Aspergillus niger Yes Yes
Aspergillus terreus Aspergillus terrestris Aspergillus terreus Yes
Botrytis cinerea Botrytis cinerea Yes Yes
Candida albicans Candida albicans Yes Yes
Chaetomium globosum Chaetomium globosum Yes Yes
Allergenic Microfungi and Human Health…
Cladosporium herbarum Byssus herbarum Cladosporium herbarum Yes Yes

Cladosporium sphaerospermum Cladosporium sphaerospermum Yes
Curvularia lunata Cochliobolus lunatus Curvularia lunata Yes
Curvularia spicifera Cochliobolus spicifer Curvularia spicifera Yes
Epicoccum nigrum Epicoccum purpurascens Epicoccum purpurascens Yes
Epicoccum purpurascens Epicoccum nigrum Epicoccum purpurascens Yes
Epidermophyton floccosum Epidermophyton floccosum Yes
Fusarium moniliforme Gibberella fujikuroi Fusarium proliferatum Yes Yes
Fusarium solani Haematonectria haematococca Fusarium solani Yes
Geotrichum candidum Geotrichum candidum Yes
Gliocladium viride Gliocladium deliquescens Gliocladium viride Yes
Helminthosporium halodes Setomelanomma rostrata Helminthosporium halodes Yes
Helminthosporium solani Spondylocladium atrovirens Helminthosporium solani Yes
431
Malassezia orbiculare Pityrosporum orbiculare Malassezia orbiculare Yes Yes

(continued)
432
Traditional scientific name Other name Current nomenclature

Genus Species In vivo (skin test) In vitro (immunocap)
Mucor plumbeus Mucor plumbeus Yes
Mucor racemosus Mucor circinelloides Mucor racemosus Yes Yes
Penicillium chrysogenum Penicillium notatum Penicillium chrysogenum Yes Yes
Penicillium glabrum Penicillium glabrum Yes
Phoma betae Pleospora betae Phoma betae Yes Yes
Rhizopus nigricans Rhizopus stolonifer Rhizopus nigricans Yes Yes
Rhizopus oryzae Rhizopus arrhizus Rhizopus oryzae Yes
Rhodotorula mucilaginosa Rhodotorula rubra Rhodotorula mucilaginosa Yes
Saccharomyces cerevisiae Saccharomyces cerevisiae Yes
Stachybotrys chartarum Stachybotrys atra Stachybotrys chartarum Yes
Stemphylium herbarum Stemphylium botryosum Pleospora herbarum Yes
Stemphylium solani Stemphylium solani Yes
Trichophyton mentagrophytes Trichophyton mentagrophytes Yes Yes
Trichophyton rubrum Trichophyton rubrum Yes Yes
Trichosporon pullulans Guehomyces pullulans Guehomyces pullulans Yes
Trichoderma harzianum Trichoderma viride Trichoderma viride Yes Yes
Trichoderma viride Hypocrea rufa (teleomorph) Trichoderma harzianum Yes Yes
Ulocladium chartarum Ulocladium chartarum Yes
M. Amado and C. Barnes
17 Allergenic Microfungi and Human Health… 433
often the new or old nomenclature is not accepted by one group or another. It can be
confusing. For example, Wallemia sebi is a Basidiomycota related to Ustilago fre-
quently identified in house dust samples by DNA-based methods. Yet, Wallemia
was not a relevant taxon in early outdoor collections and an extract for testing sen-
sitivity to this fungus is not available at least in the USA. Also, for some previously
recognized fungi, the taxonomy of the entire genus has undergone reorganization.
For example, in Rhodotorula with three commonly recognized species (R. glutinis,
R. minuta, and R. mucilaginosa), the formerly recognized R. rubra has been reclas-
sified. And, the extract formerly available under that name is actually a strain of
R. mucilaginosa. An extract is currently available under the name R. mucilaginosa,
but it is the same extract that was formerly available under the name R. rubra.
However, the use of multiple names is not unique in medical and scientific literature
and just like for cytokines and clusters of differentiation those who wish to be
knowledgeable in the field must master the multiple names.
Extracts Approved in the USA for Testing and Treatment
There is currently a move by the FDA in the USA to remove from the US pharma-
copeia those fungal extracts that do not have evidence of safety and efficacy in the
medical literature (Slater et al. 2012). Also, there is limited evidence supporting the
efficacy of immunotherapy for fungi other than Alternaria and Cladosporium
(Portnoy et al. 2015). Therefore, several currently available extracts are scheduled
to be withdrawn from the marketplace at least in the USA. And, if the major extract
providers cease producing these fungal preparations, it is unclear if the current
in vivo testing catalog will remain intact (Portnoy 2012).
Allergenic Proteins from Fungi and Cross-Reactivity
The international efforts to identify and characterize major fungal allergenic pro-
teins have made major progress over the past 15 years. Allergome (http://www.
allergome.org/), probably the most inclusive of the allergen databases, lists 258
allergenic fungal taxa containing 594 allergenic proteins. The International Union
of Immunological Societies (IUIS) database that is perhaps the most exclusive
(http://www.allergen.org/) lists 29 taxa and 100 allergenic proteins. Nearly all
allergy practitioners will agree that many fungal allergenic proteins are not unique
to individual fungal species. Fungal cross-reactivity has been observed since the
early days of allergen skin testing (Crameri et al. 2009; Dobrey 1962). Significant
fungal cross-reactivity has been demonstrated by clinical history of exposure, epi-
cutaneous skin sensitivity testing, and RAST inhibition assays (O’Neil et al. 1990;
De Zubiria et al. 1990; Bisht et al. 2002). Recent high-throughput sequence work
with fungal allergens indicates that fungi produce complex repertoires of species-
specific and cross-reactive allergens (Crameri et al. 2006).
Families of Fungal Allergic Proteins
A significant amount of sequencing work has been performed on fungal allergens

and currently they are divided into several basic families of proteins. A search of the
AllFam database (http://www.meduniwien.ac.at/allergens/allfam/) indicates that
for fungi there are 66 recognized protein families of which 28 families have been
identified in more than one fungal species. The family with the greatest number of
individual fungi contributing at least one protein is the subtilisin-like serine prote-
ase family. At least 6 Aspergillus species, 2 Cladosporium species, a Curvulata
species, and a Penicillium species have proteins identified as members of this fam-
ily. Other major families of fungal proteins include peroxisomal membrane proteins
(Asp f 3, Pen c 3, Mal f2, Mal f3), acid ribosomal proteins (Alt a 12, Clad h12, Pen
b 26, Alt a 5, Cla h 5, Asp f 8, and others), cyclophilins (Asp f 11, Asp f 27), heat
shock proteins (Alt a 3, Clad a 12, Pen c 19, Asp f 12), enolase (Alt a 6, Clad h 6,
Asp f 22, Pen c 22), aldehyde dehydrogenase (Alt h10, Cla h10), glutathione S
transferase (Pen c24), and Catalase (Pen c30, Asp n30). In addition, there are many
fungal allergens where the function or family has not been identified yet. The most
notable of these being Alt a 1.
Fungal Protein Cross-Reactivity (Theoretical and Practical)
To examine cross-reactivity in fungi, specific IgE against four fungi, Alternaria

alternata, Aspergillus fumigatus, Cladosporium herbarum, and Penicillium chrys-
ogenum, was retrieved from clinical laboratory records. Specific IgE testing results
from 6565 individuals were reviewed. A total of 2607 individuals had at least one
positive test. The highest number of positive tests was to Alternaria (2156) and the
fungus with the fewest positive tests was Cladosporium (1508). Most significantly,
of the individuals having at least one positive test nearly half (1208) tested positive
for all four fungi (Amado et al. 2014). In skin testing results, multiple-fungus sensi-
tized patients reacted in descending order to Alternaria, Candida, Cladosporium,
Aspergillus, Saccharomyces, Penicillium, and Trichophyton (Mari et al. 2003).
It is accepted that IgE cross-reactivity is an important aspect of fungal allergy
and contributes significantly to fungal allergenicity (Crameri et al. 2009). And, ele-
gant scientific investigations have been conducted using allergens produced through
recombinant protein technology (Chou et al. 2011). Because of noted extensive
fungal allergen cross-reactivity (Gutierrez-Rodriguez et al. 2011), it has been sug-
gested that a limited number of skin tests could be used to identify patients with
fungal sensitization. Once fungal sensitivity is identified, an allergist could then use
an extensive panel of fungi to identify specific species sensitization if necessary.
This might be done if immunotherapy is being considered or if an association
between exposure and symptoms due to fungi identified in a patient’s environment
is suspected (Portnoy and Jara 2015).
Certain fungal allergen cross-reactivities are well documented. Alt a 1 has been
quantified in culture filtrates of Stemphylium botryosum, Ulocladium botrytis,
Curvularia lunata, Alternaria tenuissima, C. herbarum, Penicillium chrysogenum,
and Asp. fumigatus. And, immunoblotting experiments using culture filtrate extracts
from the same fungi probed with rabbit anti-recombinant Alt a 1 serum have shown
significant amounts of Alt a 1. It has also been demonstrated (Crameri 2011) by inhi-
bition experiments using serum from mold-sensitized patients and A. alternata extract
in solid phase that IgE binding can be inhibited both by extracts closely related
(Curvularia lunata, Stemphylium botryosum, and Ulocladium botrytis) and up to 70
% by extracts of more distant fungi (Cladosporium herbarum or Aspergillus fumiga-
tus). Therefore, it has become generally recognized that this major allergen of
Alternaria alternata (Alt a 1) is expressed in other members of the Pleosporaceae
family (Saenz-de-Santamaria et al. 2006). Also, in the Pleosporaceae family it has
been documented that antigenic components ranging from 14 to 20 kDa strongly react
with specific serum raised against taxonomically related species (Saenz-de-Santamaria
et al. 2006). Yet, in spite of the extensive cross-reactivity, there are many unique fungal
allergens. Using purely bioinformatic approaches comparing structural motifs and
BLAST searches for proteins, it is estimated that the size of the allergen repertoire
necessary to represent the full range of fungal allergens could be as high as 5000
(Crameri 2011). Also, using sequencing data and literature searches at least 60 docu-
mented inter-phyla fungal cross-reactivities and 69 cross-reactivities have been docu-
mented between differing fungal phyla. There are even a remarkable number of
documented cross-reactivities with non-fungal phyla (Simon-Nobbe et al. 2008).
Exposure and Sensitization
As far as fungal exposure and sensitization are concerned, the most common prick
skin test sensitivity in many studies is Alternaria (12.6 % of atopics, 61 % of fungal
sensitized). Surprisingly, the next most sensitizing fungi was Candida (8.5 % of
atopics, 13 % of fungal sensitized) (Mari et al. 2003). In patients who were sensi-
tized to only 1 or 2 fungi, the most common consistently reactive genera were
Alternaria and Candida. The extensive Candida sensitization likely results from
cross-reacting allergens since Candida is not a common aeroallergen (Mari et al.
2003). Among asthmatics fungal sensitivity has been reported as high as 80 %
(Lopez and Salvaggio 1985). A good synopsis of fungal sensitivities can be found
in the 2008 review by Simon-Nobbe et al. (2008).
Human Exposure and Fungal Ecology
Ecology is the study of living things, their environment, and the relation between
the two. It can be extended to cover the indoor and outdoor places where airborne
fungal spores are found and where humans can come into contact with fungi.
From an allergen point of view, human fungal interaction can cause sensitization,
fungal related hypersensitivity disease, and the development of fungal related
allergy symptoms. It is very rare to find an environment devoid of fungi and their
spores. Outdoor airborne fungal spore estimations can range up to 50,000 per cubic
meter of air in the warm months of the year and even in the depths of winter a few
hundred spores per cubic meter of air can be found on most days (NAB 2000).
Individual weather conditions can have a direct and cumulative impact on outdoor
airborne spore exposure (Weber 2003). Precipitation and humidity have a tendency
to elevate airborne spore levels and snow does a marvelous job of clearing the air of
particles in general. Wind speed is important in bringing especially heavy spores
into the air, but even light winds are adequate for small very aerodynamic spores to
travel miles from their source. Thunderstorms, especially in summer, provide a
complex set of conditions that typically tend to increase airborne spore levels
(Nasser and Pulimood 2009), and dispersal of mold spores is strongly linked to
precipitation and humidity. Sexual stage ascospore and basidiospore levels can rise
strongly in relation to rainfall, and release of many taxa is triggered by high humid-
ity, whereas asexual stage ascospores are often content to grow rapidly during peri-
ods of adequate moisture only to produce spores that become airborne during dry
periods between rainfall events (Levetin 2014).
In the absence of facilitating factors that cause undue indoor amplification of
fungi, indoor airborne spore levels are controlled by outdoor airborne fungal levels
(Burge 2002). However, when facilitative factors are present in a house, elevated
indoor spore levels can result in excessive human exposure. The extent of any dis-
ease caused by this exposure has been the subject of disagreement (Bush et al.
2006). The forces arrayed on either side of the issue leave little room for a consen-
sus middle ground and often do battle in the law courts (Chapman et al. 2003).
Consultants and experts are sometimes financially conflicted and their conflicted
opinions frequently leach into the scientific literature (Bardana et al. 2003).
Facilitating factors typically involve the presence of moisture resulting from
conditions as roof leaks, basement leaks, sewage leaks, plumbing leaks, high humid-
ity, condensation, and inadequate insulation all associated with generally poor
housing. Typical soil fungi easily adapt for growth on a wide variety of building
materials. The only essential elements are a source of carbon and water. The carbon
source can be the paper facing on dry wall or the acrylates in paint (Ito et al. 2005).
Different fungal species have adapted to prefer specific moisture conditions, barely
damp (xerophilic) to very wet (hydrophilic). Traditional plaster and solid wood are
more resistant to fungal growth and chemical treatments have been used to add
additional resistance to fungal growth. However, many of the most toxic chemicals
have been justifiably removed from the market. Between 1960 and 1980 in North
America, the composition of interior building walls was shifted away from plaster
to surfaced gypsum board or drywall. As the name implies it must remain dry, for
when moist this material is susceptible to fungal growth (Flannigan and Miller
2011). Drywall along with other changes like wall-to-wall carpeting, reduced
ventilation rates, indoor appliances, and even plastic wrapping resulted in North
American housing, and that of many other parts of the world, often becoming
characterized as damp buildings. Estimates are that up to 30 % of homes in the USA

and Canada have dampness problems. Reports indicate that 10 % of US homes had
water damage from exterior leaks and 8 % had water damage from interior leaks.
The population-weighted average prevalence of dampness or mold in housing was
reported as 47 % in the USA (Park and Cox-Ganser 2011 ; Dales et al. 2008 ).
In large Canadian studies, damp spots, visible mold or mildew, water damage, or
flooding was reported by 38 % of respondents (Dales et al. 1991).
The association of damp building conditions and respiratory disease was first
strongly made in the 2004 Institution of Medicine publication Damp Indoor Spaces
and Health (Medicine Io 2004). This publication was bolstered by epidemiology
studies in Europe, Canada, and the USA that consistently show building/house
dampness and mold have been associated with increased risks for respiratory symp-
toms, asthma, and respiratory infections (Park and Cox-Ganser 2011; Mendell et al.
2011; Quansah et al. 2012). Subsequent intervention studies (Krieger et al. 2010)
continue to support these conclusions (Maheswaran et al. 2014). And, the relative
risk for increased asthma and other conditions remains after controlling for con-
founding exposures (Dales and Miller 1999). Mechanistic studies concerning the
innate immune system and fungal associated molecular patterns have shed addi-
tional light on this relationship. Damp building exposure includes fragments of
fungi (both micronic and sub-micronic), fungal allergens, bacterial endotoxins, fun-
gal glucans, mannans, and low-molecular-weight metabolites as well as other aller-
gens (e.g., pets, dust mites, roaches, etc.). It is important to realize that fungal
exposure does not occur in a vacuum but is nearly always part of a milieu of expo-
sures associated with building dampness as a facilitating factor for numerous aller-
gen sources.
In addition to allergic rhinitis and asthma, there are several other medical condi-
tions recognized to be associated with mold sensitivity. Allergic bronchopulmonary
aspergillosis (ABPA) is diagnosed in patients with asthma or cystic fibrosis. It is
characterized by pulmonary infiltrates, mucus containing Aspergillus fumigatus
hyphae, elevations of total serum IgE, specific precipitating IgG antibodies, and
eosinophils in the sputum (Greenberger 2012). ABPA is the most common form of
allergic bronchopulmonary mycosis (ABPM). It results from an allergic inflamma-
tory response to the colonization by Aspergillus in the airways although coloniza-
tion by other fungi is reported (Natarajan and Subramanian 2014; Glancy et al.
1981). Allergic bronchopulmonary mycosis (ABPM) is also reported as a
hypersensitivity-mediated disease associated with fungi other than Aspergillus. A
survey reported the commonest etiologic agents in this group to be Candida albi-
cans (60 % of cases), Bipolaris species (13 %), Schizophyllum commune (11 %),
Curvularia species (8 %), Pseudallescheria boydii (3 %) and, rarely, Alternaria
alternata, Fusarium vasinfectum, Penicillium species, Cladosporium cladosporioi-
des, Stemphylium lanuginosum, Rhizopus oryzae, Ca. glabrata, Saccharomyces
cerevisiae, and Trichosporon beigelii (Chowdhary et al. 2014). The characteristics
of ABPM include severe asthma, eosinophilia, markedly increased total IgE and
specific IgE levels, specific precipitating IgG antibodies, central bronchiectasis, and
mold colonization of the airways. The term severe asthma associated with fungal
sensitization (SAFS) has been suggested to illustrate the high rate of fungal sensi-
tivity in patients with persistent severe asthma and improvement with antifungal
treatment. Regardless of which acronym is used, the understanding of these dis-
eases is just now being developed (Knutsen et al. 2012). However, studies into these
conditions coupled with genetic studies involving mice with critical elements of the
immune system knocked out are starting to yield additional insight into the com-
plexity of the innate immune system and its response to fungi.
Allergic Fungal Sinusitis
Similar in pathogenicity to ABPA or allergic fungal pulmonary mycosis is allergic

fungal sinusitis in which an immunocompetent person presents with nasal polypo-
sis, chronic pansinusitis, and noninvasive fungal hyphae and allergic mucin on his-
topathological examination. Grossly, the allergic mucous appears tan to brown and
thick in consistency and has been described as peanut butter like. And, on micro-
scopic exam, eosinophils, Charcot–Leyden crystals, and elevated levels of eosino-
philic cationic protein are also found. Patients have IgE and IgG precipitating
antibodies to fungi, typically Aspergillus although other dematiaceous fungal spe-
cies (Alternaria, Bipolaris, Curvularia) have been identified. Treatment consists of
surgical debridement and topical and oral corticosteroids, and antifungal therapy is
advocated by some specialists. Recurrence of disease is common. In contrast, inva-
sive fungal sinusitis occurs in immunocompromised individuals such as persons
undergoing chemotherapy for malignancy. An Aspergilloma or Aspergillus fungus
ball presents usually in only one sinus (maxillary or sphenoid) without allergic
mucin. And surgical removal is curative.
Hypersensitivity pneumonitis or extrinsic allergic alveolitis is a pulmonary
inflammatory disease with several forms including acute, subacute, or intermittent
and chronic progressive disease. It is caused by inhalation of organic dusts includ-
ing molds such as farmer’s lung (Micropolyspora faeni), cheese worker’s lung
(Penicillium casei), and malt worker’s lung (Aspergillus clavatus). Fungi are not the
only cause of hypersensitivity pneumonitis and persons may present with symptoms
relating to inhaling antigens of non-fungal origin such as avian proteins (bird fanci-
ers), trimellitic anhydride, diisocyanate, and methylene diisocyanate. Unfortunately,
iatrogenic causes of invasive fungal meningitis have been recently reported second-
ary to unsterile compounding pharmacies with devastating morbidity and mortality
(Pettit et al. 2012; Smith et al. 2013; Kainer et al. 2012).
There are other diseases caused by inhalation of fungal spores in the soil.
Although not directly associated with allergic microfungi they must nonetheless be
mentioned here. Most persons who inhale fungal spores are asymptomatic or pres-
ent with mild pulmonary symptoms such as cough similar to an upper respiratory
tract infection. However, those with weakened immune systems may have dissemi-
nated disease and need treatment with antifungal medication. In the Southeastern USA,
parts of Mexico, and Central America, coccidiomycosis occurs due to inhalation of
fungal spores of Coccidioides. In the USA, this is known as Valley Fever. In the
Ohio and Mississippi valley of the USA, histoplasmosis due to the inhalation of
fungal spores associated with bird and bat droppings occurs. Blastomycosis occurs
secondary to the inhalation of Blastomyces dermatitidis. This fungus lives in moist
soil and is associated with decomposing organic matter such as wood and leaves.
Blastomycosis occurs chiefly in the east central part of the USA.
Fungi and Innate Immunity
The earliest reference to “innate immunity” that is easily found is by Miller and
Watson when they used it in the title of a 1965 article in Medical Clinics of North
America (Miller and Watson 1965). At its maximum in 2012, interest in the subject
generated 1531 review articles according to PubMed. Innate immunity, being
defined as the protection against infection that relies on mechanisms that exist
before infection, can encompass a great variety of functions (Lichtman et al. 2007).
This discussion will consider only those recently discovered elements of innate
immunity that sense molecular patterns common to fungi. Generally that means a
subset of the pattern recognition receptors (PRRs), specifically PRRs that recognize
pathogen-associated molecular patterns (PAMPs) and damage-associated molecu-
lar patterns (DAMPS) that are related to fungal presence. The major players are
Toll-like receptors (TLRs) and C-type Lectin Receptors (CLRs) that are directly
involved in fungal recognition and modulation of the innate immune response. Also
associated are the nucleotide-binding oligomerization domain (NOD)-like recep-
tors (NLRs) and the cytosolic dsDNA sensors (CDSs) that sense mostly DNA and
RNA elements some of which are fungal in origin and the retinoic acid-inducible
protein 1 (RIG) like receptors (RLRs) that play a major role in virus detection.
The chief molecular patterns found in fungi are components of the fungal cell
wall (Hardison and Brown 2012). These components include mannan (mannosyl-
ated proteins), β-glucan, and chitin. The typical picture involves an outermost layer
of proteins that are heavily modified by multiple mannose containing structures.
This outer layer is underlain by a rigid carbohydrate polymer of beta glucan sup-
ported by an even more rigid layer of polymerized n-acetyl glucosamine or chitin.
However it should be recognized that the fungal cell wall can be a dynamic structure.
It varies in composition among the numerous fungal species. And, it can change and
adapt during the various morphological transitions fungi undergo. During these
transitions and developmental stages the various elements of the fungal cell wall
can be exposed to the surface. Therefore different fungi, or even the same fungus in
differing developmental stages or grown in differing media can present a differing
immunological picture (Amarsaikhan et al. 2014).
The CLRs are a large family of transmembrane receptors that bind to carbohy-
drates and recognize many glucan and mannin structures from fungi. CLRs include
Dectin-1, Mincle (macrophage-inducible C-type lectin), DC-SIGN (dendritic cell-
specific ICAM3-grabbing nonintegrin), DNGR-1 (DC NK lectin group receptor-1),
and MBL (mannose-binding lectin). They basically recognize fungi and modulate
innate immune response. Often they are divided into type 1, type 2, and soluble
CLRs. Type I includes DEC 205 and MMR (macrophage mannose receptor), and
type 2 includes Dectin-1, Dectin-2, Mincle, DC-SIGN, and DNGR-1. Soluble CLRs
include MBL. CLRs are expressed by many cell types including macrophages and
dendritic cells.
Dectin-1 is a specific receptor for β-glucans (Brown et al. 2003) in the cell walls
of fungi. Dectin-1 has extracellular domain connected to a transmembrane region,
followed by a cytoplasmic region with an Immunoreceptor Tyrosine-based
Activation Motif (ITAM). Dectin-1 modulates expression of cytokines by inducing
Nuclear Factor of Activated T Cells (NFAT) through the Ca2+–calcineurin–NFAT
pathway (Goodridge et al. 2007). Dectin-1 signaling has been shown to collaborate
with TLR2 signaling to enhance the responses triggered by each receptor (Gantner
et al. 2003). Dectin-1 triggers phagocytosis and activation of Src and Syk kinases,
through its ITAM-like motif leading to the production of reactive oxygen species
(ROS), activation of NF-κB, and subsequent secretion of pro-inflammatory cyto-
kines (Gross et al. 2006; Dennehy and Brown 2007). ROS has a direct microbio-
cidal role in phagocytic pathways and can also affect other pathways (Kankkunen
et al. 2010).
Dectin-2 is a major PRR for fungal infection and the induction of Th17 type
immunity. It binds carbohydrates with high mannose content and is the functional
receptor for α-mannans, a major fungal cell wall component (Drummond and
Brown 2011, 2013). Like Dectin-1, Dectin-2 is part of the group of CLRs that links
pathogen recognition and adaptive immunity. Also, activation of Dectin-2 triggers
ROS leading to inflammasome activation (Ifrim et al. 2014). Mincle is another PRR
involved in the recognition of fungi (Brown 2008). Mincle binds fungal α-mannose
among other molecules (Yamasaki et al. 2009) and interacts with the Fc receptor
common γ-chain (FcRγ), triggering signaling leading to NF-κB and calcineurin-
NFAT activation. DC-SIGN is involved in the recognition of Candida species. It
activates the Raf-1-acetylation-dependent pathway and modulates TLR signaling
(Brown 2010; den Dunnen et al. 2009). Mannose-binding lectin (MBL) is a soluble
C-type lectin that has a role in innate immunity against yeast through enhanced
complement activation and uptake of polymorphonuclear cells (van Asbeck et al.
2008). MBL binds to repetitive mannose and/or N-acetylglucosamine residues on
microorganisms, producing opsonization and complement pathway activation
(Bidula et al. 2013).
Toll-like receptor (TLR) was initially identified as essential for fruit fly immu-
nity to fungi. When TLR4 was subsequently identified as an LPS receptor (Wagner
2012), it became the focus of intensive study. Up to 15 TLRs have been appreciated
in Mammals, but they are not all expressed in humans. TLRs generally form het-
erodimers that bind to PAMPS and through many subsequent steps (>25 protein
intermediates including MYD88, TRAF, IRAK) activate the immune system
through interferon regulatory factor 3 (IRF-3) and nuclear factor kappa-light-chain
enhancer of activated B cells (NF kappaB) (Villena et al. 2014). TLRs control reac-
tions to fungal-specific PAMPs. The best example is TLR 2 which heterodimerizes
with TLR 1, TOLLIP (Toll interacting protein) to recognize fungal beta-glucan

(Netea et al. 2006), and phospholipo-mannans (PLMs) unique to C. albicans
(Jouault et al. 2009). Also, TLR2/1 heterodimers recognize A. fumigatus both in
humans and mice (Rubino et al. 2012). And, TLR2/TLR1and TLR2/TLR6 het-
erodimers recognize mannans from Cryptococcus neoformans (Netea et al. 2006;
Roeder et al. 2004 ). TLR4 is activated upon ligation of O-linked mannans in
C. albicans and C. neoformans (Netea et al. 2006) (Shoham and Levitz 2005).
Other TLRs (TLR3, 9, and 7) recognize fungal RNA and DNA. The presence of
small nuclear polymorphisms (SNPs) in some TLRs raises the risk of human fungal
infection (Skevaki et al. 2015), but these specific genetic problems are only starting
to be identified.
DNA Allows Better Identification and Characterization of Fungi

and their Allergens
The advances in DNA sequencing technology have not only caused alterations in
the taxonomy of fungi, but they have also had an increasingly dramatic impact on
research involving medical aspects of fungal exposure. The advent of rapid and
high capacity genomic DNA sequencing allows much better identification and char-
acterization of fungi not only in infectious processes but also in environmental
exposures. As the biome of the trachea and lungs is being studied, new organisms
are being identified in unexpected places (Willger et al. 2014; Kolwijck and van de
Veerdonk 2014; Dickson et al. 2014). Fungal biome evaluations are also being used
to study the complexities of the hygiene hypothesis often with conflicting results
(Daley 2014).
Recombinant Allergens
The identification and characterization of fungal allergens has generally lagged

behind the characterization of plant and animal allergens. Reasons for this are
numerous including difficulty in growing fungi, the presence of materials like mela-
tonin (Paris et al. 1990) and chitin that complicate protein identification and purifi-
cation methods that were developed in animal tissue, and genetic changes in fungi
during the growing process. Several fungal allergens including Alt a1 a have been
produced through recombinant protein technology (Asturias et al. 2005). Some of
these have been found to be sufficient for diagnostic purposes. But depending on
how they are grown and the cell types used for production of these proteins (Maier
et al. 2014), they are different from the native allergens and may not always be suit-
able for diagnostic purposes. An investigation of the in vitro diagnostic value of
recombinant Alternaria allergens for the diagnosis of sensitization to Pleosporaceae
concluded that these recombinant allergens are not sufficient for a highly sensitive
diagnosis of A. alternata allergy (Postigo et al. 2011). And, although several

recombinant allergens have been suggested as potential effective agents for immu-
notherapy (Akdis 2014), no recombinant fungal allergens have been approved at
least in the USA.
Sequencing Allergenic Proteins
In spite of the difficulties involved, numerous fungal allergens have been sequenced.
The sequencing process typically has involved identification of the allergic protein
through immunoblotting followed by the determination of a portion of the sequence
through N-terminal or tryptic digest analysis. From knowledge of a portion of the
sequence, the utilization of DNA-based methods can quickly lead to the nucleotide
sequence and the deduced protein sequence. Alternatively, fungal DNA has been
“shot gun” cloned into plasmid vectors and an array of fungal proteins produced.
These proteins are then screened with human sera containing specific IgE against
fungal proteins. Once allergenic protein producing clones are identified they are
expanded and sequenced. Sequences can then be compared to large DNA databases
to identify similarities with known allergen protein families and further characterize
the protein allergens. Over 1000 fungal protein sequences are available on Allergome
(http://www.allergome.org/) and more are being added monthly. Although many of
these are redundant and represent the same protein in different organisms, it is still
a testimony to the usefulness of protein allergen sequences.
The diversity of fungal taxa in differing eco-niches has been the subject of much
attention using high-throughput DNA sequencing methods. The two main points of
focus for this fungal biome research have been house dust and areas of the human
respiratory system including both sputum and lung biopsy specimens. However, at
least in house dust, the determined microbiome was dependent on the DNA
extraction method. Three commonly used DNA extraction methodologies
(UltraClean Soil kit, High Pure PCR Template kit, and EluQuik/DNeasy kit) were
evaluated for sensitivity and susceptibility to PCR inhibitors in dust for three com-
mon fungi, Aspergillus versicolor, Rhizopus microsporus, and Wallemia sebi. The
extraction methods differed in their ability to extract DNA from particular species.
In addition, the soil DNA extraction kit showed the greatest ability to remove PCR
inhibitors from dust samples. Most importantly, the determined species composi-
tion from the sequenced clone libraries generated varied with the different DNA
extraction kits. And as has often been the case, sequencing methods produced infor-
mation concerning additional fungal species not seen in solid culture (Rittenour
et al. 2012).
In one recent sequencing study, the diversity of airborne and dust-borne fungi in
homes of asthmatic children in Kansas City, Missouri, was determined by sequenc-
ing. Sequence results were also compared to data obtained using traditional viable
and nonviable fungal exposure assessment methods. Sequencing frequently identi-
fied organisms from the Ascomycota in both air (68 %) and dust (92 %) and less
frequently organisms from the Basidiomycota and zygomycetous fungi (formerly

Zygomycota). The majority of Ascomycota clones belonged to the Pleosporales,
Eurotiales, Capnodiales, and Dothideales. Sequencing also revealed a number of
unusual Pleosporales fungi present in the samples. As seen in other studies, sequenc-
ing results identified a broader diversity of Ascomycota and Basidiomycota in
indoor environments than traditional methods (Rittenour et al. 2013).
Another study performed analysis on the indoor bacterial microbiome using
PhyloChip Array methods. They reported 1746 operational taxonomic units (OTUs)
in each sample with few genus differences between asthmatic and non-asthmatic
homes. Major phyla identified were Cyanobacteria, Firmicutes, Actinobacteria,
Proteobacteria, and Bacteroidetes. These all were more abundant in the asthmatic
homes (p = 0.001–p = 7.2 × 10–6) (Ciaccio et al. 2014). Several studies have been
performed on induced sputum samples. One case-control study identified 90 species
more common in asthma patients and 46 species more common in control subjects.
Psathyrella candolleana, Malassezia pachydermatis, Termitomyces clypeatus, and
Grifola sordulenta showed a higher frequency in the sputum of asthma patients and
Eremothecium sinecaudum, Systenostrema alba, Cladosporium cladosporioides,
and Vanderwaltozyma polyspora showed a higher frequency in the sputa of control
subjects. This study produced evidence for the widespread nature of fungi in the
sputa of healthy and asthmatic individuals, it did not answer the question whether
the organisms were colonized in the sputa or appeared there as a result of deposition
from the air (van Woerden et al. 2013). Another study of the microbiome of induced
sputa in asthmatic and non-asthmatic adults analyzed induced sputum samples
obtained from 10 non-asthmatic subjects and 10 patients with mild active asthma
(8/10 were not using inhaled corticosteroids). Proteobacteria were present in higher
proportions in asthmatic patients and firmicutes and actinobacteria were present
more frequently in samples from non-asthmatic subjects (Marri et al. 2013).
Increased Allergic and Non-allergic Fungal Disease
Both allergic and non-allergic fungi will have increased medical importance
especially as more people with altered or depressed immune systems live longer
and are integrated into the general population. A good example is allergic or acute
bronchopulmonary aspergillosis (ABPA). There are estimated to be in excess of
four million patients affected worldwide (Agarwal et al. 2013). And, a PubMed
search indicates that publication activity related to ABPA has increased steadily
since the early 1970s. Even though ABPA is the most common form of allergic
bronchopulmonary mycosis (ABPM), other fungi, including Penicillium and
Candida, have been associated (Denning et al. 2006). A higher incidence of ABPA
has been reported in compost workers, and it has been recommended that commer-
cial compost operations where high levels of A. fumigatus often occur routinely
screen workers for asthma, Aspergillus sensitivity, cystic fibrosis, bronchiectasis,
and immunodeficiency (Poole and Wong 2013). ABPA and other fungal related lung
conditions are an increasing problem in CF (Eickmeier et al. 2013). Also, if we

recognize ABPA as a specific subtype of asthma (Greenberger et al. 2014), as the
prevalence of asthma increases around the world, it is probably safe to speculate
that the prevalence of ABPA will also increase.
Evaluating the prevalence of morbidity and mortality of fungal infections is very
difficult. It is estimated that only about half of these infections are diagnosed before
the death of an individual (Dignani 2014). Invasive fungal infections are major
causes of morbidity and mortality for the immunocompromised. And those infec-
tions are especially prevalent in a growing segment of the patient population,
namely, those with oncohematological disorders, recipients of stem cell or solid
organ transplantation, and recipients of immunosuppressive agents including high-
dose steroids, antitumor necrosis factor therapies (infliximab, adalimumab, etc.), or
anti-inflammatory antibodies (Belatacept) (Ordas et al. 2012).
The use of autopsy provides probably the best indication of the prevalence of
fungal infections. Cumulative studies, in seven countries studying mainly four types
of patient populations (general, oncohematology, stem cell transplant, and acquired
immunodeficiency syndrome) (Dignani 2014), indicated that the median prevalence
of infection is 8.7 per 100 autopsies. They also found that in about half of the cases
(4.4 per 100 autopsies), there were two or more concomitant fungal organisms.
Fungal infection rates in 4 large autopsy studies of general populations ranged from
1.4 to 8.7 % (Lehrnbecher et al. 2010; Kume et al. 2011; Colombo et al. 2012;
Shimodaira et al. 2012). Whereas in reports for 5 studies of oncology and immuno-
suppressed populations percentage of autopsies with fungal infection were between
18.2 % and 30.6 % (Donhuijsen et al. 2008; Lewis et al. 2013; Alsharif et al. 2009;
Antinori et al. 2009; Sinko et al. 2008). Clearly, as more patients undergo immuno-
suppressive therapy due to transplant and oncology-related treatment and more
immunosuppressed individuals survive infancy, the overall number of fungal infec-
tions will increase.
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Chapter 18
What’s Old is New: Recognition of New
Fungal Pathogens in the Era of Phylogenetics
and Changing Taxonomy and Implications
for Medical Mycology
Nathan P. Wiederhold and Deanna A. Sutton
Introduction
Invasive fungal infections are associated with significant morbidity and mortality as
these are often difficult to diagnosis and treat. Fungi historically associated with
invasive disease in humans include the yeast within the genera Candida,
Cryptococcus, and Trichosporon, the dimorphic fungi Blastomyces dermatitidis,
Coccidioides immitis/posadasii, and Histoplasma capsulatum, and the molds,
including limited species within the genera Aspergillus, Fusarium, and
Scedosporium, and certain members of the Order Mucorales. Over the last two
decades, there has been a significant increase in the number of fungal species asso-
ciated with invasive disease in humans. Factors that have contributed to this increase
include an increase in the number of immunocompromised patients at high risk for
invasive fungal infections, including HIV-AIDS patients, those receiving immuno-
suppressive chemotherapy for malignancies, and solid organ transplant recipients,
improvements in diagnostic assays and the clinical recognition of patients with risk
factors for such infections, as well as improvements in the tools used to identify
fungal species. Unfortunately, the recognition of new etiologic agents of invasive
mycoses has surpassed the development of new diagnostic assays and treatment
strategies against these infections. The increase in the number of etiologic agents of
invasive mycoses is also method driven. Taxonomic changes due to phylogenetic
analysis have led to the reclassification of many previously recognized fungi. This
has led to a concern of nomenclature instability in medical mycology, and the clini-
cal relevance of many of the newly reclassified species is unknown (de Hoog et al.
N.P. Wiederhold, Pharm.D. (*) • D.A. Sutton, Ph.D.

Fungus Testing Laboratory, Department of Pathology,
University of Texas Health Science Center at San Antonio,
7703 Floyd Curl Drive, MC7750, San Antonio, TX 78229-3900, USA
e-mail: wiederholdn@uthscsa.edu; suttond@uthscsa.edu

DOI 10.1007/978-3-319-29137-6_18
452 N.P. Wiederhold and D.A. Sutton
2013, 2014). In this chapter, we review the identification of fungi, primarily fila-
mentous organisms, in the clinical setting, and provide examples of known and
emerging causes of invasive fungal infections in humans and changes in taxonomy
and fungal nomenclature that have occurred and are ongoing.
Fungal Identification in the Clinical Setting
The identification of fungi in the clinical laboratory has historically relied on mor-
phologic characteristics and physiologic traits. The description of the colony
appearance and the microscopic features of the organism, including the reproduc-
tive structures, has been the hallmark for fungal identification for many years.
Certain phenotypic/physiologic traits are also combined with the morphologic fea-
tures to obtain the identities of fungal isolates. For molds these include, but are not
limited to, the ability of the organism to grow at certain temperatures, tolerance to
cycloheximide and benomyl, nitrate assimilation, tolerance to different concentra-
tions of sodium chloride, growth on bromcresol purple agar, growth on trichophy-
ton agar, and growth on urea agar (Nelson et al. 1983; Pincus et al. 1988; Kane et al.
1997; Summerbell 1993). Many of these phenotypic/physiologic assays were and
still are used to identify the organism to the genus and possibly species level in
clinical microbiology and reference mycology laboratories. Identification to the
species level is clinically important as it provides the clinician with information that
may be useful in the management of patients and help guide antifungal therapy.
Indeed, early identification and the initiation of appropriate therapy have been
shown to influence patient outcomes while delaying appropriate therapy can be det-
rimental (Morrell et al. 2005; Greene et al. 2007; Garey et al. 2006; Chamilos et al.
2008). Identification to the species level is important in helping to guide appropriate
therapy, as some fungi are intrinsically resistant to certain drugs. Furthermore, some
species within the same species complex may have different antifungal susceptibil-
ity profiles and this can influence the choice of treatment that is used (Balajee et al.
2005a; Gilgado et al. 2006; Lackner et al. 2012). However, identification by mor-
phologic/physiologic characteristics alone can be time-consuming, and results may
not be available in a timely fashion for clinical decisions. Morphologic identifica-
tion can also be fraught with errors if done by those without proper training and
experience. In addition, the morphologic features of fungi may be variable (Balajee
et al. 2006, 2007). Different factors can affect these features, including the media
used for subculturing and exposure to external stressors, such as antifungal agents
prior to recovery from clinical specimens, which can often occur in patient groups
at high risk for invasive fungal infections in which empiric or preemptive antifungal
therapy is often used.
The introduction of molecular and proteomic tools, such as DNA sequence analy-
sis and matrix-assisted light desorption ionization time-of-flight (MALDI-TOF), a
relatively new diagnostic tool in the clinical microbiology laboratory, has dramati-
cally changed how fungi are identified. These methods can reduce the amount of
18 What’s Old is New… 453
Table 18.1 DNA targets Targets Genera

used for molecular sequence
Internal transcribed spacer (ITS) All genera
identification of fungi and
examples of genera these 28S rDNA large subunit (D1/ All genera
targets may be used to D2)
identify Beta-tubulin / Calmodulin Aspergillus
Calmodulin Scedosporium
Translation elongation factor Fusarium
RNA polymerase Penicillium
Glyceraldehyde-3-phosphate Curvularia
dehydrogenase
time needed to determine the identity of an organism and reduce errors associated
with morphologic variability. However, these methods have their own limitations
and have not eliminated the need for the morphologic evaluation of fungi in the clini-
cal laboratory. For both DNA sequence and protein spectrum analysis, the results
that are obtained must be compared to those deposited in databases from known
organisms in order for an identity to be obtained. For fungi, publicly available data-
bases for DNA sequence comparisons are available, including those at the National
Center for Biotechnology Information (GenBank; www.ncbi.nlm.nih.gov/gen-
bank/), the Centraalbureau voor Schimmelcultures Fungal Biodiversity Centre in the
Netherlands (CBS-KNAW; www.cbs.knaw.nl), the International Society of Human
and Animal Mycology ITS database (ISHAM; its.mycologylab.org), and the
Fusarium-ID database (http://isolate.fusariumdb.org). Reference laboratories or
clinical microbiology laboratories may also have their own databases. The use of
sequence results can be extremely useful when compared with credible deposits.
However, not all fungal deposits within databases have been confirmed to be from
accurately identified organisms (Bridge et al. 2003; Deckert et al. 2002; Crous 2002).
This can lead to erroneous results and the misidentification of the cultured specimen.
In addition, the choice of the target sequence can be critical for the proper identifica-
tion of fungi. Although the internal transcribed spacer region (ITS) has been put
forth as a universal barcode for the identification of fungi (Schoch et al. 2012; Seifert
2009; Petti 2007), this target cannot be used alone to discriminate between closely
related fungi. Several other DNA targets may be used to identify fungi in the clinical
setting (Table 18.1), and the choice of targets is dependent on the organism. Thus, an
assessment of the morphology of the organism prior to sequence analysis can pro-
vide useful information as to what DNA targets to use for identification.
Changes in Fungal Nomenclature
Under Article 59 of the International Code for Botanical Nomenclature, polymor-

phic fungi were allowed to have multiple names. These names described either the
sexual (teleomorph) or asexual (anamorph) stages of the organism’s life cycle. The
dual nomenclature system served a purpose when fungal identification was based
upon the observed morphologic features. However, it became obsolete with the
introduction of molecular tools into the field of mycology (de Hoog et al. 2014;
Hawksworth 2011). Under the newly named International Code of Nomenclature of
algae, fungi, and plants, the dual nomenclature system under Article 59 is abolished,
and as of January 1, 2013, all fungi are now to have only one correct name (Norvell
2011). Regardless of the life history stage of the type, all legitimate fungal names
are now to be treated equally for the purpose of establishing priority. While the
abandonment of the dual nomenclature system was an important first step, mycolo-
gists are now charged with implementing this change and contributing to the pro-
duction of lists of accepted and suppressed names for fungi.
The abandonment of the dual nomenclature system and the increased use of
molecular tools have implications for the field of medical mycology. Phylogenetic
studies have demonstrated that fungi are much more molecularly diverse than previ-
ously recognized, and this has led to an increase in the number of clinically recog-
nized fungi, as new species have been described and other pathogens have been
reclassified (de Hoog et al. 2013). Some examples of recent nomenclature changes
that have occurred for clinically relevant fungi are shown in Table 18.2. However,
molecular diversity does not necessarily equate to clinical diversity, and the true
clinical relevance of newly discovered species or newly reclassified organisms may
be unknown. It has been suggested that this increase in recognized fungal species,
combined with the abandonment of the dual nomenclature system, may be compro-
mising the stability of the nomenclature of medically important fungi (de Hoog
et al. 2013, 2014). Potentially detrimental aspects may be the confusion of clini-
cians who do not closely follow taxonomic changes but are responsible for the care
of patients and the impediment of navigating the literature to find clinically useful
Table 18.2 Examples of changes in fungal nomenclature of clinically relevant fungi

New species name Previous species name Invasive disease
Curvularia spicifera Bipolaris spicifera Fungal sinusitis, cerebral phaeohyphomycosis
Curvularia Bipolaris hawaiiensis
hawaiiensis
Curvularia Bipolaris australiensis
australiensis
Lichtheimia Absidia corymbifera Mucormycosis
corymbifera
Lomentospora Scedosporium Pulmonary infection (scedosporiosis)
prolificans prolificans
Rasamsonia argillacea Geosmithia argillacea Pulmonary infection
Sarocladium kiliense Acremonium kiliense Mycetoma, keratitis in immunocompetent
hosts, invasive infections in
immunocompromised individuals
Talaromyces marneffei Penicillium marneffei Disseminated infection in HIV+ individuals
Verruconis gallopava Ochroconis gallopava Cerebral phaeohyphomycosis
information about fungal pathogens and invasive mycoses due to confusion about
the name of the organism or the diseases the fungi cause. Thus, it has been proposed
that in clinical practice medical mycologists be allowed to follow taxonomic
changes and implement these more gradually (de Hoog et al. 2013). At the species
level, once the clinical relevance of molecular sibling species is determined, novel
nomenclature could be adopted. However, until such evidence becomes available,
cryptic species can be referred to as species complexes in medical practice. A poten-
tial drawback of this approach is the hindrance of gaining knowledge about the
clinicopathology of a particular organism. In order to establish a body of literature
necessary to gain an understanding of the clinical relevance of a particular fungal
species in relation to disease and its response to therapy, the true identity of the
infecting organism must be known. The rate at which such knowledge is accumu-
lated may be slowed if such similar species are lumped into species complexes
without further delineation.
Examples of Clinically Relevant/Emerging Fungi and Changes

in Fungal Taxonomy and Nomenclature
Invasive Aspergillosis and Aspergillus Section Fumigati
Invasive aspergillosis is a major invasive fungal infection and significant cause of

morbidity and mortality in immunocompromised hosts. Groups at highest risk for
the development of invasive aspergillosis include highly immunocompromised
individuals. Such groups have historically included solid organ transplant recipi-
ents, those undergoing hematopoietic stem cell transplantation, and patients receiv-
ing highly immunosuppressive chemotherapies (Wiederhold et al. 2003). Results
from the PATH Alliance Registry from 2004 to 2007, which collects information on
patients with invasive fungal infections at medical centers in the USA, demon-
strated that invasive aspergillosis was the most frequent invasive fungal infections
in patients undergoing hematopoietic stem cell transplantation ahead of invasive
candidiasis, mucormycosis, and other invasive fungal infections (Neofytos et al.
2009). Allogeneic stem cell transplant recipients who receive prolonged course of
corticosteroids for the treatment of graft versus host disease are at further risk for
invasive aspergillosis (Wiederhold et al. 2003; Baddley et al. 2010; Kontoyiannis
et al. 2010). Of the solid organ transplant recipients, individuals who receive lung
or dual heart/lung transplants are at higher risk for invasive pulmonary aspergillosis
as the primary route of entry into the body is via inhalation into the lungs (Minari
et al. 2002). Although pulmonary involvement is a major component of invasive
aspergillosis due to the route of entry into the body via the lungs, dissemination to
other organs can occur, as invasive disease has been reported in all organ systems.
The mortality rate of this disease is exceptionally high with dissemination to the
central nervous system in the setting of continued immunosuppression. In addition
to these patient populations, invasive aspergillosis has also become more important
in critically ill patients not traditionally considered at high risk, including those with
acute chronic obstructive pulmonary disease and those receiving corticosteroids
(Meersseman et al. 2004; Garnacho-Montero et al. 2005). Chronic pulmonary
aspergillosis is also a significant problem in patients with structure damage to the
lungs, such as those who have had tuberculosis or sarcoidosis (Smith and Denning
2011; Denning 2001). The prevalence of chronic pulmonary aspergillosis is esti-
mated to be approximately 3 million patients worldwide (Denning et al. 2011,
2013a, b). Treatment of these patients often involves prolonged courses of azole
therapy, which predisposes individuals to the adverse effects and drug interactions
associated with these agents and the potential development of drug-resistant organ-
isms (Howard et al. 2009).
One of the most challenging aspects of this disease is the ability to make a timely
and accurate diagnosis. The diagnosis of invasive aspergillosis involves the incor-
poration of clinical, radiological, serological, and histopathological findings.
Although studies have demonstrated the usefulness of radiographic studies, such as
chest computed tomography, in patients with risk factors for invasive aspergillosis,
the images obtained cannot conclusively rule in or rule out this fungal infection as
other pulmonary fungal infections can show similar signs (Walsh et al. 2008). Rapid
diagnosis of this disease has focused on the detection of surrogate markers of infec-
tion, including components of the cell wall within normally sterile biologic fluids.
One particular strategy that is clinically used is the detection of galactomannan, a
component of the cell wall released during growth of the organism (Latge et al.
1994). A commercially available assay, the Platelia Aspergillus ELISA kit (Bio-
Rad), uses a rat monoclonal antibody (EB-A2) directed against tetra (1 → 5)-β-D-
galactofuranoside, the immunodominant epitope in galactomannan (Stynen et al.
1992, 1995; Morelle et al. 2005). This assay has proven to be useful for the diagno-
sis of invasive aspergillosis with a high specificity (≥85 %) in patients with hema-
tologic malignancies at high risk for this opportunistic disease (Pfeiffer et al. 2006).
The detection of galactomannan using this assay now fulfills part of the diagnostic
criteria for probable invasive aspergillosis (Walsh et al. 2008). Other assays that are
clinically used to detect invasive fungal infections detect another component of the
cell wall of many pathogenic fungi, (1 → 3)-β-D-glucan. The chromogenic assay
available for clinical use (Fungitell, Associates of Cape Cod) is based on the activa-
tion of the horseshoe crab coagulation cascade and uses amebocyte enzymes from
Limulus polyphemus (Fungitell 2008). The prompt diagnosis of invasive aspergil-
losis, including the use of these surrogate diagnostic markers, can have significant
effects on patient outcomes. Early diagnosis and initiation of antifungal therapy
have been shown to reduce mortality in patients with invasive fungal infections
including invasive aspergillosis (Greene et al. 2007; Garey et al. 2006; Caillot et al.
1997). This has led to the strategy of preemptive therapy in which antifungal agents
are initiated upon the first signs of a potential infection as suggested by high-
resolution computer tomography and serial screening of surrogate markers.
However, these assays are not without their limitations. (1 → 3)-β-D-glucan is a
pan-fungal target, since many clinically relevant species contain this polysaccharide
within their cell walls (Odabasi et al. 2006). Thus, a positive result can be seen in
patients with infections caused by a variety of fungi including Aspergillus, Candida,
Fusarium, Acremonium, Trichosporon, Sporothrix, Histoplasma, Coccidioides, and
Blastomyces (Odabasi et al. 2006; Senn et al. 2008; Pickering et al. 2005; Ostrosky-
Zeichner et al. 2005). In addition, several substances can result in false-positive test
results, primarily due to glucan content. This can occur in patients receiving hemo-
dialysis with cellulose membranes, those receiving immunoglobulin products and
albumin, as well as following serous exposure to gauze, which can occur in surgical
patients (Odabasi et al. 2004, 2006). Thus, while a positive assay result does pro-
vide evidence of an invasive mycosis, it does provide information on the causative
organism, which is important for making decisions regarding appropriate therapy.
The galactomannan assay is more specific for Aspergillus than those for (1 → 3)-β-D-
glucan. However, the sensitivity of serum galactomannan may be reduced in patients
with antifungal exposure with a high degree of variability among different patient
populations (Marr et al. 2005). While the specificity of this assay is consistently
above 85 %, the sensitivity may vary considerably between patient populations with
rates reported in the literature ranging from 29 to 100 % (Pfeiffer et al. 2006;
Verweij 2005). Furthermore, reports of cross-reactivity with this assay have been
reported with other fungi, including Fusarium and Trichosporon species (Fekkar
et al. 2009; Mikulska et al. 2012; Tortorano et al. 2012). The galactomannan assay
also will not provide information on the species of Aspergillus that is causing infec-
tion, which is also important for making treatment decision in patients with invasive
aspergillosis. For example, the galactomannan assay is not able to distinguish
between A. fumigatus and A. terreus, the latter demonstrating resistance to ampho-
tericin B, a widely used antifungal agent (Steinbach et al. 2004).
Although there are over 200 individual Aspergillus species, common causes of
invasive aspergillosis in humans include A. fumigatus, A. flavus, A. niger, A. nidu-
lans, and A. terreus. Of these, A. fumigatus is the major etiologic agent of invasive
disease at most institutions (Morgan et al. 2005). This species is usually readily dis-
tinguishable from the other common causes of aspergillosis based on its morphology
(Balajee et al. 2006, 2007). However, the morphology of A. fumigatus is unstable
and by phylogenetic analysis several distinct species are now recognized within the
section Fumigati (Sugui et al. 2014). Currently, this section consists of 51 phyloge-
netically separate species, 15 of which have been reported to cause clinical disease
in humans (Sugui et al. 2014). These include A. felis, A. fumigatiaffinis, A. fumiga-
tus, A. fumisynnematus, A. lentulus, A. novofumigatus, A. parafelis, A. pseudofelis,
A. pseudoviridinutans, A. viridinutans, A. fischeri, A. hiratsukae, A. laciniosus,
A. thermomutatus, and A. udagawae. Infections that have been reported caused by
these species include invasive pulmonary aspergillosis, osteomyelitis, peritonitis,
cerebral aspergillosis, and invasive otitis (Balajee et al. 2005b; Matsumoto et al.
2002; Jarv et al. 2004; Zbinden et al. 2012; Ghebremedhin et al. 2009).
Species identification within this section is a multifaceted approach, requiring
morphologic, physiologic, and molecular sequence results. Although the morphol-
ogy and phenotypic features within this section are variable, some members are not
easily distinguished from A. fumigatus without the use of molecular sequencing.
The ITS region, although recognized as the universal barcode for fungi, is not capa-
ble of discriminating between members of section Fumigati (Samson et al. 2014).
For this purpose, sequencing of the beta-tubulin region is recommended. Thus, here
is the potential for misidentification of members of this section in clinical laborato-
ries that do not routinely use sequence analysis for identification. The need for cor-
rect identification of the species causing infection is important since several species
within this section are refractory to antifungal therapy and may cause more chronic
infections than A. fumigatus (Zbinden et al. 2012; Barrs et al. 2013; Vinh et al.
2009a). These include A. lentulus, A. felis, A. parafelis, A. pseudofelis, A. pseudo-
viridinutans, N. pseudofischeri, and N. udagawae (Balajee et al. 2005a; Sugui et al.
2010, 2014; Vinh et al. 2009b; Khare et al. 2014). Clinical failures have occurred in
patients treated with antifungals for infections caused by these species that were
misidentified as A. fumigatus (Balajee et al. 2005a, b, 2007; Zbinden et al. 2012;
Vinh et al. 2009a; Khare et al. 2014).
Scedosporiosis and Pseudallescheria/Scedosporium
Another group of fungi recognized to cause invasive infections for which there has
been significant taxonomic change is the genus Scedosporium. Members of this
genus are ubiquitous ascomycetes found in soil, polluted water, sewage, and manure
and are capable of causing many different types of infections in humans (Cortez
et al. 2008; Walsh et al. 2004; Guarro et al. 2006). Invasive infections have been
reported primarily in immunocompromised hosts, and these fungi are recognized as
the second most common fungal colonizers in cystic fibrosis patients behind
Aspergillus species (Blyth et al. 2010). Because the route of entry is similar to that
of Aspergillus species, Scedosporium species can cause sinopulmonary disease
that is difficult to distinguish from disease caused by Aspergillus and other molds
that may be more amendable to antifungal therapy (Walsh et al. 2004; Bouza and
Munoz 2004). Such infections can be especially devastating in lung transplant
recipients (Johnson et al. 2014), and because these fungi can so adversely affect
patient outcomes, colonization of the lungs may be a contraindication to lung trans-
plantation (Raj and Frost 2002; Morio et al. 2010). Scedosporium species have also
been recognized as causes of breakthrough infections in persistently neutropenic
and/or lymphopenic patients receiving antifungal therapy (Lamaris et al. 2006;
Nucci 2003). In patients whose immune system fails to recover disseminated infec-
tions may occur, which portends a poor prognosis. Infections can also occur in
immunocompetent individuals. Near-drowning victims can develop pulmonary
infections with dissemination to the brain, which are difficult to treat and associated
with significant morbidity and mortality (Guarro et al. 2006; Buzina et al. 2006;
Nakamura et al. 2013). Mycetomas, chronic, tumor-like infections of subcutaneous
tissue and contiguous bone with draining sinuses, can also occur in otherwise
healthy hosts secondary to traumatic inoculation (Cortez et al. 2008; Walsh et al.
2004; Guarro et al. 2006).
Over the last decade, the taxonomy of Scedosporium has changed markedly. Due
to the ability to develop sexual structures on route culture media, members of this
genus were identified by clinical microbiology laboratories as Pseudallescheria
boydii when the teleomorph was present and as Scedosporium apiospermum when
only the anamorph was found. However, with the use of molecular phylogeny it was
subsequently determined that P. boydii (anamorph Scedosporium boydii) and
Pseudallescheria apiosperma (anamorph S. apiospermum) were indeed separate
species (Gilgado et al. 2008, 2010). Other species that have been discovered through
the use of molecular phylogenetics, but which are morphologically identical to
these sibling species, include S. aurantiacum, P. minutispora, P. desertorum, and S.
dehoogii (Gilgado et al. 2005, 2008; Lackner et al. 2014a). Recently, due to the
abolishment of Article 59 of the Code of Botanical Nomenclature of algae, fungi,
and plants, it has been proposed that Pseudallescheria should be treated as a syn-
onym of Scedosporium and that Scedosporium be given precedence as it is the old-
est valid generic name (Lackner et al. 2014a). The morphologically distinct species
Scedosporium prolificans, which in contrast to other members of the Scedosporium
genus, is a phaeoid mold with inflated versus tubular conidiogenous cells and has
been renamed Lomentospora prolificans based on significant phylogenetic differ-
ences (Lackner et al. 2014a). The distinction between this species and Scedosporium
species is clinically relevant, as L. prolificans is highly resistant to multiple antifun-
gal agents (Lackner et al. 2012, 2014b; Cortez et al. 2008; Walsh et al. 2004; Lewis
et al. 2005; Wiederhold and Lewis 2009), and infections caused by this organism
are extremely difficult to treat and are associated with poor patient outcomes (Cortez
et al. 2008).
For the members of the genus Scedosporium, it has been suggested that for the
routine identification in clinical microbiology laboratories, these fungi might be
identified as members of the Scedosporium apiospermum complex since the sibling
species S. apiospermum and S. boydii are without medically relevant differences
(Lackner et al. 2014a). However, there is some evidence that differentiation among
the members of this complex may be clinically important, and differences in anti-
fungal susceptibility have been reported among these species. For example, S. apio-
spermum isolates have been reported to be less susceptible to posaconazole than
those of S. boydii (Lackner et al. 2012). In addition, several studies that have evalu-
ated the in vitro potency of clinically available antifungals have demonstrated that
S. aurantiacum isolates are resistant to these agents with the exception of the tri-
azole voriconazole (Lackner et al. 2012, 2014b; Tintelnot et al. 2009;
Alastruey-Izquierdo et al. 2007), which is currently the drug of choice for the treat-
ment of infections caused by Scedosporium species (Tortorano et al. 2014). Since
many of the S. aurantiacum isolates included in these studies have been of clinical
origin, this may be of clinical significance. Some of the species within this genus do
have reduced susceptibility to voriconazole, including S. dehoogii, and the recently
renamed Lomentospora prolificans (formerly S. prolificans), mentioned earlier,
which is resistant to all clinically available antifungals (Lackner et al. 2012). While
the clinical relevance of reduced susceptibility to antifungal agents is not fully
understood, with the exception of the resistance observed with L. prolificans, fur-
ther insights into the clinicopathology of infections caused by different Scedosporium

species may be difficult to ascertain if these fungi are routinely lumped into a spe-
cies complex.
Mucormycosis and the Order Mucorales
Pathogenic fungi of the Order Mucorales are capable of causing invasive infections
termed mucormycosis. Organisms that have been associated with infections in
humans include members of the genera Rhizopus, Rhizomucor, Mucor,
Cunninghamella, Lichtheimia (formerly Absidia), Saksenaea, and Apophysomyces
(Kontoyiannis and Lewis 2006; Mendoza et al. 2014; Kwon-Chung 2012; Petrikkos
et al. 2012). Mucormycosis is a highly aggressive angioinvasive fungal infection
that primarily occurs in immunocompromised hosts, including solid organ trans-
plant recipients, hematopoietic stem cell transplant recipients, and hematologic
malignancy patients receiving immunosuppressive chemotherapy (Kontoyiannis
and Lewis 2006; Petrikkos et al. 2012). In addition, diabetic patients with poorly
controlled disease have also been shown to be at risk for infections caused by mem-
bers of the Order Mucorales. Mucormycosis has also been reported in otherwise
healthy individuals following traumatic inoculation (Hospenthal et al. 2011; Neblett
Fanfair et al. 2012). Infections caused by these fungi include rhino-orbital and
rhino-cerebral disease, pulmonary, gastrointestinal, and cutaneous infections.
Disseminated infections can also occur and are associated with high mortality rates
(Kontoyiannis and Lewis 2006; Petrikkos et al. 2012). Aggressive treatment is
needed in patients with mucormycosis, and this often involves multiple modalities
including high-dose antifungal therapy and surgery when possible to remove
infected and necrotic tissue (Kontoyiannis and Lewis 2006). However, in the setting
of continued immunosuppression, clinical outcomes may be poor even with aggres-
sive treatment. Furthermore, these species are also resistant to several antifungals,
including voriconazole, the azole frequently used to treat other invasive mold infec-
tions such as invasive aspergillosis and scedosporiosis, and the echinocandins
(Kontoyiannis and Lewis 2006; Almyroudis et al. 2007).
The name used to describe an infectious disease caused by members of the Order
Mucorales has also been subject to change. In 1885, the first well-documented case
was published by the German pathologist Paltauf, who used the term mycosis
mucorina to describe a systemic infection with rhino-cerebral and gastrointestinal
involvement (Kwon-Chung 2012; Paltauf 1885). The use of mucormycosis as the
disease name was first used by Baker to describe an infection caused by members
of the Order Mucorales in the 1950s (Baker 1956, 1957). Ajello et al. subsequently
proposed the term zygomycosis to include infections caused by species from two
separate orders: (1) Mucorales, including infections caused by species within the
genera Rhizomucor, Rhizopus, Mucor, Lichtheimia (Absidia), Apophysomyces, and
Saksenaea, and (2) Entomophthorales, due to Conidiobolus and Basidiobolus spe-
cies (Ajello et al. 1976). Until recently, zygomycosis was the term frequently used
synonymously in the clinical literature with mucormycosis to describe infections

caused by members of the Order Mucorales, in animal models of invasive infec-
tions caused by them, as well as in vitro antifungal susceptibility results against
these organisms. However, there are major differences between species classified in
the Orders Entomophthorales and Mucorales, including morphologic and epidemi-
ologic differences, and in the types of infections that they cause (Mendoza et al.
2014; Petrikkos et al. 2012). While the pathogenic species within Mucorales cause
acute invasive infections in immunocompromised hosts, chronic subcutaneous
infections are often caused by the Entomophthorales and can often occur in immu-
nocompetent individuals (Mendoza et al. 2014; Kwon-Chung 2012). In addition,
the phylum Zygomycota has been eliminated, as it was found to be polyphyletic and
had also not been validly described (Hibbett et al. 2007). Thus, mucormycosis and
entomophthoromycosis have again been put forth as the proper terms used to
describe infections caused by these different groups of fungi (Kwon-Chung 1994).
Phylogenetic studies within the Order Mucorales have resulted in several name
changes in pathogenic species. Some examples of these include Lichtheimia corym-
bifera (formerly Absidia corymbifera), Mucor circinelloides f. janssenii (formerly
Mucor velutinosus), Mucor ardhlaengiktus (formerly Mucor ellipsoideus), and
Mucor irregularis (formerly Rhizomucor variabilis) (Walther et al. 2013; Alastruey-
Izquierdo et al. 2010). Some clinical laboratories that do not routinely use sequence
analysis for identification of fungi attempt to discriminate between species within
the genera Rhizopus and Mucor by the presence or absence of rhizoids. However,
these morphologic features are not specific to Rhizopus species and can be found in
several Mucor species, including the clinically relevant species M. circinelloides.
There is also some controversy regarding the name of one of the most prevalent
causes of mucormycosis, Rhizopus arrhizus or Rhizopus oryzae. Although the
names are synonymous, some have suggested that R. arrhizus is the correct species
name since it was used previous to that of R. oryzae (1892 vs. 1895) (Dolatabadi
et al. 2014). The name that is used may be of consequence as R. oryzae is more
frequently used in the medical literature, and there is some suggestion that there
may be differences in in vitro potency between the triazoles isavuconazole and
posaconazole (Verweij et al. 2009; Gonzalez 2009; Thompson and Wiederhold
2010; Chowdhary et al. 2014). This is significant as both agents can be orally
administered, thus possibly avoiding the need for long-term intravenous therapy
with the nephrotoxic agent amphotericin B in patients with mucormycosis caused
by this species. There is also some controversy as to whether or not the similar
species Rhizopus delemar is a separate species or a variety of R. arrhizus. By
molecular analysis using ITS alone and with multiple loci, several authors had dem-
onstrated that R. delemar is indeed a phylogenetically distinct species (Dolatabadi
et al. 2014; Abe et al. 2006, 2007). In addition, phenotypically R. delemar lacks the
ldhA gene and is unable to produce lactic acid and instead forms fumaric and malic
acid, while R. arrhizus contains two genes for lactate dehydrogenase and is there-
fore able to produce lactic acid (Abe et al. 2007; Saito et al. 2004). The genome
sequence of a R. delemar strain previously classified as R. oryzae that was obtained
from a fatal case of mucormycosis is also available (http://www.ncbi.nlm.nih.gov/
bioproject/13066) (Ma et al. 2009). However, zygospore formation has been

reported in crosses between R. delemar and R. arrhizus strains, which has led some
to suggest that these are the same species, even though the viability of the progeny
could not be demonstrated (Dolatabadi et al. 2014). It is currently unknown if there
is a clinical difference between R. delemar and R. arrhizus.
Conclusion
The introduction of molecular tools for the identification and classification of fungi
has led to significant changes in fungal taxonomy and nomenclature. These changes
have major implications for the field of medical mycology, which may be both ben-
eficial and detrimental in the clinical setting. The correct identification of the spe-
cies that is causing infection is important and can help guide therapy and ensure the
use of appropriate antifungal agents. However, the rapid changes in fungal taxon-
omy may also lead to nomenclature instability in the clinical setting, and there is
concern that this may impede access to clinically relevant literature. The changes in
taxonomy and nomenclature are also currently outpacing our understanding of the
clinical significance of newly classified cryptic species as well as how to effectively
manage patients with infections caused by these organisms.
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Chapter 19
Mycotoxins in Food and Feed: A Challenge
for the Twenty-First Century
J. David Miller
By the turn of the nineteenth century, it was recognized that microfungi produced
compounds deleterious to animal and human health, although much of this literature
was from the USSR and Japan and not accessible to the English-speaking world
(Ceigler and Bennett 1980). From the first use in the literature (circa 1960), the term
“mycotoxin” refers to secondary metabolites produced by microfungi that are capa-
ble of causing disease and death in humans and other animals. By convention, this
excluded mushroom toxins (Bennett and Klich 2003; Miller 1995). When the con-
ditions favor the growth of toxigenic species on crops or food, it is an invariable and
unfortunate rule that one or more of the compounds for which the fungus has the
genetic potential are produced. A great deal is known about the impact of the agri-
culturally important mycotoxins on public health and the economics of farming
(Pitt et al. 2012; Miller et al. 2014).
Mycotoxins have affected human populations since the beginning of organized
crop production. Ergot of rye is mentioned several times in various Biblical texts
(Schiff 2006). Epidemics of ergotism were reported in Western Europe from about
800 AD. The screams of the victims, the stench of rotting flesh, extremities falling
off, and death all feature in the descriptions of the disease (Van Rensburg and
Altenkirk 1974). In 1630, Dr. Thuillier was the first to prove that consumption of
ergoty rye caused ergotism. He observed that the intensity of the malady was in
proportion to the amount of ergoty grain consumed and that people with more
diverse diets suffered less or not at all. He fed ergot sclerotia to chickens, geese, and
pigs and they all died. Unfortunately, he did not publish his results. It was left to his
son, also a physician, working with the Paris Academy of Sciences to repeat the
experiments (and publish). More than a century after this, L’Abbé Tessier proposed
cultivation of potatoes instead of rye, improved drainage, and the enforced cleaning
of grains. About the same time, Johann Taube eliminated ergotism in patients by
J.D. Miller, Ph.D. (*)

Department of Chemistry, Carleton University, Ottawa, ON, Canada K1S 5B6
e-mail: david.miller@carleton.ca

DOI 10.1007/978-3-319-29137-6_19
470 J.D. Miller
controlling the quality of bread in hospitals in Gottingen (Stroup 1990; van Dongen
and de Groot 1995).
There are only five agriculturally important mycotoxins with worldwide distri-
bution: aflatoxin, deoxynivalenol, fumonisin, zearalenone, and ochratoxin. In cool
temperate and generally wet areas, T-2 toxin /HT-2 toxin can be a problem.
Similarly, although ergot sclerotia are efficiently removed during milling, ergot
alkaloids can occur in processed cereals in affected regions. Excepting in the case
of mycotoxins in corn that is nixtamalized (De La Campa et al. 2004), all of these
toxins are stable in the processes typical of food and feed processing whether in
cereals or nuts (Bullerman and Bianchini 2007; Kabak 2009; Park 2004). Pork
products can be a minor dietary source of ochratoxin. For the remaining toxins,
animal sources are not important under normal circumstances (Perši et al. 2014;
Prelusky 1994). Milk can contain aflatoxin M-1 a mammalian metabolite of afla-
toxin B1 albeit with much lower potency (IARC 2012).
Additionally, there are a number of Penicillium toxins that affect animal health
that occur in silage (Nielsen et al. 2006; Rasmussen et al. 2010; Sumarah et al.
2005; Tangni et al. 2013), mold-damaged recycled food (Rundberget et al. 2004),
and sometimes corn left in the field after harvest (Sumarah et al. 2005).
Deoxynivalenol, Nivalenol, and Zearalenone
“Red mold poisoning” was reported in rural Japan throughout the 1950s. Eventually,
deoxynivalenol (DON) was discovered by Japanese researches from grain that had
made humans ill (Morooka et al. 1972). The same chemical was subsequently
re-reported as “vomitoxin” from Fusarium graminearum-contaminated corn in 1973
(Vesonder et al. 1973). Large-scale acute human toxicoses from deoxynivalenol have
occurred in modern times in India (Bhat et al. 1989), China, and Korea among other
countries (Beardall and Miller 1994; Miller 2008). Corn contaminated by F. gra-
minearum was associated with estrogenic symptoms particularly in swine from the
1920s. An active fraction was isolated from corn and ultimately the chemical structure
of zearalenone was reported in 1966 (Caldwell et al. 1970).
The Fungi
These toxins occur when wheat, barley, corn, and sometimes oats and rye are
infected by Fusarium graminearum and F. culmorum or F. asiaticum. Incidence of
Fusarium Head Blight is most affected by moisture at anthesis under warm condi-
tions (Schaafsma and Hooker 2007; Sutton 1982). F. culmorum is associated with
cooler growing conditions (Miller 1994). The same fungi cause a similar disease in
corn called Gibberella or pink ear rot. Disease incidence is most affected by mois-
ture at flowering/silk emergence. In the USA and Canada and southern China,
19 Mycotoxins in Food and Feed: A Challenge for the Twenty-First Century 471
Fusarium head blight is typically caused by F. graminearum, which is common in

wheat grown in North America and China. In the 1980s, F. culmorum was the domi-
nant species in cooler wheat-growing areas, such as the UK, Finland, France,
Poland, and The Netherlands (Daamen et al. 1991; Miller 1994), but in recent years,
warmer summers have resulted in dominance by F. graminearum (van der Fels-
Klerx et al. 2012; Xu et al. 2005).
F. graminearum comprises a number of closely related clades (O’Donnell et al.
2004), but F. graminearum sensu stricto and F. asiaticum appear most common.
Morphologically identical isolates of F. graminearum sensu lato and F. culmorum
can produce either deoxynivalenol (DON) and zearalenone or nivalenol and zeara-
lenone as the principal toxic metabolites that accumulate in grain. Within the former
group, some strains produce DON by the 3-acetylated precursor and others make
the 15 acetylated precursor. Historically, DON-producing strains with the
15-acetylated precursor dominated in Canada, the USA, Mexico, and South America
(Miller et al. 1983a, 1991; Mirocha et al. 1989; Piñeiro et al. 1995). The Asian and
New World strains are genetically distinct (O’Donnell et al. 2000). In China and
Japan, strains that produced DON generally did so via 3-ADON (Miller et al. 1991;
Yoshizawa and Jin 1995). There are few older data from Europe, but these suggest
that 3ADON chemotypes were more common (Logrieco et al. 1988; Miller et al.
1991). Recent surveys in Europe indicate that most (80 %) isolates of F. gra-
minearum produce DON via 15-ADON, but most isolates of F. culmorum produce
nivalenol and those that produce DON do so via 3-ADON (Somma et al. 2014;
Vogelgsang et al. 2009). So far, nivalenol-producing strains of F. graminearum are
rare in Canada (Tanaka et al. 1988) and the USA (Gale et al. 2011; Schmale et al.
2011). The toxin is not found in Canadian grain (Tittlemier et al. 2013). In addition,
nivalenol- producing F. asiaticum occurs in the southeast USA. This had hitherto
only been known in Japan and China (Gale et al. 2011).
The Asian chemotypes were probably introduced to eastern Canada from wheat
cultivars imported from Europe to the Maritimes in the 1970s and 1980s. The first
3-ADON strain in the Canadian National Mycological Herbarium (DAOM) was
deposited in 1969. Another source of the strains was from breeding material brought
in from China and Europe. By 2007, nearly 100 % of the strains in Atlantic Canada
were Asian as opposed to the native 15-ADON-producing strains. A similar situation
has emerged in Manitoba and Saskatchewan. As of 2007, the 3-ADON genotype
accounted for ca. 70 % of a large number of strains examined (Kelly et al. 2015). In
this study, 5.8 % of the strains tested were from Ontario and, where given, were
mainly from soft wheat. Where the county of harvest was provided (Kelly et al.
2015), these are soft wheat, shorter season areas. F. graminearum strains were iso-
lated in a survey in 2010 of 24 fields in Essex, Chatham-Kent, Hamilton-Wentworth,
Brant, Elgin, Middlesex, Huron, Perth, Wellington, and Lambton Counties. Almost
all (97 %) of 155 isolates of F. graminearum were of the 15-ADON chemotype; the
remainder were the 3-ADON genotype (Tamburic-Ilincic et al. 2015). In upstate
New York, the 15-ADON genotype also predominated, but there were important
differences: some 92 % (652/709) at near Lake Ontario, 78 % (332/379) somewhat
inland in New York, and 53 % (167/319) at a location near Lake Champlain.
472 J.D. Miller
The authors suggested that “regional populations may be differentiated based on

selection associated with climatic or landscape features not currently identified”
(Kuhnem et al. 2015a). A companion study indicated that host (corn, wheat) “did not
appear to structure the populations” examined (Kuhnem et al. 2015b).
Isolates from the 3-ADON chemotype produce significantly more
DON + 3-ADON and are more fecund and have higher growth rates than isolates
from the 15-ADON chemotype (Ward et al. 2008). These conclusions have been
confirmed in a number of field studies in wheat in Canada (Amarasinghe et al. 2015:
Gilbert et al. 2002, 2010; Tamburic-Ilincic et al. 2008; von der Ohe et al. 2010) and
in the adjacent USA (Schmale et al. 2011). Clear et al. (2013) reported studies that
may provide some information of the impact of this in practical terms. They inocu-
lated a barley field with strains of the two chemotypes. Three years later, the preva-
lence of the two chemotypes changed such that the 3-ADON chemotype dominated.
Further, the highest kernel DON concentrations were associated with the increased
frequency of the 3-ADON chemotype. In general though, the aggressiveness
between the two strains appears to be similar (Spolti et al. 2014; Kuhnem et al.
2015b). Thus, it appears that modest differences in temperature and perhaps rainfall
play a role in the distribution of the two important clades (Backhouse 2014; Del
Ponte et al. 2015; Zhang et al. 2012), but this remains unclear (e.g., Gilbert et al.
2014; Kuhnem et al. 2015a; Panthi et al. 2014).
However, genetic changes are also occurring. When the two chemotypes were
recognized in 1983 (Miller et al. 1983a, 1991), the two populations were genetically
homogeneous (O’Donnell et al. 2000); however, a shift has taken place towards a
population of genetically divergent strains (Gale et al. 2007; Mishra et al. 2009).
In China, it appears that this is manifest by a rapid shift under way to more virulent
populations of F. graminearum sensu lato (Zhang et al. 2012). Strains that are crosses
of the two ADON chemotypes but produce 15 ADON are more virulent than either
parent (Foroud et al. 2012). In addition, the so-called Northland population origi-
nally isolated and first described in the Midwest USA does not produce DON or
nivalenol but a related trichothecene, 3ɑ-acetoxy, 7ɑ,15-dihydroxy-12,13-
epoxytrichothec-9-ene (Varga et al. 2015). So far, this population appears uncom-
mon, with isolates collected in Minnesota, North Dakota, and South Dakota (Liang
et al. 2014) as well as Québec, Ontario, PEI, and Saskatchewan (Kelly et al. 2015).
The Toxins
Deoxynivalenol (DON) Because there can be high human exposure to this toxin, it
has been a serious global challenge for four decades. Consequently, it has been
necessary to carefully understand the toxicity of DON. In relevant animal models,
DON is excreted rapidly in the urine, and depending on dose, a small amount may
be excreted in feces after conjugated. DON is not found in meat, milk, or eggs
(Miller 2008; Pestka 2010). The Joint Expert Committee on Food Additives and
Contaminants of the FAO and WHO (JECFA) Provisional Maximum Tolerable
Daily Intake (PMTDI) is based on weight reduction in a 2-year study in male and
female B6C3F1 mice (Iverson et al. 1995; JECFA 2011). At high doses, DON
results in emesis and anorexia in humans, swine, and mink (Miller 2008). The mini-
mum oral dose required for emesis in swine is in the order of 100 mg kg/bw (Pestka
et al. 1987). The emetic response in dogs appears to occur at a similar dose (Ueno
1983). The mechanism for the emetic effect appears to be mediated by the effect of
DON on peptide YY3-36 and 5-hydroxytryptamine (Wu et al. 2013). There are a
number of mechanisms that result in DON-induced anorexia. DON modulates the
insulin-like growth factor acid-labile subunit expression (Amuzie and Pestka 2010).
Additionally, DON results in neuroendocrine signaling at the enteric and central
levels (Pestka 2010). DON crosses the blood–brain barrier resulting in nausea
resulting from inflammation and resulting cytokine upregulation in the appetite cen-
ter (Bonnet et al. 2012). Chronic exposure above the PMTDI results in loss of intes-
tine cell wall integrity, mucosal immune function, and immunological impairment
(Pestka 2010; Pinton and Oswald 2014).
The established provisional maximum tolerable daily intake limit (PMTDI) for
DON is 1 μg/kg body weight/per day on the basis of the NOAEL of 100 μg/kg bw
per day in the Canadian 2-year feeding study of mice and a safety factor of 100
(JECFA 2001). This was modified in 2010 to include both acetates (3- and 15-ADON)
for a group PMTDI (JECFA 2011). In 1993, IARC classified DON as a category 3,
which is not classifiable as to its carcinogenicity to humans, and no data have emerged
to change this determination (IARC 1993; JECFA 2001, 2011).
Nivalenol Much less is known about the toxicity of nivalenol, although it is broadly
assumed to be similar to that of DON (Pestka 2010; Sugita-Konishi and Nakajima
2010). Its emetic potential is ca. 1/10th that of DON (Wu et al. 2013). There are a
number of chronic studies in mice that have been conducted that have provided low-
est observed adverse effect levels (LOEAL) but not NOAELs. EFSA and the Food
Safety Commission of Japan set TDIs for nivalenol 1.2 μg/kg bw/day and 0.4 μg/kg,
respectively (FSCJ 2010; EFSA 2013).
Zearalenone This compound is an estrogen analogue and causes hyperestrinism in

female pigs and is a potent reproductive toxicant; the dietary no effect level is < 0.1
mg/kg bw/day. Cows and sheep are also sensitive to the estrogenic effects of this
toxin with depressed ovulation and lower lambing percentages. The dietary
NOAELs in domestic animals other than pigs are not clearly known (Fink-Gremmels
and Malekinejad 2007; Metzler et al. 2010; Prelusky et al. 1994). Zearalenone
efficiently binds to both mammalian estrogen receptors in a manner similar to
17β-estradiol (Takemura et al. 2007). Zearalenone has been implicated in several
incidents of precocious pubertal changes in girls in Europe and South America
(Falkay et al. 1993; Schiefer 1990). There is some evidence that zearalenone expo-
sure affects reproductive health in young women. Massart et al. (2008) found that
girls living in a part of Italy where corn can be contaminated by zearalenone had
precocious puberty. In a small cohort, this was related to plausible serum
concentrations of zearalenone in their serum (based on the pharmacokinetics and
474 J.D. Miller
contamination levels in the region). There were other possible factors identified in
the study, but a role of zearalenone could not be ruled out (Massart and Saggese
2010). Similarly, a role for zearalenone in the same phenomenon could not be
excluded from a subsequent study in China (Deng et al. 2012). EFSA (2011a) con-
cluded that there were a number of reports linking zearalenone to human disease
that are biologically plausible, but the data are inconclusive. The PMTDI for zeara-
lenone is 0.5 μg/kg bw based on the NOEAL of a 15-day study in pigs and a safety
factor of 100 (JECFA 2000). The EU Scientific Committee on Food (SCF) estab-
lished a temporary TDI of 0.2 μg/kg per day (EFSA 2011a).
Aside from the regulated toxins, DON, or nivalenol and zearalenone, F. gra-
minearum and related species produce a wide variety of metabolites from several
biosynthetic families (Miller et al. 1991). This includes other apotrichothecenes
(Greenhalgh et al. 1989), calonectrins (Greenhalgh et al. 1985, 1986), sambucinol,
sambucoin (Greenhalgh et al. 1986), and culmorins (Kasitu et al. 1992) and
butenolide.
Fumonisin
The Fungi
Discovered only in 1988, fumonisins (Marasas 2001) are produced by Fusarium

verticillioides (formerly F. moniliforme), F. proliferatum, and F. fujikuroi as well as
some uncommon species, F. anthophilum, F. dlamini, F. napiforme, and F. thapsi-
num (Rheeder et al. 2002; Suga et al. 2014). Fumonisin can be a contaminant of rice
resulting from infection by F. proliferatum and F. fujikuroi (Uegaki et al. 2014).
However, most fumonisin exposure results from consumption of corn affected by
the disease Fusarium Kernel Rot. Fusarium verticillioides or F. proliferatum occurs
systemically in leaves, stems, roots, and kernels and can be recovered from virtually
all maize kernels worldwide including those that are healthy (Miller 2001, 2008).
Fumonisin can only accumulate in stressed or senescing kernel tissue under warm
conditions and dry conditions between silking and early grain fill (Miller 2001).
Insect damage is associated with increased fumonisin concentrations under permis-
sive conditions. There is a strong, consistent relationship between insect damage
and Fusarium kernel rot, although the insect species can vary by location (De La
Campa et al. 2005; Parsons and Munkvold 2010, 2012). Maize genotypes contain-
ing the Bt protein have reduced amounts of fumonisin compared to non-Bt geno-
types (De La Campa et al. 2005; Hammond et al. 2004). There are a number of
naturally occurring fumonisins. In order of decreasing concentration in affected
corn, these are fumonisin B1, FB2, FB3, and FB4 (Pitt et al. 2012).
Fumonisin B2 and B4 are produced by Aspergillus niger. These toxins have been
found in grapes (Knudsen et al. 2010) and other dried fruits (Somma et al. 2012),
notably figs (Heperkan et al. 2012; Moretti et al. 2010), and in wine (Logrieco et al.
2010; Mogensen et al. 2010).
The Toxins
Consumption of corn contaminated by F. verticillioides (previously F. monili-

forme) by horses was long associated with equine leukoencephalomalacia (ELEM),
a liquefactive necrosis of the brain leading to death. It was not until the discovery
of fumonisin that the cause was identified (Marasas 2001; Wilson et al. 1990).
Aside from ELEM, fumonisin causes porcine pulmonary edema in pigs (Haschek
et al. 2001). FB1 is toxic to the liver in all species and the kidney in a range of labo-
ratory and farm animal species, causing apoptosis followed by mitosis in the
affected tissues. FB1 is also toxic to the cardiovascular system in pigs and horses.
FB1 and other fumonisins inhibit ceramide synthase in all species including labo-
ratory and farm animals and disrupt sphingolipid metabolism, a process underlying
the mechanism of toxicity and pathogenesis of fumonisin-related diseases (Voss
et al. 2007; Voss and Riley 2013). In most assays, FB1 is most potent followed by
FB2, FB3, and FB4.
The discovery of fumonisin resulted from studies of an area of South African
with high corn consumption and very high rates of esophageal cancer. Fumonisin
proved to be a potent cancer promoter and is a rodent carcinogen. The linkage
between fumonisin and human cancer remains unclear (Bulder et al. 2012; Marasas
2001). Fumonisin has also been associated with neural tube birth defects in several
populations mainly in Africa, China, as well as parts of Latin America and Mexico.
Based on studies in two strains of rats and two non-human primate species, fumoni-
sin does not cross the placenta (IPCS 2000). There is, however, limited epidemio-
logical evidence suggesting that fumonisin exposure during early gestation could be
involved in the increased incidence of neural tube defects (NTD) in areas of the
world where maize is consumed in large amounts and diets are likely to be deficient
in critical micronutrients and vitamins necessary for normal neural tube closure
(Gelineau-van Waes et al. 2009; Marasas et al. 2004; Suarez et al. 2012). These
epidemiological data are supported by studies in mice which have provided a partial
mechanistic basis. Low folate is a risk factor for neural tube birth defects. Fumonisin
in the serum prior to the development of the placenta blocks the transport of folate
into the cells of the developing embryo and affects the neural crest cells (Gelineau-
van Waes et al. 2005, 2012; Marasas et al. 2004; Voss et al. 2014). However, at the
time of writing, there is no direct evidence that fumonisin causes NTD in humans.
Two studies from Tanzania suggest that fumonisin exposure may be associated
with stunting in children. In this prospective cohort study, infants were enrolled at
6 months of age and followed until 12 months of age. Exposure was categorized as
high or low, using the JECFA provisional maximal tolerable dietary intake (PMTDI).
By 1 year, the highly exposed infants were significantly shorter and lighter than the
105 infants with low exposure, after controlling for total energy and protein intakes,
gender, and village (Kimanya et al. 2010). Shirima et al. (2014) measured fumonisin
and aflatoxin exposure. They reported that there was an inverse association between
urinary FB1 and growth after 1 year. In this study, the aflatoxin exposures did not
result in stunting. Fumonisin is known to affect gut cell function (Bouhet and Oswald
2007), which may provide support for these epidemiological data.
476 J.D. Miller
In 2012, the WHO/FAO Joint Expert Committee on Food Additives (Bulder

et al. 2012) evaluated the existing human epidemiology studies linking fumonisin
exposure to NTDs and concluded that the results in combination with what is known
about the toxicology of fumonisin “…indicates that fumonisin exposure in pregnant
women may be a contributing factor to increased NTD risk in their babies.”
Fumonisin B1 (FB1) has been classified as a Group 2B carcinogen, possibly carci-
nogenic to humans (IARC 2002). The established provisional maximum tolerable
daily intake limit for the fumonisins (FB1, FB2, and FB3) from all sources is 2 μg/
kg body weight/per day on the basis of the NOEL of 0.2 mg/kg bw per day and a
safety factor of 100 (JECFA 2011).
Fumonisin and aflatoxin frequently co-occur and concurrent exposures can be
very high in parts of the world dependent on maize as a staple food including for
example Nigeria (Adetuniji et al. 2014), Kenya (Mutiga et al. 2014), and Guatemala
(Torres et al. 2014). There are a number of implications of this noted following.
Aflatoxin
The Fungi
Aflatoxin is produced by 13 species of fungi, most of which are of no relevance to

agriculture. This list currently includes Aspergillus pseudotamarii, A. nomius, A.
bombycis, A. parvisclerotigenus, A. minisclerotigenes, A. arachidicola, A. ochra-
ceoroseus, A. rambellii, Emericella astellata, E. venezuelensis, and E. olivicola
(Varga et al. 2009). Aspergillus flavus was first recognized to cause aflatoxicosis in
domestic animals (Wogan 1966) and is the most important species. A. flavus pro-
duces aflatoxin B1 and B2 and is a problem in many commodities, but most human
exposure comes from contaminated corn, peanuts, and rice. The second important
producer of aflatoxin, A. parasiticus, produces aflatoxin B1, B2, G1, and G2 and is
primarily associated with peanuts in the Americas but also can occur on corn, figs,
and pistachios (Horn 2003).
A. flavus infection of corn or peanuts results from (1) airborne or insect-
transmitted conidia that contaminate the silks and grow into the ear when the maize
is under high temperature stress and (2) insect or bird damaged kernels that become
colonized with the fungus and accumulate aflatoxin. Drought-, nutrient-, or
temperature-stressed corn or peanut plants are more susceptible to colonization by
A. flavus or A. parasiticus (Guo et al. 2008; Horn 2007; Pitt et al. 2012; Wu et al.
2011). Studies in the USA Corn Belt have shown that, typically, A. flavus-infested
kernels are randomly distributed in ears resulting mainly by insect damage (Payne
and Widstrom 1992; Smart et al. 1990). The kernels that were the sites of initial
infection have very high aflatoxin contents. In contrast, the ecology of A. flavus in
corn in subtropical regions of Asia and the USA is different from that in the
Midwest USA. In subtropical areas, apparently healthy kernels are infected (Miller
1995; Pitt et al. 2012).
It has been long suggested that many if not most species of Aspergillus have a
tropical to subtropical distribution or more precisely abundance peaks in the sub-
tropics (Christensen and Tuthill 1985). A systematic review of the prevalence of 52
species from plating data indicated that 30 occurred above expected frequencies in
the tropical latitudes and hence were less common in higher latitudes. The data were
separately considered by ecozone: forest, grassland, desert, and cultivated land.
Of these, grassland had the lowest diversity (Klich 2002).
The genetics of Aspergillus flavus and A. parasiticus has been studied in great
detail. In the past few years, this information has enabled informative studies on
the ecology of these species. Studies of mating type distribution, female sterility,
and aflatoxigenicity in A. flavus and A. parasiticus done on a global level revealed
that in cooler wet areas, atoxigenic strains predominate in clonal A. flavus popula-
tions and toxigenic strains predominate in warm dry areas (Olarte et al. 2012).
In fertile strains, there is both qualitative and quantitative variation in AF chemo-
types (B1, B2, G1, G2, o methyl sterigmatocystin, and cyclopiazonic acid; Moore
et al. 2013; Olarte et al. 2012, 2015).
This modern evidence of the high prevalence of A. flavus and A. parasiticus in
warm and dry areas supports older data resulting from traditional plating methods.
Propagule densities are much higher in cultivated fields and desert ecosystems are
than in forests and prairies (Horn 2003; Klich 2002). In a study in Missouri (39°
N), the maximum number of A. flavus and A. parasiticus propagules was found in
soil cropped to a rotation of wheat, red clover, and corn using conventional tillage
practices. No isolates were observed in a virgin prairie soil (Angle et al. 1982).
Apart from that, the prevalence of aflatoxigenic strains is known to be associated
with latitude. Both the occurrence of the two species and the percentage that pro-
duced aflatoxin were examined in a transect from northern Japan to Indonesia. A
modest percentage of strains of the two species were isolated in soil from the north
of Japan (43° N) and, of these, a negligible percentage produced aflatoxin. In con-
trast in Indonesia (6.6° S), a high percentage of soil fungi were A. flavus and almost
all were good producers of aflatoxin (Manabe and Tsuruta 1978). In warm areas,
as fields are converted to corn and peanuts, the prevalence of aflatoxigenic species
increases (Horn 2003).
Aflatoxin is not a problem in colder corn-growing areas as, for example, Canada.
There are few data on the occurrence of A. flavus in Canadian soils. Bisby et al.
(1933) reported the isolation of kojic acid-producing strains from Manitoba soils. A
study of soil fungi in eastern Ontario from fields planted to alfalfa and old agricul-
tural soils did not report A. flavus (Keller and Bidochka (1998). Soil fungi were
plated from a growing corn field in Ottawa (see Miller et al. 1983b) and A. flavus
was found as an occasional component of the mycoflora (Miller unpublished data).
Corn feed samples collected in seven midwest states in 1988–1989 were analyzed
for fungi and incubated under adverse storage conditions followed by analysis for
mycotoxins. Corn from Michigan did not contain aflatoxin initially or after storage
as above. Samples were positive in two states just south of Ontario, Ohio, and
Minnesota (Russell et al. 1991).
478 J.D. Miller
The Toxins
There are many detailed reviews of the toxicology of aflatoxin including that of
IARC (Pitt et al. 2012). Aflatoxin B1, the most toxic of the aflatoxins, causes a vari-
ety of adverse effects in different animal species, especially chickens. In poultry,
these include liver damage, impaired productivity and reproductive efficiency,
decreased egg production in hens, inferior egg-shell quality, inferior carcass quality,
and increased susceptibility to disease (Wyatt 1991). Swine are somewhat less sen-
sitive than poultry species with the LD50 being perhaps half of that of chickens.
Aflatoxin is hepatotoxic and its acute and chronic effects in swine are largely attrib-
utable to liver damage (Armbrecht 1978). In cattle, the primary symptom is reduced
weight gain as well as liver and kidney damage. Milk production is reduced (Keyl
1978). Aflatoxin is also immunotoxic in domestic and laboratory animals with oral
exposures in the ppm range. Cell-mediated immunity (lymphocytes, phagocytes,
mast cells, and basophils) is more affected than humeral immunity (antibodies and
complement; Bondy and Pestka 2000). The effects of aflatoxin on laboratory animals
have been exhaustively reviewed by IARC (Pitt et al. 2012). Cyclopiazonic acid is
also produced by most strains of A. flavus and some related species and accumulates
in the crop along with aflatoxin. This compound is toxic and immune suppressive in
various strains of mice and rats as well as swine and poultry (Burdock and Flamm
2000; de Waal 2002; King et al. 2011).
Naturally occurring mixtures of aflatoxins were classified as class 1 human car-
cinogens and aflatoxin B1 is also a class 1 human carcinogen. There is inadequate
evidence of the human carcinogenicity of aflatoxin M1, the metabolite of aflatoxin
B1 found in human and animal milk (IARC 2012). Aflatoxin exposure explains
approximately 25 % of liver cancer globally notably in Africa (40 %) and Asia (27
%; Liu and Wu 2010). Many people in developing countries are seropositive for
hepatitis B and C which are also liver carcinogens. Although aflatoxin is a potent
chemical carcinogen, its ability to alter response to the hepatocarcinogenic viruses
is perhaps of greater importance. The relative rates of liver cancer in hepatitis B
positive populations are an order of magnitude greater (~60×) when exposed to
aflatoxin. This is because the toxin interferes with the processing of the virus. Thus,
reducing aflatoxin exposure to non-detectable levels could reduce HCC cases in
high-risk areas by ~25 % (Liu et al. 2012).
Aside from the carcinogenicity of aflatoxin, exposure may be associated with
child stunting. Two studies have been reported involving 680 children living in
West Africa. Height and weight for age were lower in a dose-dependent fashion for
increasing aflatoxin exposures as measured by the aflatoxin–albumin adduct
(AF-alb) in serum (Gong et al. 2002). In a longitudinal study, over a period of 8
months, children with the highest aflatoxin exposures had the smallest gains in
height (Gong et al. 2004).
As noted above, in Africa and parts of Latin America, co-exposure to aflatoxin
and fumonisin at multiples of the acceptable limits is common. AFB1 is a potent
mutagen and DNA-reactive carcinogen, while FB1 is an effective cancer promoter

(Gelderblom et al. 1988) with a non-genotoxic mechanism of action (Bulder et al.
2012). It is, therefore, reasonable to believe that co-exposure is likely to enhance
hepatoxicity and hepatocarcinogenicity in humans.
Ochratoxin
The Fungi
In cereals, Penicillium verrucosum is the sole producer of ochratoxin (OTA). P.

nordicum is known to produce OTA on dried salted meats (Cabañes et al. 2010;
Sonjak et al. 2011. Many species of Aspergillus produce OTA. In the section
Circumdati, some 13 species are good producers of ochratoxin A: A. affinis, A. cre-
tensis, A. fresenii, A. muricatus, A. occultus, A. ochraceopetaliformis, A. ochraceus,
A. pseudoelegans, A. pulvericola, A. roseoglobulosus, A. sclerotiorum, A. steynii,
and A. westerdijkiae. Some other species are variable producers of this toxin.
Agricultural products damaged by A. ochraceus, A. steynii, and A. westerdijkiae are
of greatest concern for OTA contamination (Visagie et al. 2014). The geographic
distribution of some of these species remains unclear. OTA contamination of cocoa,
coffee, and grapes and wine results from A. carbonarius and some strains of
A. niger and this is an important problem mainly in warm grape-growing areas. The
other species in section Nigri that produces OTA is A. sclerotioniger (isolates from
coffee; Varga et al. 2011).
Ochratoxin in coffee and cocoa can be minimized by good agricultural practices
during harvest, drying, and processing followed by sorting out damaged beans
(Copetti et al. 2013; Taniwaki 2006). Preventing OTA contamination in grapes and
wine is somewhat more difficult. The occurrence of the relevant species of black
aspergilli is affected by several factors including weather, agronomic practice, and
fungicide use (Battilani et al. 2003; Hocking et al. 2007). Grape damage from
insects (Cozzi et al. 2006) and fungal diseases (e.g., mildew, Botrytis bunch rot) and
rain damage predispose the grape to colonization by A. carbonarius and A. niger.
Harvesting grapes with minimal damage, rapid processing, and good sanitation
practices reduce the risk of OTA (Hocking et al. 2007; Visconti et al. 2008).
A few percent of surface-disinfected wheat and barley kernels collected at harvest
in the UK, Denmark, and Sweden were contaminated by P. aurantiogriseum and P.
verrucosum and this was similar in studies done in western Canada over many years.
Infestation of some kernels by the ochratoxin-producing fungus P. verrucosum
occurs from anthesis and surface contamination is common at harvest. The absolute
level of pre-harvest infestation varies according to site and season (Miller 1995;
Elmholt 2003).
480 J.D. Miller
The Toxin
Ochratoxin is a potent nephrotoxin in swine and causes kidney cancer in male

Fisher 344 rats (Benford et al. 2001). Pigs are affected at low exposures in terms of
kidney damage, but typically there are no overt signs or biochemical/hematological
changes. At higher concentrations (>2 μg/g), decreased weight grains occur
(Prelusky et al. 1994; Stoev et al. 2002). Poultry are similarly affected with reduced
growth rate and egg production at low ochratoxin concentrations > 2 μg/g. Higher
dietary ochratoxin concentrations are often fatal. Cattle are resistant to the ochra-
toxin concentrations typical of naturally contaminated grain (Prelusky et al. 1994).
Ochratoxin is often found with other toxins such as citrinin, penicillic acid, and the
naphthoquinone mycotoxins from Penicillium aurantiogriseum (Krogh 1991;
Vrabcheva et al. 2000). Citrinin mimics the effects of ochratoxin, although it is less
potent (Krogh 1991). The naphthoquinones xanthomegnin and viomellein from
P. aurantiogriseum are nephrotoxic (Carlton et al. 1976). Interactions have been
reported between ochratoxin and citrinin and ochratoxin and penicillic acid on renal
toxicity in swine (Prelusky et al. 1994; Stoev et al. 2001).
Human exposure to ochratoxin primarily occurs from whole-grain breads. Some
exposure comes from the consumption of animal products, especially pork and pig-
blood-based products (Kuiper-Goodman and Scott 1989; Kuiper-Goodman et al.
2010). Ochratoxin has been long suspected as the cause of urinary tract cancers and
kidney damage in areas of chronic past chronic exposure in parts of Eastern Europe
(Anonymous 1977; Kuiper-Goodman and Scott 1989; IARC 1993). After investiga-
tions of Balkan Endemic Nephropathy (BEN) began in the late 1960s, various eti-
ologies were suggested including viral disease. With the recognition that ochratoxin
could produce serious renal pathology in swine, the ochratoxin hypothesis was
strongly advanced by researchers (e.g., Krogh and Elling 1976; Pfohl-Leszkowicz
et al. 2002). Various mechanisms have been advanced to explain the carcinogenic-
ity including genotoxicity and the putative linkage to BEN (Haighton et al. 2012;
Pfohl-Leszkowicz and Manderville 2011).
However, after 50 years, there is little evidence for OTA as causing human dis-
ease (Bui-Klimke and Wu 2014a). Historically, OTA has been a common exposure
in parts of Eastern Europe where BEN occurs; exposure to aristolochic acid also
may be common in some years. This is a potent renal toxicant and IARC class 1
human carcinogen. The literature suggested that Aristolochia was not an important
weedy species in the region. However, that is the case (Dimitrova 2009; Markovic
et al. 2005; Mehmeti et al. 2009). Seed set occurs just prior to harvest; thus, exposure
to aristolochic acid can occur in the region where BEN has been seen. Aristolochic
exposure results in tumors with a characteristic genetic signature which are seen in
at least some BEN patients (Grollman et al. 2007; Savin et al. 2014). Bui-Klimke
and Wu (2014b) concluded that on a weight of evidence basis, aristolochic acid is
the probable cause of BEN (see also Wu and Wang 2013).
The last IARC evaluation of ochratoxin determined it to be a possible human
carcinogen (IARC 1993). In 1995, the JECFA established PTWI of 100 ng/kg bw
per week (Benford et al. 2001). In reaching this conclusion, “the Committee noted
the large safety factor applied to the NOEL for nephrotoxicity in deriving the PTWI,
which corresponds to a factor of 1500 applied to the NOEL for carcinogenicity in
male rats, the most sensitive species and sex for this end-point.” The last JECFA
evaluation retained the PMTI which was supported using a study demonstrating the
LOEAL in swine based on nephrotoxicity (Stoev et al. 2002). The JECFA panel
remarked that “Although an association between the intake of ochratoxin A and
nephropathy in humans has been postulated, causality has not been established.”
The current PTWI is 100 ng/kg bw (Benford et al. 2001).
Other Mycotoxins that Can Occur in Food or Feed
T-2 Toxin
T-2 toxin was isolated from “strain T-2” of F. sporotrichioides mis-identified as

F. tricinctum isolated from corn associated with cow mortalities (Ueno 1983; Marasas
et al. 1984). F. sporotrichioides also produces HT-2 toxin and typically both are pres-
ent in affected commodities along with various other compounds (Greenhalgh et al.
1988). T-2 is also produced by F. acuminatum (Miller 1994). T-2 toxin has been the
subject of considerable toxicological study (IARC 1993) because it is easy to isolate
and purify. During World War II, there were large-scale poisonings of the rural popu-
lation in the former Soviet Union caused by the consumption of overwintered grains
left in the field over winter (estimates range to 1,000,000 victims). The disease was
called Alimentary Toxic Aleukia (Joffe 1971). Samples of extracts made at the time
were shown to contain T-2 and related toxins (Mirocha and Pathre 1973). In cool
temperate and generally wet areas, T-2 toxin /HT-2 toxin can be a problem (Bertuzzi
et al. 2014; Miller 1995; Pettersson et al. 2011). The toxicology of these trichothe-
cenes toxin is similar in character to that described above for deoxynivalenol and is
reviewed extensively by EFSA (2011b).
Patulin
Patulin is primarily found in apple and grape juices where it occurs from the growth
of Penicillium expansum on rotted fruit (Menniti et al. 2010). Aside from patulin, this
fungus produces citrinin, chaetoglobosins, communesins, roquefortine C, and expan-
solides A and B (Andersen et al. 2004). Patulin is easily controlled by removing
rotted fruit (Menniti et al 2010) and is further reduced by processing (Welke et al.
2009). The sparse toxicological data on this compound have been reviewed by Puel
et al. (2010) and Brandon et al. (2012). The JEFCA PMTI is 400 ng/kg BW day
which is based on growth retardation in mice (JECFA 1995).
482 J.D. Miller
Ergot Alkaloids
Ergot alkaloids seldom appear in meaningful concentrations in food samples in the

North America or Europe because the presence of Claviceps sclerotia in grains is a
grading factor. Ergotism in humans has only rarely been reported in modern times in
France, India, and parts of Africa (Beardall and Miller 1994; Belser-Ehrlich et al.
2013). Ergot sclerotia are efficiently removed during milling. Thus, the majority of
products made from wheat or rye contain traces of ergot alkaloids in affected regions
(Malysheva et al. 2014; Scott 2009; Scott et al 1992). Infections by ergot alkaloid-
producing fungi remain common in the sense that plants on the edges of rye, barley,
and wheat fields are often infected. There is some sense that ergot is getting more
common again in parts of Europe (Krska and Crews 2008) and western North
America (Menzies and Turkington 2014). Grains downgraded to animal feed can
contain ergot; thus, this remains an animal health issue (Belser-Ehrlich et al. 2013).
Cattle are more sensitive than mice, sheep, or swine and sclerotial contents of ca. 0.1 %
appear to be tolerable. In cattle, the symptoms include lameness, and in other domes-
tic animals, reduced weight gain can be expected (Prelusky et al. 1994).
Future Prospects
Balbus et al. (2013) reviewed the implications of climate change in relation to man-
agement of human health risks of chemicals in the environment. They identified 8
contaminants as high risk of getting more serious, due to climate change, one of
which was mycotoxins. Importantly, the global population has converged on a few
sources of dietary starch (Khoury et al. 2014) most of which are rather susceptible
to the toxins discussed here. Various authors have provided opinions on the poten-
tial relationship between climate change and mycotoxins (Magan et al. 2011;
Miraglia et al. 2009; Paterson & Lima 2011; Wu et al. 2011) and food security
(Marroquín-Cardona et al. 2014).
There are some examples of climate variability affecting the distribution of
mycotoxins. Climate variability has played a role in the presence of mycotoxins in
corn grown in Ontario. In the period 1972–1981, the prevalence and concentrations
of zearalenone in corn were quite high (Andrews et al. 1981; Scott 1997). Sutton
et al. (1980) found that zearalenone was associated with rainfall in August, but only
moderately or weakly with rainfall for July, September, and October, and occurred
during the latter part of the crop year. During 1970–1980, the weather was cooler.
However, from 1980 to the present mean daily temperatures measured in several
sites in Southwestern Ontario have increased (Environment Canada data for London
airport; Hussell 2003, his figure 2). The biosynthesis of zearalenone has a require-
ment for high oxygen tension. As noted, zearalenone is typically accumulated in
corn in the late summer when the crop is drying. Oxygen tensions are higher than
in living plants and later in the season it is cooler than in July. Oxygen solubility
in water is increased in colder versus warmer water. In contrast, deoxynivalenol is

produced under conditions of low oxygen tension. Deoxynivalenol is seen in corn
kernels concurrent with development of the infection when the plant is living and
little zearalenone is being produced (Miller 2001). This warmer period has seen a
possible change in the presence of fumonisin in corn. Analysis by Hooker and
Schaafsma (2005) of 856 randomly selected Ontario corn fields between the years
of 1993–2000 showed a total incidence of 23 % for fields contaminated with 1.0
FB1 ppm or greater. There was a large year-to-year variation, with the lowest inci-
dence in 1993–1996. The highest incidence, 56 %, was observed in 1999. Mean
concentrations varied between 1.0 ppm in 1994 and 2.3 ppm observed in 1995. The
highest maximum concentration was seen in 1995 (7.0 ppm; Hooker and Schaafsma
2005). In 2012, summer mean temperatures were higher than the normal values in
the Ontario corn-growing areas by as much as 4.3 °C (Environment Canada). In a
sample of 84 corn samples taken at harvest 75 % were positive and six were >1
ranging to 4.2 μg/g (De Lamay Rios & Schaafsma unpublished data).
A more recent example has been the dramatic rise of aflatoxin in corn grown in
parts of Europe. Prior to the early 2000s, aflatoxin was virtually unknown in
European grown maize. In the last decade, this has changed such that in 2013,
aflatoxin contamination became a substantial problem in some EU countries
(Perrone et al. 2014).
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Chapter 20
Inhalation Exposure and Toxic Effects
of Mycotoxins
Harriet M. Ammann
Introduction
The respiratory system differs from ingestion as a toxic exposure route because of
its intimate, direct relationship with the blood in the general circulation through its
gas exchange region. The nose and sinuses, with their baffle-like structures, are very
good at trapping large particles to eliminate them via ciliated transport of mucus to
be swallowed. However, because of nasal and sinus structure, particles, including
mold spores and bacteria, can become trapped and colonize their mucous mem-
branes. Additionally, the nose has axonal nerve endings that are directly connected
with their cell bodies in the brain, along which particles can travel, while most
nongaseous contaminants are excluded from the brain by the blood–brain barrier.
Potency of toxic inhalants is greater than those ingested due to the structure and
function of the respiratory system.
Toxic symptoms in man and farm animals have been known to be caused by
molds for a long time before the identification of mycotoxins in 1960 in England
when over 100,000 turkeys died as a result of “Turkey X disease” which was later
found to be caused by the mycotoxin aflatoxin (UNJR 1970). Intense research on
mycotoxin effects on crops and food animals and on human health from consump-
tion of mycotoxin-containing foods has resulted due to the estimated mean annual
cost of mycotoxin-induced losses of $932 million in food losses, $466 million in
mitigation loss, and $6 million in livestock loss (pre-2003 dollars) (CAST 2003).
While human disease impacts have also been investigated, most of the research on
human health has focused on ingestion of contaminated food. Early reports from the
Soviet Union had tracked a noninfectious disease of horses caused by feeding
moldy straw, and a number of studies detailed local and systemic toxic effects in
H.M. Ammann, Ph.D. (*)

Department of Environmental and Occupational Health Sciences, School of Public Health
and Community Medicine, University of Washington, Seattle, WA, USA
e-mail: h.ammann@comcast.net

DOI 10.1007/978-3-319-29137-6_20
496 H.M. Ammann
humans also exposed to aerosols of toxic strains of Stachybotrys chartarum

(reported as S. atra) (Forgacs 1972). Controlled animal experiments using the inha-
lation route of exposure are more difficult to do and generally more expensive than
ingestion studies, and exposure through mycotoxin-contaminated animal feed and
human food was thought to be a more significant source of damage exerted by
mycotoxins. For these reasons relatively few inhalation experiments on animals
were done in the 1960s and 1970s.
Ueno (1984) found that low-level T-2 toxin (140 ppb) inhalation exposure for
30 min killed mice within several days. In the 1980s and 1990s, the US Army
Medical Research Institute of Infectious Diseases (USAMRIID) supported research
on the inhalation and skin exposure for certain mycotoxins, especially the simple
trichothecene T-2 toxin produced by certain Fusarium species, because of interest
in the potential use of such a toxin as a battlefield weapon that could incapacitate
enemy troops through aerosol dispersion. A seminal paper (Croft et al. 1986) drew
attention to the potential exposure to mycotoxins from indoor mold growth, specifi-
cally that of Stachybotrys atra (=S. chartarum), with subsequent health effects. The
energy crisis of the 1970s brought about a change in building construction, a tight-
ening of buildings without sufficient consideration for ventilation needs, and the use
of new, more moisture susceptible building materials, with the result that dampness
and mold growth were recognized as an increasing problem as health effects in
damp buildings were reported (IOM 2004; WHO 2009; NIOSH 2012). Considerable
research on molds and mycotoxins in the built environment has been done (Nielsen
and Frisvad 2011) and has recently been reviewed by Miller and McMullin (2014).
The work sponsored by USAMRIID used high concentrations of T-2 toxin in
inhalation studies on mice, rats, guinea pigs, and swine to determine acute effects,
including lethality, to determine utility of mycotoxins as a warfare agent. In addi-
tion to determining acute lethality and target tissues, these studies determined inter-
esting differences between exposure routes.
For instance, inhalation of pure T-2 toxin in rats was 20 times more potently
lethal at 10-min high-level exposures than other routes of exposure, including der-
mal application, ingestion, and systemic injection (Cresia et al. 1990). Because the
swine respiratory system is more like humans than those of rodents, swine were also
tested. Inhalation exposure of swine to T-2 toxin was as potent as intravenous injec-
tion for a number of the end points examined (Pang et al 1987, 1988), indicating
direct access of the toxin from the lung to the circulation.
The primary function of the respiratory system is the efficient exchange of oxy-
gen and carbon dioxide across the membranes of the alveoli and the capillaries
which surround them. The blood carries oxygen (and other absorbed substances) to
tissues throughout the body and carbon dioxide from those tissues back to the lung
to be eliminated. However, the air is not composed of only nitrogen, oxygen, and a
number of noble gases, but contains many other gases and particles produced by the
natural environment and by human endeavors. The respiratory system takes in
whatever is in the air, and the tissues ideal for necessary gas exchange present little
barrier to small molecules, including mycotoxins, and, unlike the digestive system,
allows direct entry into the circulatory system and the blood and thus to systemic
20 Inhalation Exposure and Toxic Effects of Mycotoxins 497
target organs. Ingested materials are absorbed primarily through the small intestine
into the enterohepatic circulation which routes blood to the liver where many poi-
sons are detoxified, before blood is returned to the systemic circulation.
The nature of inhaled contaminants, especially the size and aerodynamic diam-
eter of particles, determines where in the respiratory system particles land and what
happens when they interact with the tissues there. Mycotoxins are not considered to
be volatile and are associated with spores and other particles, which serve as a car-
rier into the respiratory system.
Mycotoxins were found in and on spores from molds, and because spores from
many fungal genera are dispersed through aerosolization, it was assumed that mea-
surement of spores in air was a means of determining exposure to mycotoxins. In
itself, measurement of spores in air in buildings is fraught with difficulty since air
moves through natural and mechanical ventilation, and its content of spores varies
with both building and human activity. Those molds that disperse their spores
through the air do so periodically through “blooms,” and some of them show tem-
poral patterns. Thus, concentrations of spores captured during quiescence can be
very low, and those captured during blooms can be many orders of magnitude
higher.
Mycotoxins are located not only in and on spores however but in and on mycelia,
on fragments of both spores and mycelia, and on dust settled on surfaces on which
molds grow (Palmgren and Lee 1986; IOM 2004). Mycotoxins are exotoxins,
secreted onto the surfaces on which molds grow. These toxins are secondary metab-
olites, not needed for maintaining life of the fungal cells, yet costing the organism
considerable metabolic energy (Bennett 1983). Secretion of exotoxins by molds
onto the surfaces where they (and some bacteria) grow is thought to be a means by
which bacteria and molds compete for resources that they need for survival, and this
competition is one of the primary stimuli for toxin production in mixed cultures of
microbes (Nielsen 2003). Temperature, water activity (aw), and nature of the sub-
strate are other major influences (Moss 1991; Nielsen et al 2004; Frazer et al 2012).
While inhibiting the growth of competing microbes provides advantage to produc-
ers, other organisms such as vertebrates, including humans, can be harmed when
exposure inhibits life processes, such as protein, RNA, or DNA synthesis, among
others, that microbes share with other life forms. To distinguish secondary metabo-
lites of molds that primarily affect other microbes (antibiotics) or plants (phytotox-
ins), mycotoxins are defined as low molecular weight secondary metabolites
produced by molds that can harm animals, including humans, at low levels of expo-
sure (Bennett and Klich 2003; Miller and McMullin 2014).
Inhalable Particles and Mycotoxins
Because mold spores and mycelia fragment into smaller particles as spores germi-
nate and mycelia grow and because small particles can dislodge from substrate,
particles and dust with adsorbed toxins can be aerosolized through building and
498 H.M. Ammann
human activity; attempts to determine inhalation exposure need to account for all
these potential exposure agents (Palmgren and Lee 1986). Mycotoxins are generally
not known to be volatile, although some water-soluble toxins can form droplet aero-
sols (Pestka et al. 2008). Jarvis et al. (1998) noted that satratoxins H and G produced
by highly toxic strains of S. chartarum are exported to the surface of spores where
they become water soluble, probably because they are imbedded in surface
polysaccharides.
Spores of molds can be relatively large compared to fragments of spores and
mycelia. For instance, the aerodynamic diameter of Aspergillus versicolor spores is
2.5 μm, those of Cladosporium cladosporioides 1.8 μm, and that of Penicillium
melinii 3.0 μm (Górny et al. 2002). S. chartarum spores are about 5 μm in aerody-
namic diameter (Sorenson et al. 1987). Spore and mycelial fragments 0.3 μm (the
limit of detection of the optical particle counter used) and smaller outnumbered
spores by as many as 320 times in an experiment that studied the release of particles
from moldy ceiling tiles from vibrations normally occurring in buildings, under
various airflow conditions (Cho et al. 2005,2007; AIHA 2008). For S. chartarum,
the number of fragments was 540 times that of spores.
Seo et al. (2007) developed a new field-compatible collection system for small
fungal particles that could divide particles into three size fractions: spores (greater
than 2.5 μm), fragment/spore mixtures (1.0–2.5 μm), and submicron fragments (less
than 1.0 μm). This system was used in measuring fungal fragments in moldy houses
during a field study in homes in New Orleans and Southern Ohio (Reponen et al.
2007) which found that the fungal fragment to spore ratio in actual moldy homes
was much greater than had been determined under laboratory conditions by Cho
et al. (2005). The spore to fragment ratio would be 103 for the 0.3-μm fragment size
and 106 for the 0.03-μm fragment size, very much higher than the estimates from the
earlier laboratory experiments. The authors hypothesize that naturally occurring air
currents in homes and vibrations from activity and other disturbances account for
the difference. Because sampling times ranged from 2 to 3 h, aerosolized spores
could also have settled out, increasing the particle to spore ratio. Small particles,
including mold fragments and dust from surfaces on which mold grows, are more
important from a mycotoxin exposure perspective than mold spores, because small
particles have a much larger aggregate surface area per unit mass than larger parti-
cles such as spores do. Small particles may convey much larger exposure to myco-
toxins (and allergens) than spores and larger fragments do (AIHA 2008). Small
particles are more buoyant and remain suspended in air for longer periods of time
than large particles do and penetrate deeply into the lung, where natural defenses
are fewer (IOM 2004; AIHA 2008).
Mycotoxins in settled dust and particulate air samples from damp indoor envi-
ronments have been measured in a number of studies. Sterigmatocystin has been
measured on building materials and dust (Andersson et al. 1997), in carpet dust
(Englehart et al. 2002), and in moldy building (Bloom et al. 2007, 2009a, b). Toumi
et al. (2000) found that of 79 bulk samples from moldy buildings, 43 % contained
one or more mycotoxins and 15 % contained trichothecenes. The most prevalent
toxin was sterigmatocystin, which was detected in 19 samples. The HITEA study
(Health Effects of Indoor Pollutants: Integrating microbial, toxicological, and epi-

demiological approaches) in Spain, the Netherlands, and Finland (Täubel et al.
2011) found a large number of fungal and bacterial secondary metabolites, includ-
ing mycotoxins in dust samples from water-damaged schools.
Brasel et al. (2005a, b) found airborne particles smaller than conidia containing
macrocyclic trichothecene mycotoxins from particle samples from damp indoor
environments. Dustborne and airborne fungal particles were found to differ with
differing housing characteristics, and mold, fungal particles, and endotoxin were
found in homes investigated after hurricanes Katrina and Rita (Chew et al. 2003,
2006; Rao et al 2007). Spores, fragments, and dust particles can be re-entrained into
the breathing space of occupants of damp buildings and be inhaled. Toivola and
Nevalainen (2004) assessed personal exposures to microbes and particles and com-
pared them to stationary particle monitors to obtain a better understanding of per-
sonal versus surrogate exposure. Particles from combustion (black smoke) correlated
with personal monitors at home and at work. However, the stationary time-weighted
microenvironmental model underestimated personal exposure to particle mass, via-
ble fungi, total fungi, and total bacterial concentrations. Personal monitors gave
better estimations of exposure to biological particles than the stationary monitors.
The size of particles determines their aerodynamic behavior and their deposition
in the respiratory system. Cho et al. (2005) affirmed this for deposition of fungal
fragments in the lung. Particles are deposited into the respiratory system depending
on their size (studied as mean mass aerodynamic diameter or MMAD), the anatomy
of the respiratory system, and air flow patterns that differ for nose, naso-oral or oral
breathing, and respiratory rate and depth. Deposition is not homogenous, and larger
particles (2–5 μm) tend to concentrate via impaction and deposition in the nose and
upper airways of the lung (Balásházy et al. 2003).
The lower respiratory system branches out from the trachea below the glottis into
two large bronchi and then into multiple generations of bronchiolar tubes (the num-
ber of which depends on the species of animal), ending in closed air sacs, the alve-
oli. All of the conducting airways are lined with ciliated epithelium and secretory
cells that produce mucus, enzymes, and defensive molecules. Cilia beat in the direc-
tion of the glottis, moving a particle-trapping mucous blanket upward toward the
esophagus where it is swallowed. The respiratory bronchioles nearest the alveoli
lack cilia (St. George et al. 1993), but have outpouching alveoli in humans. As
described above, the alveoli are a very fragile barrier between the air and the blood
and are a portal to the bloodstream, which can carry contaminants to systemic target
organs.
The MMAD determines whether particles deposit on the respiratory epithelium
through impaction, gravitation, or diffusion. Large particles (˃5 μm) will mostly
deposit in the nasopharyngeal region. About a 16 % fraction of 0.01-μm-size parti-
cles will also deposit in this region and about 40 % of 0.05-μm-size particles as
well. The majority of submicron particle deposition occurs in the pulmonary region
of the respiratory tract, that is, in the respiratory bronchi and alveoli (Bond 1993).
Impaction occurs as larger particles are pulled into the nose and down into the
lung. Because of their inertia, particles encounter surfaces of the nasal turbinates,
500 H.M. Ammann
the bronchi, and bronchial junctions in the tracheobronchial area of the lung. Smaller
particles deposit through the effect of gravity, settling on junctions where bronchi-
oles branch in the lower airways (Kleinstreuer et al. 2008). Foci of impaction and
sedimentation can represent hot spots of exposure to mycotoxins carried by parti-
cles, since they lodge there in larger numbers and may remain for longer periods of
exposure (Balásházy et al. 1999, 2003; Miller and Ammann 2005). Such hot spots
in the tracheobronchial regions can deliver large particle doses to small numbers of
epithelial cells. Balásházy et al. (1999) calculated that as particle diameters increase
from 0.01 to 10 μm, a small area of 0.1 by 0.1 mm can receive 50 to more than a
hundred times the particle deposition than the average cell of the airway. Noting
that neoplastic lesions of smokers are seen predominately at the bifurcations of the
central airways, Churg (2000) has noted that particles landing at these junctions
were slow to clear and were taken up by mucosal tissues in animals. They noted that
in human autopsy sections of lungs, particles were concentrated in an almost 10:1
ratio at the branching of bronchioles compared to uptake in the straight, tubular por-
tion of the airways.
Very fine particles (less than a micron in MMAD) enter the entire respiratory
system, but especially the alveoli, through diffusion, behaving as a gas throughout
the respiratory space until they eventually touch surfaces of the respiratory tract. In
the alveoli they can be pinocytosed by alveolar cells or even absorbed directly
through the two thin layers of alveoli and capillary squamous epithelium and enter
the bloodstream of the general systemic circulation and thus reach specific target
organs such as neural, immune or cardiac, and other tissues outside the lung
(Oberdörster et al. 2002, 2004; Peters et al. 2006).
Only a fraction of particles inhaled deposit on respiratory surfaces; some fraction
is exhaled again. Of the amount deposited, some will be trapped in the mucous of
the mucociliary escalator and be transported to the oropharynx and swallowed into
the digestive tract. Many mycotoxins have been shown to inhibit the function of
ciliated cells or cause apoptosis of these, pulmonary macrophages, and mucus-
producing cells, thus delaying or preventing clearance and prolonging exposure to
the tissues where they land (Sorenson et al. 1986; Jakab et al. 1994; Amitani et al.
1995; Pestka et al. 2008).
Rate and efficiency of clearance mechanisms must also be considered. The
mucociliary clearance mechanism works quite efficiently to trap larger particles in
the nose and upper and tracheobronchial regions of the respiratory tract down to the
respiratory bronchioles. Alveolar macrophages can capture some particles in the
alveoli. The respiratory bronchioles in humans are not ciliated, and alveolar macro-
phages are generally not found there, so that area is especially vulnerable to particle
deposition, longer-term residence time, and damage from toxic effect (Lippman
et al. 1980; St. George et al. 1993; Oberdörster et al. 1994). Rate of ciliary beat and
mucus movement, as well as uptake by alveolar macrophages, can vary depending
on both physiological and toxicological influences.
In the alveolar area, alveolar macrophages can take up particles and remove
them either to the interstitium of the lung or to the lymphatic system. Some macro-
phages may also crawl up to the mucociliary escalator and ride it to the oropharynx,
where they are swallowed. Rates of particle uptake by macrophages, as well as

movement and disposal, are influenced by the toxic potency of the particles taken
up by these cells. Many mycotoxins have been shown to be cytotoxic, specifically
to macrophages and other cells of the lung (Sorenson et al. 1986; Sorenson 1993,
1999; Yang et al. 2000). Toxic influences on the mucociliary escalator and on mac-
rophages affect clearance. Longer residence time of particles on respiratory sur-
faces can allow for greater dose rate through leaching of adsorbed toxins off the
particles into the interstitium, lymph or blood circulation, or increase direct damage
to cells on which particles land, and into which they may be taken up. The effect on
such cells for many mycotoxins, especially the trichothecenes, is apoptosis or pre-
mature cell death.
High particle concentrations of any nature, including those that are biologically
derived, can have an effect on both mucociliary and macrophage particle clearance.
High loads can inhibit the function of both or even damage the cells involved. Very
high fine particle accumulation can lead to retention of particles in the interstitial
tissues of the lung (Oberdörster et al. 1994).
Respiration rate and mode of breathing, that is, nasal, oronasal, or mouth breath-
ing, can also affect not only the amount of particle deposit but also its location
within the respiratory system. For instance, extrapolation of animal data on respira-
tion to human must take into account such differences since particles inhaled by a
two-legged human, with upright stance, will result in particles depositing in differ-
ent areas of the lung than that which occurs with a four-legged animal. Rodents
generally are obligate nose breathers, while humans can breathe through their nose
at rest, their nose and mouth, or their mouth predominantly, depending on activity
level and whether they are talking, singing, or crying or whether they are suffering
some impairment due to allergy or disease (Pope 2000; American Academy of
Pediatrics 2004; Salvi 2007).
For human children mouth breathing is more common than in adults. Human
children (as well as the young in animal experiments) are more exposed than adults
because they breathe more air per unit body weight than adults do. Children there-
fore are both more exposed to air contaminants than adults and more susceptible to
toxins. Their respiratory system, immune system, and blood–brain barrier are not
completely formed until months to years after birth, and defenses against inhaled
toxicants are less effective than in adults (Pope 2000; American Academy of
Pediatrics 2004; Salvi 2007).
Determination of dose to the respiratory surface from particles themselves is
calculated by subtracting clearance from deposition. Attempts to determine dose
from biological particles such as mold spores, hyphal fragments, or dust particles
must take factors described above into account, no matter what health point is being
investigated. However, residence time, solubility of toxic substances on and in
spores, spore and hyphal fragments, and dust become an important consideration in
determining the effects of mycotoxins that can be introduced to the respiratory sys-
tem through inhalation of particulate matter.
502 H.M. Ammann
Nasal Exposure
The nose is the portal to the respiratory system. Its mucous membrane-lined baffles
moisten and warm the air, and its mucus traps particles that can be cleared from the
respiratory to the digestive system, through ciliated cells moving the particle-
containing mucus downward to the oropharynx, where it can be swallowed.
However, particles in inhaled air also move upward in the nasal passages and can
enter the paranasal sinuses. Mold spores have been shown to colonize the mucous
membranes of the sinuses, but may or may not elicit an immune response and play
a role in the development of chronic fungal rhinosinusitis (Ponikau et al. 1999).
However, in following 210 patients with chronic rhinosinusitis, Ponikau et al.
(1999) found that 202 of the 210 patients tested positive for culturable fungi.
Allergic fungal sinusitis was diagnosed in 94 of 101 consecutive surgical cases with
chronic rhinosinusitis, based on histology and culture results. Polzehl et al. (2005)
used both culture methods and polymerase chain reaction (PCR) in examining nasal
lavage samples of patients with chronic rhinosinusitis and used the two methods to
detect fungi in 50 % of patients examined that had been diagnosed with chronic
rhinosinusitis.
Chronic rhinosinusitis can also involve bacterial colonization, or a mixture of
microorganisms, and is characterized by prolonged inflammatory mucosal response
(Harvey and Lund 2007). Chronic rhinosinusitis is often resistant to treatment
through antibiotics, and surgical intervention, after which the condition frequently
returns. Evidence exists for the formation of a biofilm on sinus mucous membranes
as a part of this condition (Harvey and Lund 2007; Foreman et al. 2009, 2011, 2012;
Suh et al. 2010; Boase et al. 2013). A biofilm is described as complex surface-
associated populations of opportunistic or pathogenic microorganisms that are
embedded in an extracellular matrix, whose members are different from free-living
microorganisms (Harvey and Lund 2007; Hall-Stoodley and Stoodley 2009).
Bacteria such as Staphylococcus aureus, Haemophilus influenzae, and Pseudomonas
aeruginosa have been found in biofilms in patients suffering from this illness, as
have a number of fungi such as yeasts of Candida species, Aspergillus fumigatus, A.
versicolor, Chaetomium, and Trichoderma, among others (Foreman et al. 2009;
Muszkieta et al. 2013).
Infections that involve biofilms are important clinically because bacteria and
fungi that are part of a biofilm are recalcitrant against both antibiotic treatment and
host defenses (Healey et al. 2008; Hall-Stoodley and Stoodley 2009). Biofilms pro-
tect bacteria against through a number of mechanisms, while the same bacteria as
free-living organisms are susceptible (Parsek and Singh 2003). Similar mechanisms
for fungal biofilm resistance are described by Ramage et al. (2012) for a number of
nosocomial infections. Successful treatment of chronic rhinosinusitis with certain
antifungal agents amphotericin B (Anyanwu et al. 2004; Ponikau et al. 2005) and
itraconazole (Ponikau et al. 2006; Seiberling and Wormald 2009; Denning et al.
2009) has emphasized fungal role in this yet poorly understood disease.
Biofilms that contain mixed cultures of fungal and/or bacterial species may like-
wise be protected and may represent a means for increased mycotoxin production
as a result of competition among such organisms within a biofilm. Bruns et al.

(2010), using genomic techniques, showed that primary metabolism was reduced
in mature biofilms containing Aspergillus fumigatus, but biosynthesis of second-
ary metabolites, especially gliotoxin, was upregulated. It is generally accepted
that genes of fungi and bacteria that are silent in laboratory culture can be upregu-
lated to produce secondary metabolites in mixed cultures (Schroeckh et al. 2009;
Nützmann et al. 2011) as well as in biofilms (Zavahir and Seneviratne 2007; Bruns
et al. 2010; Morales and Hogan 2010). With the sequencing of a number of fungal
genomes, it has become obvious that genes coding for many secondary metabo-
lites are only expressed when the organisms coexist in mixed microbial cultures,
provided that water activity and temperature are favorable (Nielsen 2003;
Schroeckh et al. 2009; Nützmann et al. 2011). The term “genome mining” is being
used in efforts to awaken silent gene clusters through microbial cross-talk in the
search for new antibiotics (Brakhage and Schroeckh 2010; Kück et al 2014;
Seneviratne et al 2008).
Mycotoxins have been measured in patients with chronic rhinosinusitis
(Lieberman et al. 2011). The role for particular secondary metabolites including
particular mycotoxins in the formation of biofilms as part of chronic fungal sinusitis
is being studied (Wargo and Hogan 2006; Zavahir and Seneviratne 2007; Shank and
Kolter 2009). It is known that many mycotoxins have a modulating effect on
immune function (Bondy and Pestka 2000; WHO 2009), which may in itself play a
role in infection and the formation of biofilms in sinuses and in the nasal mucosa.
Uptake from the Nose into the Brain
The nose not only is the portal to the respiratory system but also serves as a gateway
to the nervous system. The olfactory nerve (cranial nerve I, the sense of smell) sen-
sory receptors are located in the olfactory epithelium, and its afferent fibers lead to
the olfactory bulb of the brain through the sieve-like bony cribriform plate of the
ethmoid bone of the skull, located above the nose. The trigeminal nerve (cranial
nerve V or common chemical sense that responds to pungency) also has fibers that
innervate the nasal epithelium and dendrites that terminate in the olfactory bulb and
elsewhere in the brain (Thorne et al. 2004; Brand 2006; Silver and Finger 2009).
It has long been known that a number of metals, small peptides, small particles,
and viruses and a number of drugs can enter the brain directly via nasal neural trans-
port (Shipley 1985; Tallkvist et al. 2002; Oberdörster et al. 2004; Lewis et al. 2005).
These pathways are currently being explored to find means of treating central ner-
vous system (CNS) disorders that are recalcitrant to treatment systemically because
they are barred from the brain by the blood–brain barrier (Hanson and Frey 2007;
Scranton et al. 2011). Axonal transport of particles, as described by Fechter et al.
(2002) and Oberdörster et al. (2004), is especially relevant to mycotoxin exposure
of the brain since currently inhalation exposure to mycotoxins is estimated to be
primarily from small particles (Rand and Miller 2011).
504 H.M. Ammann
Tracing of radiolabeled particles administered intranasally demonstrates that

such small particles not only travel to the olfactory bulb of the brain but are distrib-
uted to the brain in general (Oberdörster et al. 2004; Scranton et al. 2011). The
rostral migratory stream (RMS) is a structural entity that connects the olfactory bulb
to periventricular regions of the brain. It is a pathway in mammals (though not well
described in humans) several millimeters in length that connects the subventricular
zone (SVZ) with the olfactory bulb, which has been largely studied in its transport
of neuroblasts from the SVZ to the olfactory bulb (Scranton et al. 2011). Scranton
et al. (2011) used low molecular weight fluorescent probes and radiolabeled ligands
administered intranasally in mice to show that fluorescence and radioactive signal
was found throughout the brain, but not in peripheral tissues like the lungs or the
blood. Surgical interruption of the RMS prevented uptake into the brain.
Islam et al. (2006) instilled the macrocyclic trichothecene satratoxin G (SG) pro-
duced by certain strains of S. chartarum into the noses of mice and found that SG
specifically induced apoptosis in olfactory sensory neurons in the olfactory epithe-
lium in a dose–response manner. They found a lowest-effect level 24 h after expo-
sure to be 25-μg/kg body weight and a no-effect level to be 5-μ/kg body weight,
with severity increasing with dose. Time course of a single instillation of 500 μ/kg
showed that maximum damage to the olfactory epithelium occurred at 3 days; expo-
sure to lower doses (100 μg/kg) for 5 consecutive days resulted in the same degree
of atrophy and apoptosis, indicating that the damage was cumulative. SG also
induced neutrophilic rhinitis from the sensory epithelium, an indication of inflam-
mation, and inflammatory cytokines were found in the olfactory bulb and within the
brain. Satratoxins are not commercially available and are difficult to purify from
fungal culture, so Islam et al. (2007) used roridin A (RA), a structurally related
trichothecene produced by the fungus Myrothecium that is commercially available,
and found similar neurotoxic effects in their mouse model. They also determined
the kinetics of RA transport. RA-induced apoptosis of olfactory sensory neurons
was observed 24 h after instillation and was maximal after 72 h. Atrophy of the
olfactory nerve layer of the olfactory bulb in the brain occurred concurrently.
Concomitant exposure to endotoxin (lipopolysaccharide found in cell walls of
Gram-negative bacteria) potentiated the effect both in sensory neurons and the
olfactory bulb. Certain Gram-negative bacteria grow in damp indoor spaces along
with a number of mold species, including S. chartarum, so co-exposure to bacterial
and fungal fragments is common. Microbial communities that include competing
fungal and bacterial organisms also show upregulation and production of mycotox-
ins and bacterial exotoxins and are more likely to be sources of exposure via
inhalation.
Kinetics of SG tissue distribution following intranasal exposure in mice was
determined by Amuzie et al. (2010). SG was rapidly taken up from nasal tissues and
distributed to tissues involved in respiratory, immune, and neuronal function, then
cleared. The nasal turbinates retained a significant amount of the toxin, which may
account for the toxin’s ability to cause sensory neuron death. The toxin was rapidly
cleared from plasma and low levels remained in blood. This can be explained by
efficient distribution to various organs such as the kidney, lung, spleen, thymus,
heart, olfactory bulb, and brain. As the authors point out, selective targeting of neu-
rons in the nose and olfactory bulb of the brain is intriguing because olfactory func-
tion loss often occurs in the early stages of degenerative illnesses such as Parkinson’s
and Alzheimer’s disease.
Rodents and humans differ in their breathing habit and have differences in the
structure of their noses. Their nasal turbinates are complex and branching, and they
have large amounts of olfactory epithelium which covers about 50 % of their intra-
nasal surface. Humans and other primates such as rhesus monkeys have simple
turbinate structures (Harkema et al. 2006), and their olfactory epithelium is only a
small part of the nasal cavity, about 15 % of the total intranasal surface.
Carey et al. (2012) developed a young adult rhesus monkey model, whose nasal
structures and airways more closely resemble that of humans, to study acute and
repeated SG exposure from intranasal instillation. They found that low-dose (5 μg)
exposure to SG caused neutrophilic rhinitis and apoptosis of olfactory sensory neu-
rons in monkeys, similar to the injury described above in mice at comparable expo-
sure concentrations. Four repeated daily doses of SG at the low concentration
caused more damage than a single high dose of 20 μg, indicating cumulative dam-
age. These experiments demonstrated that injury to the olfactory sensory epithelium
and brain by SG occurs in primates whose nasal anatomy is similar to that of humans
so that correlations to human injury can be made.
It has been argued by some authors that the number of Stachybotrys spores
required to cause adverse human health effects in damp indoor spaces would be
very large to be comparable to these pure toxin exposures (Chapman et al. 2003;
Hossain et al. 2004; Kelman et al. 2004; Hardin et al. 2009). However, since SG is
not only found in and on spores but in nonviable fungi, fungal fragments, and dust
from surfaces on which Stachybotrys is growing, spores are not the only exposure
agents to be considered. Concentrations of fungal fragments of S. chartarum, aero-
solized through a fungal spore source strength tester (FSSST) and collected with an
electrical low-pressure impactor (ELPI) that measured size distribution, were 514
times greater than that of spores (Cho et al. 2005). The particles were aerosolized
from agar plates within the FSSST and thus were limited to fragments of S. charta-
rum spores and mycelia and did not include fine dust particles containing the exo-
toxins that could be aerosolized from moldy surfaces in the built environment.
Bloom et al. (2007) demonstrated that mycotoxins, such as the macrocyclic tricho-
thecenes SG and satratoxin H (SH), sterigmatocystin produced by A. versicolor, and
citrinin, gliotoxin, and patulin produced by Aspergillus and Penicillium species,
could be isolated from dust above floor level from moldy buildings. Gottschalk
et al. (2008) used LC-MS/MS to measure airborne SG (0.25 ng/m3) and SH (0.43
ng/m3) in a water-damaged building. Satratoxin-equivalent concentrations ranging
from 2 to 330 ng/m3 have been estimated to occur in some water-damaged rooms
within a home (Yike et al. 1999; Vesper et al. 2000; Carey et al. 2012).
About 70–90 % of inhalation exposure to molds is thought to be from fungal
fragments measured as β-D-glucan, a structural molecule from fungal cell walls
(Salares et al. 2009). Small particle air pollutants have been shown to translocate to
the brain (Oberdörster et al. 2004) through the olfactory pathway and have been
506 H.M. Ammann
linked to cognitive deficits and brain abnormalities in children and dogs (Calderon-
Garcidueñas et al. 2008). Epidemiological studies have linked traffic-related fine
particles to cognitive decline in elderly men (Power et al. 2011) and women (Weuve
et al. 2012), with mechanisms of oxidative stress and inflammation similar to those
modeled in human neurological cells by Karunasena et al. (2010) for satratoxin H,
at toxin levels found in wet and damp indoor spaces. The effect modeled in human
cells, together with the neurological damage, demonstrated in mice and rhesus mon-
keys that satratoxins specifically target neural tissue in the nose and brain, and the
neurological deficits reported in some occupants of moldy built environments
(Johanning et al. 1996; Gordon et al. 2004) provide support that the nose–brain con-
nection is an important route of inhalation exposure for mycotoxins.
Uptake by the Lung
Mycotoxin effect on the respiratory system in general, and the lung in particular,
has been studied intensively since an outbreak of pulmonary hemorrhage in 34
infants, 10 of whom died, in 1994–1998 in Cleveland, Ohio, in the USA (Dearborn
et al. 1999; AIHA 2008). Because of the epidemiologic link of this outbreak to S.
chartarum and subsequent case reports linking this mold with bleeding lungs in
infants and children, much of this research has focused on the effect from
Stachybotrys toxins (Nikulin et al. 1996, 1997; Jarvis et al. 1998; Rao et al. 2000;
Gregory et al. 2004; Dearborn et al. 2002; Mader et al. 2007; Pestka et al. 2008).
Direct damage to various cells of the lung, as well as the effect of compromise of
mechanical and immunological lung defenses, has been explored for macrocyclic
trichothecenes from S. chartarum. Other toxins, for example, gliotoxin produced by
A. fumigatus and some other molds, have been implicated as a virulence factor in
aspergillosis, and its role in damage to ciliated respiratory cells, with decrease in par-
ticle clearance of the lung, has been studied (Amitani et al. 1995; Lewis et al. 2005;
Amitani and Kawanami 2009). Aflatoxin B1, produced by A. flavus and A. parasiticus,
also has ciliostatic effects and impairs phagocytosis of particles (including viruses and
bacteria) by alveolar macrophages and suppresses antibody response (Jakab et al. 1994).
Absorption of mycotoxins within the respiratory system is largely a factor of
particle distribution since mycotoxins are not considered to be volatile (although
some are semi-volatile). As described above, mycotoxins are in and on spores, on
fragments of spores and mycelia. Because they are exotoxins secreted by fungi onto
the surfaces on which molds grow, they are also found on dust associated with the
moldy surfaces (Englehart et al. 2002; Nielsen 2004; Bloom et al. 2009a, b).
Particles therefore act as carriers for the toxins. Some mycotoxins, such as the satra-
toxins, are water soluble and can form liquid aerosols and can be leached off parti-
cles into cells (Pestka et al. 2008). Whether and to what degree absorption of
mycotoxin-associated particles occurs depends on a number of factors, of which the
most important are the morphology and cellular makeup of the tissues where parti-
cles land, the physical and chemical nature of the toxins, and the ability of cells to
take up toxins (Dahl and Gerde 1994; Miller 1999).
Recent epidemiological meta-analyses and reviews have linked exposure to

damp indoor environments with worsening and development of new asthma and
other respiratory health effects (Fisk et al. 2007; Mendell et al. 2011; Quansah et al.
2011). Respiratory exposure to mold and other microbes was implicated in these
disease outcomes, although the role of specific mycotoxins had not been studied.
Mycotoxins and their effects associated to the lung and cells of the lung in cul-
ture of human and animal cells of the lung, and in animals, have recently been
reviewed (Rand and Miller 2011; Miller and McMullin 2014). Evidence from such
studies, especially those by Miller et al. (2010), indicates that low molecular weight
compounds, including mycotoxins, produced by molds that grow in damp indoor
environments are strongly pro-inflammatory and modulate genes in the lungs of
mice for inflammatory cytokines and mucus production analogous to those in
humans related to nonallergic, that is, toxin-induced, respiratory health effects.
There is ample evidence from animal studies that mycotoxins have systemic
effects aside from damage to the route of entry. Early inhalation studies (Cresia
et al. 1987, 1990; Pang et al. 1986, 1987, 1988) indicate that systemic effects occur
more directly from this exposure route. Cresia et al. (1990) found that inhalation
exposure of rats to the simple trichothecene T-2 toxin produced significant lesions
to immune tissues throughout the body of these animals, while the lung remained
intact. Myocardial and pancreatic lesions were demonstrated in swine (Pang et al.
1986, 1987) after intravenous administration of sublethal doses of T-2 toxin.
Inhalation was equivalent to intravenous administration in later experiments in
swine (Pang et al. 1988). Okai et al. (2008) found that inhalation of S. chartarum
caused pulmonary arterial hypertension in mice.
Di Paolo et al. (1993) describe a case of acute renal failure from ochratoxin
exposure in a woman who inhaled ochratoxin-containing wheat dust while working
for 8 h in a granary that had been shut for several months. Guinea pigs and rabbits
were experimentally exposed to the same dust and were shown to have kidney and
liver lesions. The patient recovered with treatment after several months.
Concentrations of toxins in the wheat dust were not determined.
Experimental exposures of humans to mycotoxins are not permissible, as myco-
toxins are defined as causing harm to animals and humans at low doses, and some
mycotoxins that have been isolated from damp indoor spaces are known to be
potently toxic at low exposures. While there are some putative biomarkers for
human exposure, such as DNA adducts for some genotoxic mycotoxins, it may be
possible to determine exposure by measuring DNA adducts in bodily fluids, but not
differentiate ingestion from inhalation or dermal exposure.
Complexity of Damp Indoor Spaces
A review of the complexity of the damp indoor environment by Thrasher and

Crawley (2009) discusses nine types of biocontaminants: indicator molds, Gram-
negative and Gram-positive bacteria, microbial particulates, mycotoxins, volatile
508 H.M. Ammann
microbial compounds (both of microbial and nonmicrobial origin), proteins, galac-

tomannans, 1 → 3-β-D glucans, and lipopolysaccharides (LPS), endotoxins, and
toxic or health effects that have been associated with each category of contaminant,
when such information was available. They could also have included allergens,
which may exacerbate effects, or through inflammation, allow more exposure to the
contaminants they discuss. People breathing air indoors are also exposed to particu-
lates from combustion and animal detritus, VOCs from building materials, cleaning
agents, paints, and odorants, among other air contaminants; exposure to many, or all
of these, may have overlapping symptoms with those from biocontaminant expo-
sure. The complexity of microbial growth in damp indoor environments also makes
it difficult to identify specific agents related to disease. When health effects in damp
structures are discussed, occupants’ symptoms are often described as “nonspecific,”
when in fact the symptoms are specific to individual contaminants, but there is over-
lap of symptoms from exposure to multiple contaminants. There are many air con-
taminants that invoke “irritation of mucus membranes,” for instance, and this may
be related to a number of disease entities. Their combined exposure can exacerbate
symptoms or worsen specific diseases, but the symptoms and disease are still spe-
cific to each contaminant exposure. What is “nonspecific” is that the description of
exposure is incomplete.
Microbial communities in damp buildings are part of a larger ecosystem where
populations of bacteria, fungi, and protozoans change over time in response to
changes in resources and ambient conditions. For example, increases in moisture
will allow molds that require a high water activity (aw) to predominate, while xero-
philic molds do not grow well. The reverse could prevent molds such as S. charta-
rum from growing. Specific nutrient availability will select for microbes in the
building just as they do in laboratory media. Some molds and bacteria working to
survive in a particular ecologic niche can produce secondary metabolites such as
toxins to compete for resources. Water activity (aw) and temperature changes allow
different microbes to thrive (Nielsen et al. 2004).
The difficulty in establishing what the exposure agents from microbial growth in
buildings is illustrated by Tuomi et al. (2000). They analyzed bulk samples from
building materials from moldy interior surfaces in buildings with moisture damage
for mycotoxins in Finland. They found sterigmatocystin, satratoxins G and H,
diacetoxyscirpenol (DAS), deoxynivalenol (DON), and verrucarol in varying
amounts in many samples. They also cultivated the samples on 2 % malt extract
agar at 25 °C for 7 days before identification of fungal species. They found
Aspergillus versicolor, a producer of sterigmatocystin, but also found this myco-
toxin in samples that did not reveal this fungus through culture. Some
sterigmatocystin-containing samples did not yield Aspergillus species, yet yielded
Penicillium species; the majority of the samples containing Penicillium species
yielded neither sterigmatocystin nor citrinin, the latter of which was found in a few
of the Penicillium spp. samples. Satratoxins were found only in samples from which
S. chartarum was grown, with one exception. Tuomi et al. (2000) point out that it is
not surprising to find toxins without finding the known producer fungal species or
to find the fungal species without finding the toxin, because of the dynamic nature
of microbial communities in building ecosystems. The authors note that toxigenic

species have different growth requirements and that the lack of correlation of spe-
cies isolated with mycotoxins known to be produced by various mold species may
also be due to the researcher’s procedure of using a single growth medium and
temperature for all their samples.
Instead of recognizing that a damp building is a complex ecosystem that changes
with water, temperature, and microbial interactions, some authors have misinter-
preted the paper by Tuomi et al. (2000) as stating that fungi growing on building
materials do not always produce mycotoxins. This became a common view that has
now been realized to be erroneous (Miller and McMullin 2014). The presence of
other microbes competing for vital resources is one of the strong influences for
toxin production in both microfungi and bacteria capable of producing exotoxins
(Nielsen 2003; Wargo and Hogan 2006; Schroeckh et al. 2009; Bruns et al. 2010;
Nützmann et al. 2011; Frey-Klett et al. 2011).
There is evidence that the presence of mixed microbial communities sends sig-
nals that activate genes coding for metabolites that are silent when isolated bacteria
or fungi are grown in culture. Various techniques that take advantage of microbial
interactions to awaken silent genes to produce secondary metabolites that might be
useful in finding antibiotics are currently being exploited (Bergmann et al. 2010;
Brakhage and Schroeckh 2010; Frey-Klett et al. 2011; Kück et al. 2014).
The complex spectrum of secondary metabolites measured in damp indoor
spaces was recently elucidated as part of the HITEA study of schools in Finland,
Spain, and The Netherlands. Using a multi-analyte, HPLC tandem-mass spectros-
copy methodology to analyze metabolites in damp school environments, Vishwanath
et al. (2009) were able to determine 159 fungal and 27 bacterial metabolites, many
of which had never before been identified in samples from actual damp environ-
ments. In a subsequent study, the same methodology was applied to 69 samples
from severely moisture-damaged homes, and these were found to have at least 1 of
186 targeted metabolites. Thirty-three bacterial metabolites were found co-occurring
with mycotoxins. The bacterial compounds monactin, nonactin, staurosporine, and
valinomycin were found in moist structures, and chloramphenicol was found in
settled house dust. These highly bioactive compounds are produced by Streptomyces
spp., bacteria which are considered to be indicators of dampness in buildings
(Täubel et al. 2011).
Kirjavainen et al. (2015) used LC-MS/MS methodology to analyze dust sam-
ples from the living rooms of 93 homes of 1-year-old children in Finland, of
which 15 had moisture damage, for 330 secondary metabolites. The purpose of
this study was to characterize the presence of microbial secondary metabolites
and to determine whether there was an association with molds and moisture dam-
age and with the development of asthma. The study found that secondary metabo-
lites were ubiquitously present in low concentrations in home floor dust, even in
the absence of moisture damage. Forty-two different metabolites were found. The
total load of metabolites in living room floor dust tended to be moderately
increased if there was moisture damage in the living room or mold odor anywhere
indoors, but this increase did not seem to have an adverse effect, but may rather
510 H.M. Ammann
have had a protective association with asthma in children. Of the children resident
in the homes, 8 children had active asthma at age 6, and 15 had lifetime doctor
diagnosed asthma. The macrocyclic trichothecene metabolites of S. chartarum,
such as the satratoxins and verrucarol, which have been detected in dust samples
from severely water-damaged homes and implicated in adverse health effects
elsewhere (Bloom et al. 2007, 2009a, b; Peitzsch et al. 2012) were not detected,
but the macrocyclic strains of S. chartarum are rarely found in the Nordic climate.
Eleven of the samples in this study did contain stachybotrylactam, a toxic metab-
olite of S. chartarum, but this metabolite was not associated with moisture dam-
age or asthma. The apparent protective finding of these low concentrations of
secondary fungal and bacterial metabolites is of interest in view of the hygiene
hypothesis, but the number of asthmatic children in the study is small, and further
investigation must be done.
Some toxicological studies have attempted to determine the adverse effects of
exposure to more than one mycotoxin, or combinations of bacterial toxins and
mycotoxins, but none have been able to mirror the complexity of exposure described
above (Speijers and Speijers 2004). Studies using the mouse macrophage cell line
RAW 264.7 to investigate effects from S. chartarum and Streptomyces californicus
that were grown in coculture showed synergistically increased markers of inflam-
mation, cytotoxicity, and immuno- and genotoxic effects (Huttunen et al. 2004;
Penttinen et al. 2005; Murtoniemi et al. 2005; Markkanen et al. 2009). Tissue cul-
ture evaluation of toxic interactions can determine additivity, antagonism, and syn-
ergism in such a model system. Relatively few mycotoxins have been investigated
for additive or synergistic effects or antagonistic inhalation effects on animals, and
critical effects have generally not been determined.
Risk Assessment
Risk assessment is generally used to determine allowable levels of exposure to toxi-

cants for public health protection or regulatory efforts (Ammann 2012). Calls for
risk assessment of airborne mycotoxins have been heard for many years. The
American Conference of Governmental Industrial Hygienists (ACGIH) Bioaerosols
Committee issued a statement in 1999 that described why the organization consid-
ered a threshold limit value (TLV) for worker protection to be inappropriate for the
complex mixture of bioaerosols found in the built environment as the result of
dampness (ACGIH 1999; Ammann 1999). In 2004, the Institute of Medicine of the
National Academies of Science, in its report “Damp Indoor Spaces and Health”,
made this recommendation: “Animal studies should be initiated to evaluate the
effects of long-term (chronic) exposures to mycotoxins via inhalation. Such studies
should establish dose–response, lowest-observed-adverse-effect levels, and no-
observed-adverse effect levels for identified toxicologic endpoints in order to gener-
ate information for risk assessment that is not available from studies of acute,
high-level exposures.” No scientifically credible risk assessment for inhalation of
any mycotoxins associated with damp indoor environments has been possible,
because these kinds of studies are not currently available. Assessment, especially of
the more potent mycotoxins from molds that thrive in damp indoor environments,
may still be useful for immediate hazard assessment in building investigations and
contribute to public health actions.
Guidance for risk assessment has been put forward by a number of public health
and regulatory agencies. The risk assessment paradigm developed by the USEPA
for allowable inhalation exposure levels of humans of various susceptibilities to
single non-cancer-causing air toxicants (reference concentrations or RfCs) is used
for regulatory purposes in the USA under the Clean Air Act and is generally
accepted as a useful means of limiting toxic exposures. Carcinogenic air toxics are
analyzed differently through probabilistic models to determine risk with 1 in a mil-
lion chance of cancer considered a threshold for allowable emissions.
According to USEPA guidance, four specific steps are involved in RfC
development:
1. Hazard evaluation identifying a critical effect and a critical study from an exten-
sive review of the human and animal literature. Various tissues have different
susceptibilities, so it is important to elucidate effects of toxins in whole animals
in order to determine a critical effect, that is, the effect that occurs in the most
sensitive animal and the most sensitive animal tissue, at the lowest level of expo-
sure. The assumption in determining critical effect is that protecting against the
most sensitive toxic end point will also protect against damage occurring in less
sensitive tissue.
Because of route of entry and physiological effects, including barriers to
toxin access and the ability of different tissues to detoxify or to bind or eliminate
toxins, such considerations must also be explored. Route of entry, i.e., oral, or
inhalation methodologies differ because potency to system tissues varies by
route of entry. Ability to repair damage also differs in tissues, so, for instance,
lesions to the nervous system may have an overall more profound effect on the
organism than damage to the lung. Developmental effects also differ profoundly
depending on the stage of development when toxic impact occurs.
2. Dose–response assessment to determine no-observed-adverse-effect levels
(NOAELs) and lowest-observed-adverse-effect levels (LOAELs), usually from
chronic animal inhalation studies if no human data are available. Conversion of
animal to human data through dosimetry adjustments produces a human equiva-
lent concentration (HEC).
3. Exposure assessment consisting of exposure measurements.
4. Risk characterization results in an RfC after the HEC has been divided by appro-
priate uncertainty factors. The RfC is also ranked as having levels of confidence
in the data available and the confidence in the RfC itself (USEPA 1994; Ammann
2012). The RfC, as defined by USEPA, “is an estimate (with uncertainty span-
ning perhaps an order of magnitude) of a continuous inhalation exposure to the
human population (including sensitive subgroups) that is likely to be without
appreciable risk of deleterious non-cancer health effects during a lifetime
512 H.M. Ammann
(USEPA 1994).” In regulating air toxics, the RfC value is divided into modeled
or measured concentrations in air produced by an industry to determine risk to
populations near facilities. Development of RfCs for individual mycotoxins may
help in determining relation of those toxins to health, but exposure in damp
buildings is not to individual toxins.
New understanding of exposure through fine particles, assessment technology,
genomics and proteomics tools, and knowledge of microbial interactions in the pro-
duction of toxins by both fungi and bacteria indoors shows that exposure is even
more complicated than previously thought. To date, attempts at risk assessment for
microbial exposure indoors have failed to account for the complexity and variability
of organisms and their products, which are currently being revealed. Long-term
inhalation studies have not been performed, and no critical effects from inhalation
of mycotoxins have been determined.
Screening tools suggested to be used in risk assessments for low levels of indus-
trial emissions (Drew and Frangos 2007), such as the concentration of no toxico-
logic concern (CoNTC), are not applicable for mycotoxins, as proposed by Hardin
et al. (2009). Unlike industrial emissions, toxin concentrations are not reliably mea-
surable in air, nor are they predictable, and the large number of secondary metabo-
lites and their toxicological and physiological interactions are unknown. The
decision tree as developed by ILSI (International Life Sciences Institute) Europe
(2005) for additives in food, and suggested as a screening tool for air toxics by
Drew and Frangos (2007), also forbids the use of the process for genotoxic com-
pounds and aflatoxin-like compounds and recommends compound-specific toxicity
data be used. Sterigmatocystin is an aflatoxin-like carcinogenic compound fre-
quently isolated from damp indoor spaces where its primary producer, Aspergillus
versicolor, is considered an indicator of dampness (Toumi et al. 2000; Englehart
et al. 2002).
Hardin et al. (2009) includes sterigmatocystin, aflatoxins B1 and B2, as well as
other genotoxic and carcinogenic mycotoxins, such as ochratoxin A and citrinin
that require toxin-specific analysis under the ILSI decision tree. Hardin et al. (2009)
quotes ACOEM (2002) which “estimated that 1010 spores/m3 (of S. chartarum)
would be required to achieve a 1 mg satratoxin/m3 which was the no-effect concen-
tration of T-2 toxin in 10-min rat inhalation exposures (Cresia et al. 1987, 1990).”
In fact, the Cresia et al. 1990 study cited in the ACOEM and in Hardin et al. (2009)
determined an inhalation LC50 (lethal concentration50) of 0.02 mg/L (20 mg/m3) of
air for T-2 toxin, not satratoxins G or H, which are more potent macrocyclic tricho-
thecenes than the simple trichothecene T-2 toxin. A no-effect concentration cannot
be determined from an LC50 experiment. The number 1 mg/m3 for T-2 toxin
described as a no-effect concentration for a 10-min exposure to T-2 in Hardin et al.
(2009) is in fact the concentration at which no rats died within 24 h after expo-
sure. No lethality after 24 h is not equivalent to “no effect” unless effect is defined
as death.
The authors of the ACOEM position paper also cited papers by Nikulin et al.
(1996) regarding satratoxin G and H concentrations in S. chartarum (reported as
S. atra) spores to “provide perspective relative to T-2 toxin, 1.0 mg satratoxin would
require 1010 (ten trillion) S. chartarum spores/m3.” The comparison was between
pure T-2 toxin concentrations for “no mortality in rats” and concentration of S. char-
tarum spores containing satratoxins G and H calculated from Nikulin et al.’s (1996)
concentrations of toxins of S. chartarum (cited as S. atra) spores. There was no
consideration of the relative toxicity of T-2 toxin and the satratoxins in the determi-
nation of the number of S. chartarum spores that would be equivalent to the T-2
toxin “not dead” concentration. In citing Rao et al. (2000a, b), the ACOEM authors
state “A range of doses was administered in rat studies and multiple, sensitive indi-
ces were monitored, demonstrating a graded response, with 3 × 106 spores/kg being
a clear no-effect level.” Actually, Rao et al. (2000a, b) do not state that a “clear no-
effect level” exists in either paper. Changes in numbers of neutrophils (polymorpho-
nucleocytes), macrophages, albumin, and lactic acid dehydrogenase concentrations
(all signs of inflammation) and increases in hemoglobin (a sign of bleeding) in
bronchoalveolar lavage (BAL) fluid of rats exposed to “toxic” S. chartarum spores
bear out the conclusion of Rao et al. (2000a) that “our data indicate that direct pul-
monary exposure to S. chartarum spores can cause severe inflammatory effects in
the lungs.”
Acute studies of satratoxins in animals have primarily focused on damage to the
lung. In contrast, the Cresia et al. (1990) LC50 study found that T-2 toxin caused no
respiratory lesions in the exposed rats, but necrosis of cells in immune tissues was
observed, especially in the spleen and thymus gland. T-2 toxin and the satratoxins
seem to have different target tissues, as well as significantly different potencies. No
long-term inhalation studies at low enough exposures that could determine a
NOAEL for satratoxins or other mycotoxins, suitable for risk assessment, have been
performed to date. Kelman et al. (2004) also describes the concentration of T-2
toxin at which no rats died in the Cresia et al. (1990) LC50 study as “the 10-min no-
observed-effect level (NOEL)” in using it to substitute T-2 toxicity data for that of
trichoverrols A and B. They repeat this description in Table 20 of the paper. The
paper states: “Because we were unable to identify any toxicity studies done on
mammals with these mycotoxins (trichoverrols A and B), we chose to compare
them to T-2 toxin, a trichothecene mycotoxin produced by Fusarium species and
purified for use as a biological warfare agent” (Kelman et al. 2004). The same paper
states, “A maximal airborne mold spore concentration (N) of 200,000 spores/m3
was assumed based on our experience collecting air samples in indoor environ-
ments with abundant visible surface mold.” No published data are cited to support
this number. The complexity of exposure to mycotoxins, especially from fragments
and dust with adsorbed toxins, was not considered.
The descriptions of risk from the ACOEM position paper (Kelman et al. 2004,
and Hardin et al. 2009) lack scientific credibility. Credible assessments of risk from
inhalation exposure to individual mycotoxins from appropriate studies are lacking.
Risk from the complex and variable mixture of mold and bacterial spores, frag-
ments, and products is even more problematic to assess.
In the absence of the ability to evaluate risk from complex exposures of micro-
bial toxins and the ability to determine what “safe” levels of exposure to such toxins
514 H.M. Ammann
are, what can be done to protect the public from exposure to airborne mycotoxins
indoors? The ACGIH in 2008, in its book Recognition, Evaluation, and Control of
Indoor Mold, described the processes that cause buildings to be wet (with resultant
microbial growth) and those building and maintenance practices that keep buildings
dry and clean. For the present, prevention of exposure is the best option for avoiding
health effects from mycotoxins via inhalation in damp indoor spaces, but also for
attendant allergens and other contaminants found there.
Exposure to damp spaces is a significant public health issue, for which upper and
lower respiratory tract illness, in particular to the development and exacerbation of
asthma, has been related causally in epidemiologic studies (Fisk et al. 2007; Mendell
et al. 2011; Quansah et al. 2011). The relationship, however, is to dampness and
mold, and the specific causal agent or agents are still to be identified. Only about 50
% of asthma is related to allergy worldwide (Douwes et al. 2002). Work performed
by workers at the National Institute for Occupational Safety and Health (NIOSH)
has shown that healthy nonallergic workers, who move into damp and moldy non-
industrial workplaces such as offices, can develop asthma, which implies that the
causative agents are toxic singly, or in combination, and not allergens (Park et al.
2008; Cox-Ganser et al. 2009). Other systemic effects from inhaled mycotoxins
have not been as well investigated in humans, but have been reported in numerous
clinical papers.
Reduction in microbial numbers and fragments has resulted from targeted reno-
vation (Huttunen et al. 2008). The emphasis on prevention is supported by several
well-designed and controlled intervention studies, in which sources of dampness
and mold were remediated, moldy and damaged materials removed, and cleaning of
visible mold accomplished, with a dramatic reduction in symptoms and worsening
of asthma (Kercsmar et al. 2006; Krieger et al. 2010).
Summary
Inhalation exposure of mycotoxins differs from ingestion and dermal exposure

because inhalation allows direct access of gases and small particles to the blood-
stream for distribution to all of the systemic circulation without passage of blood
through the liver, the major detoxifying organ of the body. It also allows direct
access to the brain by passage of particles and toxins along the olfactory and tri-
geminal cranial nerves, which are not protected by the blood–brain barrier.
While exposure to aerosolized spores from molds growing indoors has in the
past been thought to be the way in which occupants of damp buildings were exposed
to mycotoxins, it is now known that the primary agents of exposure are small
particles that are fragments of spores and mycelia and re-entrained dust from sur-
faces onto which the fungi secreted their exotoxins. The aggregate surface area of a
given mass of small particles is much greater than for it is an equal mass the size of
spores, and this large surface area allows the particles to carry greater amounts of
toxins to the lower respiratory tract, as well as to the nasal sensory epithelium,
where the olfactory and trigeminal nerves can carry them into the brain.
The morphology and physiology of the respiratory system prevent homogeneous

distribution of gases and particles, which allows accumulation of toxin-carrying
particles at “hot spots” where damage and long-time residence can be greater. Very
small toxin-containing particles act like a gas and are distributed through diffusion
to the deep parts of the lung, the respiratory bronchioles, and alveoli, from where
they can directly enter the bloodstream.
It is clear from inhalation studies in animals that mycotoxins cause systemic
effects in target organs outside the lung. Few studies have attempted to trace such
effects in humans.
Secondary metabolites, including mycotoxins, are more numerous and their mix-
tures more complex in damp buildings than was previously realized. Microbial
interactions within human tissues, and within the built environment, appear to be a
prime stimulus for both toxigenic microfungi and toxigenic bacteria to produce tox-
ins in ecologically competitive growth. The additive, synergistic, or antagonistic
effects of co-exposure to these metabolites are not known, except for a few highly
toxic mycotoxins whose interactions and effects on the health of agricultural ani-
mals have been studied.
Prevention of exposure through building and maintenance practices that keep
buildings dry and clean is currently the best action to prevent health effects in build-
ing occupants.
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Chapter 21
Fungi in Fermentation and Biotransformation
Systems
Carla C.C.R. de Carvalho
Introduction
Several microfungi species have been used successfully throughout human history
to produce foods and beverages, such as bread, cheese, soy sauce, tea, beer, wine,
and sake. Fungal metabolism and metabolites play fundamental roles in the manu-
facture of, e.g., ethanol, citric acid, and pharmaceutical drugs, as well as in the
production of biocontrol agents, enzymes, and pigments. It has also been suggested
that fungal enzymes are the most efficient lignocellulose degraders, allowing the
conversion of biowaste and agriculture crop residues into, e.g., biochemicals and
bioenergy (Lange 2010). Conventional production of bioethanol, the most common
biofuel in use, from lignocellulosic material may apply fungal cellulases for bio-
mass hydrolysis and yeast fermentation of the resulting glucose. Since the mono-
saccharides resulting from the hydrolysis of cellulosic materials cause feedback
inhibition of the hydrolases used, it has been proposed to simultaneously perform
hydrolysis and fermentation, thus preventing accumulation of glucose and disac-
charides. Few individual microorganisms able to carry out simultaneous saccharifi-
cation and fermentation have been reported, including the thermotolerant yeast
strain Kluyveromyces marxianus CECT 10875 (Ballesteros et al. 2004). In simulta-
neous saccharification and cofermentation, the use of an organism able to ferment
both glucose and pentoses released from biomass will result in an increased ethanol
yield. In 1989, the cellulase hyperproducing strain Fusarium oxysporum F3 was
reported as being able to ferment glucose, xylose, cellobiose, and cellulose directly
to ethanol, reaching a maximum ethanol concentration of 14.5 g/L from 50 g/L of
cellulose (53.2 % of the theoretical yield) in 6 days (Christakopoulos et al. 1989).
C.C.C.R. de Carvalho, Ph.D. (*)

iBB-Institute for Bioengineering and Biosciences, Department of Bioengineering, Instituto
Superior Técnico, Universidade de Lisboa, Av. Rovisco Pais, Lisbon 1049-001, Portugal
e-mail: ccarvalho@ist.utl.pt

DOI 10.1007/978-3-319-29137-6_21
526 C.C.C.R. de Carvalho
Nevertheless, the very low number of microorganisms able to carry out the entire
process makes simultaneous saccharification and fermentation/cofermentation pro-
cesses using enzymes for the saccharification step, or those using mixed cultures for
the degradation of hexoses and pentoses, more common. A consolidated biopro-
cessing comprising (1) the production of saccharolytic enzymes, (2) the hydrolysis
of the polysaccharides in the pretreated biomass, and (3) the fermentation of both
hexose and pentose sugars in a single bioreactor, will most likely require synthetic
biology techniques as no single microorganism with the desired features has been
found. Among the candidates to be developed are Saccharomyces cerevisiae and
Kluyveromyces marxianus (Karimi et al. 2006; Millati et al. 2008; Karimi and
Zamani 2013). Mucor indicus is also able to ferment lignocellulosic hydrolysates,
both hexoses and pentoses, while tolerating inhibiting compounds resulting in high
yields of ethanol (Karimi et al. 2006; Millati et al. 2008). This species is an example
of a microfungus with several applications from fish feed to wastewater treatment,
being able to produce commercially interesting products from chitosan to polyun-
saturated fatty acids (Karimi and Zamani 2013).
In wastewater treatment, fungal biomass has been used to reduce organic matter
(at low pH where bacterial growth is inhibited), to sequester and adsorb suspended
solids, to degrade recalcitrant pollutants, and to perform denitrification (More et al.
2010; Ryan et al. 2005; Mannan et al. 2007). However, the production of bioactive
compounds is probably the most important feature of fungi. Terrestrial, marine, and
plant endophytic fungi are a source of unique metabolites with, e.g., antimicrobial,
insecticidal, and antitumour activities (Debbab et al. 2010; Wang et al. 2014).
To reach high titres of secondary metabolites during fermentation, a combina-
tion of conditions, including medium composition, substrate(s) type and concentra-
tion, aeration, stirring conditions, and addition of inducers, have to be found,
implemented, and maintained. Process performance in filamentous fungi is clearly
affected by strains and inocula, morphology, and rheology (Posch and Herwig
2014; Posch et al. 2013). In stirred tank reactors, microfungi are particularly sensi-
ble to shear force, but vigorous agitation is usually required to surpass the high
viscosity and oxygen demands observed during submerged growth (Kelly et al.
2006; Ranjan 2008). Shear forces are probably responsible for differences in mor-
phology observed during cell growth between bioreactor scales, making it neces-
sary to use methodologies, such as microscopy and flow cytometry, soft sensors,
and mathematical modelling to predict broth rheology and fungal morphology dur-
ing scale-up of bioprocesses (Posch et al. 2013).
Fermentation
In nature, most fungal genera evolved on moist, solid substrates. They should,
therefore, be more apt to produce secondary metabolites and enzymes under these
conditions than in liquid media. Solid-state fermentation (SSF), by simulating natu-
ral microbiological processes and by using the ability of fungal cells to adhere to
21 Fungi in Fermentation and Biotransformation Systems 527
solid particles, should result in higher productivities. The main SSF advantages
when compared to submerged processes are the reduced bacterial contamination
level, due to the low water activity used, the simple bioreactors necessary, and the
low installation and running costs (Cannel and Moo-Young 1980; Viniegra-
Gonzalez et al. 2003). It is particularly convenient when the solid phase is simulta-
neously the substrate, such as cellulose, lignin, or agricultural residues (Thomas
et al. 2013; Pérez-Guerra et al. 2003). On the other hand, submerged fermentations
allow better process control since parameters, such as temperature, pH, aeration,
and mixture, are monitored and controlled in the bioreactor, allowing also a better
scale-up (Papavizas et al. 1984; Soetaert and Vandamme 2010).
Solid-State Fermentation
After centuries of being used for the production of, e.g., bread and cheese, SSF
systems were considered “low-technology” processes by the end of the twentieth
century (Pandey et al. 2000). However, during this century, SSF has received
renewed attention since it allows stable productions in smaller fermenters, with less
energy requirements, and causes less impact on the environment than liquid fer-
mentations (Pérez-Guerra et al. 2003). SSF is defined as a culture where microbes
thrive on the surface and at the interior of a solid matrix, in the absence of free water
(Viniegra-Gonzàlez 1997; Lonsane et al. 1985). The system comprises solid, liquid,
and gaseous phases, but the low amount of water in the system reduces the risk of
bacterial contamination.
Depending on the nature of the substrate, two SSF systems may be considered:
SSF on natural solid substrates and SSF on impregnated inert supports (Barrios-
González 2012). Although the former should be a cheaper process, the latter may be
an advantage for (1) the production of high-added-value products, as medium costs
are usually only a fraction of the overall production costs (Barrios-González and
Mejýa 2008), and (2) to study the system since the absorbed medium composition
may be designed and monitored during fermentation (Barrios-González 2012).
Since the majority of fungal SSF are batch processes, the morphology and physi-
ology of fungal cells will change with time. Studies aimed at understanding the
biological aspects should be conducted, but most of the research already carried out
aimed at the production of commercially interesting products from cheap substrates
and at the optimisation of the bioprocess (Barrios-González 2012). Biomass has
been estimated by, e.g., respirometry, infrared spectroscopy, and image analysis,
whilst mass, water, and heat balances have been determined to accurately simulate
and characterise SSF processes (Papavizas et al. 1984; Barrios-González and Mejýa
2008). Bioprocess engineering, biochemistry, molecular biology, and microbiology
have to be successfully combined to help the understanding of these complex fer-
mentations and to overcome the critical parameters affecting them (Table 21.1).
To understand the effect of the parameters and to optimise the design and opera-
tion of SSF, mathematical models have been developed. In these models, kinetics
Table 21.1 Critical parameters influencing fungal morphology and productivity in solid-state
fermentations
Parameter Considerations Example
Strain selection Natural production ability of the strain; Vu et al. (2010)
possibility to improve it
Solid substrate Usually a natural agricultural or agro- Mitchell et al. (2004)
industrial by-product/residue, or an inert
synthetic material
Aeration Oxygen is crucial for fungal growth, Rahardjo et al. (2006),
production rates, and yields Coradin et al. (2011)
Water activity Depends on the retention capacity of the Casciatori et al. (2014),
substrate; influences fungal morphology Mariano et al. (1995)
Physical parameters Temperature, pH, and other environmental Rodrigues et al. (2009)
conditions influence fungal performance
Medium composition Concentration and type of, e.g., phosphorous Senthilkumar et al.
and nitrogen sources and minerals added (2005), Liu et al.
should be optimised (2007)
may be described by empirical equations or by complex equation systems addressing

micro-scale effects that affect bioreactor performance. Among the effects to include
in a mathematical model are temperature, pH, aeration, water activity, and the
nature of the solid substrate (e.g. chemical composition, mechanical properties, par-
ticle size) (Thomas et al. 2013). In SSF, mathematical models have to overcome the
heterogeneity of fungal growth, substrate, and medium, which may result in heat
and mass transfer limitations, inaccurate and non-reproducible measurements of
process variables, and difficulties during scaling-up and bioreactor control. Since in
SSF there is no convective transport of substrates and products, the necessary gra-
dients to supply nutrients and remove the products may affect cellular growth and
production and require models coupling reaction and diffusion phenomena
(Rahardjo et al. 2006). Coradin and co-workers were able to develop a three-
dimensional model, simulating the growth of the aerial hyphae in random direc-
tions, to study phenomena occurring at micro-scale and to help the preparation of
substrate and fermenter operation (Coradin et al. 2011). The growth of aerial hyphae
in the space between particles increases the pressure drop through aerated beds and
may decrease oxygen transfer when agglomerates are formed.
When studying the impact of moisture content and packing technique on parti-
cle and bulk densities and porosities of beds packed with sugar cane bagasse, wheat
bran, orange pulp and peel, and mixtures of them, the authors found that the mac-
roscopic properties of the bed mixtures could be estimated as a weighted average
of the properties of each individual material (Casciatori et al. 2014). Besides, the
mycelial growth of Myceliophthora thermophila I-1D3b and Trichoderma reesei
QM9414 did not affect the bed properties, but the cellulase yield of the latter was
strongly affected by the ratio sugar cane bagasse/wheat bran and by the moisture
content.
According to a mathematical model relating the growth of Aspergillus niger, in

a packed bed with amberlite, with variables, such as sugar, water content, oxygen
and carbon dioxide concentrations, and bulk temperature, the rate-limiting step for
mycelial growth is sugar depletion (Mariano et al. 1995). The model was effective
in predicting biological parameters difficult to determine experimentally, but failed
when product formation occurred.
To improve xylanase production by A. fischeri in SSF with wheat bran,
Senthilkumar et al. used response surface methodology and central composite
rotary design (Senthilkumar et al. 2005). The analysis, involving the effects of four
variables (sodium nitrite, potassium dihydrogen phosphate, magnesium sulphate,
and yeast extract), allowed the optimisation of culture media for maximum xyla-
nase production and minimum protease activity.
SSF has been found to be particularly useful for enzyme production, espe-
cially of ligninases, xylanases, and pectinases, to increase the value of agricultural
by-products. The low-technology necessary allows its application in farms and
could provide animal feeding in developing countries (Graminha et al. 2008).
Submerged Fermentation
During submerged fermentation (SF) of filamentous fungi, the morphology plays a

critical role (Gibbs et al. 2000). Several parameters influence the growth of fungi as
free mycelia or pellets, such as shear stress, which should be high in stirred tanks
(Kelly et al. 2006; Ranjan 2008; Xu et al. 2006). High viscosities are often observed,
resulting in, e.g., decreased oxygen transfer, formation of nutrient gradients, high
power requirements, and reduced productivity. Several methodologies have been
proposed to prevent increased viscosity, including pulsed addition of the limiting
carbon source (Bhargava et al. 2003), which results until the point of conidial for-
mation (Bhargava et al. 2005). Since filamentous fungi require simultaneously low
stirring speeds and high amounts of oxygen, while viscosity increases as a result of
cellular growth, optimisation of fermentation conditions should be assessed for
each strain and bioreactor (Coradin et al. 2011; Cho et al. 2006). If very high air
flow rates and low stirring speeds are combined, air dispersion is reduced and
impeller “flooding” (where air flows mainly up the stirrer shaft) results in poor mix-
ing and reduced oxygen transfer rates (Michelin et al. 2011; Doran 1995).
Albaek et al. developed a mathematical model to simulate a 550 L pilot-scale
fed-batch fermentation of recombinant A. oryzae at different stirring speeds and
aeration rates, comprising the reaction equation stoichiometry, a mass transfer cor-
relation, and viscosity prediction (Albaek et al. 2011). Two impeller types were
tested in the 20 fermentations used to develop the model: a Rushton disc turbine
and a hydrofoil Hayward Tyler B2. Contrarily to what was expected, analysis of the
energy dissipation/circulation function (EDCF) showed that the apparent viscosity
was lower when the B2 hydrofoil was used, whilst the lower number of blades and
associated vortices would result in higher shear forces. Heo and co-workers also
showed that the impeller type affects mycelial morphology: cells of A. oryzae
grown in a submerged culture with propeller agitation grew in the form of a pellet
whilst those in a bioreactor with turbine agitation grew as freely dispersed hyphae
and in a clumped form (Heo et al. 2004). The former morphology allowed the high-
est protein production levels for both intracellular heterologous protein
(β-glucuronidase) and the extracellularly homologous protein (α-amylase). Further
improvement could be achieved by supplying the carbon source through pulsed
feeding.
When the effects of increased agitation power on enzyme expression of A. oryzae
were studied in an 80 m3 fermenter operated in fed batch, it was found that increased
power improved bulk mixing but resulted in lower recombinant enzyme productiv-
ity (Li et al. 2002). Biomass assays and image analysis showed that slower growth,
altered morphology, or increased hyphal fragmentation were not responsible for the
reduced productivity observed at higher power inputs. Since the impeller power was
increased by 50 %, by increasing 10 % the impeller diameter while operating at
lower stirring speeds, EDCF values dropped linearly from 40 to 15 kW m−3 s−1 dur-
ing “high power” batches. In control fermentations the EDCF values were nearly
constant at 25 kW m−3 s−1. The results thus showed that oxygen transfer was less
efficient when higher agitation power was applied because of the lower impeller
speed, making it important to study how power is applied to the media during scale-
up of viscous fungal fermentations.
Low-shear environments may be provided by running fermentations in airlift
bioreactors. Airlifts do not have mechanical stirrers, decreasing the risk of contami-
nation, energy demand, and costs (Michelin et al. 2011, 2013). Enzyme productivi-
ties and production rates are, in general, higher in airlifts than in stirred tanks: lipase
productivity by Geotrichum candidum was ca. 60 % higher (Burkert et al. 2005),
xylanase production was higher, and both xylanase and β-xylosidase were produced
at a faster rate by Aspergillus terricola (Michelin et al. 2011). However, higher
xylanase levels were produced by A. niger in a stirred tank although when the enzy-
matic production was compared for the two types of bioreactors, at the same kLa
values, both xylanase and β-xylosidase productions were higher in the airlift
(Michelin et al. 2013). During exopolysaccharide (EPS) production by Paecilomyces
tenuipes C240, it was found that the specific production rate was significantly higher
in the airlift reactor, but the final concentration of the mycelial biomass was much
lower, whilst the carbohydrate composition of EPS produced in each reactor was
quite different (Xu et al. 2006). On the other hand, the specific productivities and
yield coefficients of both biomass and EPS of a submerged mycelial culture of the
mushroom Tremella fuciformis were higher when the cells were grown in a 5 L
airlift as compared to a 5 L stirred tank reactor (Cho et al. 2006). The results obtained
in the different studies show that there is a link between mycelial morphology,
which is affected by several parameters, such as shear stress and available oxygen
concentration, and metabolic activities in filamentous fungi.
Table 21.2 Examples of bioreactors used for fungal fermentations

Bioreactor type Remarks Reference
Airlifts Lower power requirements for the same kLa than Cho et al. (2006), etc
mechanically stirred tanks and lower shear stress group (2013)
Bag reactors Cell aggregation may be prevented; allow the Jonczyk et al. (2013)
growth of shear stress sensitive fungi
Laterally aerated Can perform batch and continuous SSF Wong et al. (2011)
moving bed
Membrane Use the natural ability of fungal cells to adhere to Hevekerl et al. (2014),
reactors surfaces Linde et al. (2014)
Microtiter plares Allows to simplify and expedite process Hevekerl et al. (2014),
optimisation Linde et al. (2014)
Packed bed High substrate/volume ratio allowed; forced Casciatori et al. (2014)
aeration; porosity affects directly fluid dynamics
and heat and mass transfer
Rotary drum Allows mixing with control of rotation speed Diaz et al. (2009)
Stirred tanks High biomass concentration possible but may Gibbs et al. (2000),
affect rheology, mixing, and oxygen transfer; Gabelle et al. (2012)
good control of parameters (temperature, pH, etc.)
Tray bioreactors Popular bioreactor for industrial SSF; scale-up Bhargav et al. (2008),
achieved by increasing number of trays but Byndoor et al. (1997)
difficult to predict cell behaviour; limited heat and
mass transfer
Bioreactor Design
As mentioned previously, the performance of fungi in bioreactors depends greatly

on the rheological properties of the broth, which are also influenced by the concen-
tration of biomass and cellular morphology. The relations between rheology and
morphology should be studied and taken into consideration during the design of the
bioreactor. Several types of reactors have been proposed for SSF and SF (Table
21.2), but the resulting productivities and yields obtained in each type vary consid-
erably depending on fungal strains and products.
Tray bioreactors are possibly the simplest type of reactor for SSF, requiring low
costs and operation skills, but the amount of substrate is limited and the number of
trays necessary for scale-up requires space (Thomas et al. 2013). Heat and mass
transfer in tray bioreactors may also be limited by intraparticle oxygen diffusion,
growth rate of the fungus, and absence of heat exchange processes (Bhargav et al.
2008). Nevertheless, in tray bioreactors, the cells are not under mechanical stress;
support agglomeration may be prevented by keeping the layer of substrate thin, and
the low aeration may prevent fungal overgrowth (Rosales et al. 2007). For these
reasons, laccase production by Trametes hirsuta is higher in this type of bioreactor
than when the fungus was grown in Erlenmeyer flasks or on a fixed-bed tubular bio-
reactor (Rosales et al. 2007). In fact, tray reactors have been particularly successful
for the production of industrial enzymes, such as phytases, pectinases, and cellulases
(Thomas et al. 2013). To improve pectinase production from lemon peel pomace by
A. niger, Ruiz et al. developed a column-tray bioreactor containing eight perforated
base trays inside a vertical cylinder (Ruiz et al. 2012). The system used a compressor
for forced aeration and a water jacket for temperature control.
To allow the mixing of the solid substrates in SSF, several groups have suggested
rotating drum bioreactors. However, this reactor type seems to provide better results
when static or low agitation conditions are implemented. The maximum hydrolytic
enzyme activities from a mixture of 1:1 (w/w) of grape pomace and orange peels
using Aspergillus awamori were attained in static conditions or an agitation as low
as 1 min/day (Diaz et al. 2009). The production of cellulases and hemicellulases by
the thermophilic microfungus Thermoascus aurantiacus was carried out in a drum
bioreactor rotating at 10 rpm for 1 min every 3 h (Kalogeris et al. 2003).
In packed-bed reactors, the solid humidified substrate is retained on a perforated
base through which air is forced to pass. To the glass or plastic body of the reactor,
a water jacket may be added to control the fermentation temperature. However, in
these reactors, non-uniform growth may be observed; the product may be difficult
to recover and heat transfer and scale-up problems may occur (Couto and Sanromán
2006). When comparing coal biosolubilisation in a stirred tank, in a fluidised bed,
and in a fixed-bed bioreactor, Oboirien and co-workers found that in the packed-bed
bioreactor the biosolubilisation was minimal probably due to clogging of the bed
particles by fungi which leads to unpredictable internal mass transport and due to
the large size of the coal particles used (Oboirien et al. 2013). In this case, the stirred
tank allowed the best fungal performance whilst the fluidised bed reactor demanded
the highest aeration rates since air is also responsible for mixing. In fluidised beds,
continuous agitation with forced air prevents adhesion and aggregation of the sub-
strate particles, helping mass and heat transfer, but cellular damage and increased
heat due to shear forces may decrease the expected product yield (Couto and
Sanromán 2006).
Although stirred tank reactors may cause high shear stress to filamentous fungi,
these reactors often overcome limitations caused by insufficient mixing and mass
and heat transfer observed in other types of reactors. For example, high lactic acid
concentration (85.7 g/L) and yield (86 %) were attained from waste potato starch in
a mechanically stirred bioreactor using acid-adapted Rhizopus arrhizus (Zhang
et al. 2008). Since mixing is the most important feature of stirred tanks, the type of
impellers should be studied. During the fermentation of digested solka floc using a
recombinant strain of Zymomonas mobilis, the bioreactor was tested with a Rushton
turbine and a marine impeller, with and without wall baffles (Um and Hanley 2008).
At 120 rpm, the enzymatic saccharification for glucose production attained much
higher concentration of glucose in reactors with Rushton turbines than those with
marine impellers. Furthermore, the presence of baffles in the reactor walls improved
the fungal fermentation when the system was mixed by Rushton impellers but not
when marine impellers were used. Since Rushton turbines performed poorly in a
75 L fermenter developed for the conversion of lignocellulosic agricultural materi-
als by Neurospora sitophila, they were replaced by suitable axial flow impellers
(Chisti and Moo-Young 1994). Although no significant increase in fungal growth

rate was observed between the two impellers, cellulose utilisation was ca. 86 %
higher with the axial flow impeller as no agitation-associated mechanical damage
was observed in the mycelia.
One of the advantages of using submerged cultures of filamentous fungi for the
production of commercially interesting enzymes is the possibility to use gas–liquid
mass transfer predictions to scale-up the system (Gabelle et al. 2012). A power law
model to simulate the rheology of the model dependent on the biomass concentra-
tion could be determined during the growth of Trichoderma reesei (Gabelle et al.
2012). A model including the effects of dissolved oxygen, viscosity, temperature,
pH, and dissolved carbon dioxide to describe penicillin fermentation by Penicillium
chrysogenum could be developed to improve control and optimisation of biomass
and penicillin production (Goldrick et al. 2015).
To prevent cell damaging in shear stress sensitive basidiomycetes such as
Flammulina velutipes, a disposable bag bioreactor developed for cultivation of
mammalian cells may be used (Jonczyk et al. 2013). Contrarily to what was
observed for stirred tank reactors, dispersed pellets were observed and F. velutipes
reached higher biomass concentrations and twofold higher peptidolytic activities.
During cultivation of filamentous fungi, the initial low viscosity Newtonian
medium usually becomes a highly viscous non-Newtonian fluid and airlift bioreac-
tors could be an efficient alternative to mechanically stirred reactors. In airlift reac-
tors, the parameters affecting product formation are gas hold-up, liquid circulation
velocity, and mixing (Kang et al. 2001). When studying an airlift bioreactor with
internal recirculation loop for the production of a biopolymer with Sclerotium glu-
canicum NRRL 3006, Kang et al. noticed significant differences between experi-
mental “real” fungal culture medium and simulated systems (Kang et al. 2001).
While the latter are homogeneous, simple systems, real fungal cultures are highly
heterogeneous, presenting several morphologies, and cells attached to substrate par-
ticles. Recirculation in the airlift may also be affected by biomass concentration: a
fast growth rate of Geotrichum candidum observed at high kLa values blocked bio-
mass recirculation (Burkert et al. 2005). Nevertheless, the productivity in the airlift
reactor was 60 % higher than that attained with a stirred fermenter.
What the published works demonstrate is that due to the heterogeneity of fungal
strains, and the numerous types of morphological responses to the reaction condi-
tions, several studies are required to select the best reactor design for each system
as no simple prediction of cellular behaviour should be possible.
Biotransformation Systems
Biotransformations using fungal cells allow the production of compounds that may
be classified as “natural” products by the European and American food legislations
(Krings and Berger 1998), reaching much higher market value than chemically syn-
thesised compounds. One of the best examples is the production of vanillin
(3-methoxy-4-hydroxybenzaldehyde), the flavour of vanilla. Natural vanillin,

extracted from the seed pods of the vanilla orchid, represents ca. 0.25 % of the
global market (approximately 40–50 tons per year) and costs ca. US$ 4,000 per kg,
whilst around 16,000 tons per year of synthetic vanillin are produced and sold at
US$10–20 per kg (etc group 2013; Hansen et al. 2009). Solvay, the world’s leading
vanillin producer, now produces Rhovanil® Natural by bioconversion of ferulic
acid, a natural organic compound found in rice bran (Solvay SA 2013). The Swiss
biotech company Evolva developed a yeast-based fermentation route to produce
both vanillin and other vanilla flavour components (evolva 2015).
A de novo pathway enabling one-cell microbial generation of vanillin from glu-
cose was recently developed in the fission yeast Schizosaccharomyces pombe and
also in baker’s yeast, Saccharomyces cerevisiae (Hansen et al. 2009). The engi-
neered pathways developed involved the incorporation of 3-dehydroshikimate
dehydratase from Podospora pauciseta, of an aromatic carboxylic acid reductase
from a bacterium of the Nocardia genus, and of an O-methyltransferase from Homo
sapiens. The productivities reached 65 and 45 mg/L after introduction of 3 and 4
heterologous genes, respectively.
Several fungi, including species of Aspergillus, Fusarium, Polyporus, and
Rhodotorula have been found able to convert ferulic acid, which results from lignin
degradation of common agricultural residues like cereal brans and sugarbeet pulp
(Rosazza et al. 1995). The fungus Sporotrichum thermophile produces vanillic acid
during ferulic acid degradation via the propenoic chain degradation (Topakas et al.
2003). When the fungus was grown on glucose for 3 days, it was able to convert 80
mmol/L of ferulic acid with a molar yield of 35 %, reaching 4.8 g/L of vanillic acid.
Around one-third of the percentage of papers published in the first decade of this
century on the production and/or biotransformation of terpenes were based on stud-
ies with fungi (de Carvalho and da Fonseca 2006). Terpenes are widely distributed
in nature and some such as limonene and α-pinene are inexpensively available in
large quantities, whilst their oxygenated derivatives terpenoids have been used as
fragrances and flavours for centuries. Fungal biotransformations may provide terpe-
noids for the food, cosmetic, and pharmaceutical industries (Table 21.3) with high
value to the increasingly demanding consumers looking for natural and healthy
products. Furthermore, the compounds may be produced with high stereo-specificity
and selectivity, under mild conditions (de Carvalho and da Fonseca 2006; de
Carvalho 2009).
Fungal cells have also been a good source of interesting enzymes. The GRAS
(generally regarded as safe) fungus Aspergillus niger was found as a good source of
the enzyme tannase, produced by SSF from cheap tea by-products (Ni et al. 2015).
After treating a tea infusion with tannase, the tea presented significant increases in
clarity, 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulphonate) and 2,2-diphenyl-1-
picryl-hydrazyl inhibition activities, and hydroxyl radical scavenging capacity.
Chitosanases are able to catalyse the endohydrolysis of β-1,4-glycosidic bonds
of partially acetylated chitosan to release chitosan oligosaccharides and are unable
to hydrolyse chitin (Thadathil and Velappan 2014). The bioconversion of chitinous
wastes from, e.g., the seafood processing industry to bioactive compounds seems
promising. Among the fungi reported as chitosanase producers are strains belonging
Table 21.3 Examples of biotransformations of terpenes carried out by fungi

Fungus Biotransformation Reference
Aspergillus niger Hydroxylation reactions of acyclic Madyastha and Murthy
monoterpene alcohols (1988)
Aspergillus niger (+)-limonene to terpineols and carveols Garcia-Carnelli et al.
(2014)
Botrytis cinerea α-santonin and sclareol Farooq and Tahara (2000)
Cyathus africanus Insecticidal sesquiterpenes cadina-4,10(15)- McCook et al. (2012)
dien-3-one and aromadendr-1(10)-en-9-one
Mortierella (+)-limonene to perillyl alcohol and perillyl Trytek et al. (2009)
minutissima aldehyde
Mucor plumbeus Conversion of sesquiterpenoid (−)-maaliol Wang et al. (2009)
to (+)-7,8-didehydro-9β-hydroxymaalioxide
and
(−)-7,8-didehydro-1β-hydroxymaalioxide
Penicillium and Biotransformations of (RS)-linalool, Rueda et al. (2013)
Fusarium strains (S)-citronellal, and sabinene
Penicillium D-limonene to α-terpineol Lindmark-Henriksson
digitatum et al. (2004), Ni et al.
(2015)
Penicillium Conversion of geraniol, nerol, “citrol”, citral Demyttenaere and De
digitatum to 6-methyl-5-hepten-2-one Kimpe (2001)
Pichia and Limonene, α- and β-pinene Bier et al. (2011)
Rhizopus strains
Picea abies β-pinene to trans-pinocarveol and minor Lindmark-Henriksson
products et al. (2004)
to the genera Aspergillus, Gongronella, and Trichoderma (Thadathil and Velappan

2014).
White-rot fungi have also been used to produce laccases, since they are the only
known organisms able to fully mineralise all components of lignin, although yeasts
have been used as suitable hosts for heterologous laccase production (Couto and
Toca-Herrera 2007). Laccases have been mainly applied in the decolouration of
dyes in the textile, dye, or printing industries and in the delignification and bleach-
ing of paper pulps, with other applications including organic synthesis, the manu-
facture of biodevices, or the detoxification of pollutants (Couto and Toca-Herrera
2007; Cañas and Camarero 2010).
Bioremediation
The enormous number of enzymes and substrates metabolised by fungi also make
these microorganisms attractive for the bioremediation of recalcitrant compounds
(Potin et al. 2004; Leyval et al. 1997; Gray 1998; Zhdanova et al. 2000).
As mentioned, white-rod fungi possess ligninolytic enzymes with broad sub-
strate specificity, being able to degrade pesticides, polychlorinated biphenyls, poly-
cyclic aromatic hydrocarbons, synthetic dyes, synthetic polymers, and wood

preservatives (Pointing 2001). Strains of Phanerochaete chrysosporium, Pleurotus
ostreatus, Phellinus weirii, and Polyporus versicolor have been shown able to min-
eralise DDT, whilst P. chrysosporium, P. ostreatus, and Trametes versicolor are
capable of degrading polychlorinated biphenyls and polycyclic aromatic
hydrocarbons.
Other fungi have been used for the bioconversion of soluble/insoluble (organic)
substances in, e.g., activated domestic sludge: Penicillium corylophilum and
Aspergillus niger increased filterability/dewaterability while decreasing consider-
ably COD and turbidity (Mannan et al. 2005). Using response surface methodology,
COD removal by P. corylophilum could be improved to 98.5 % by using an incuba-
tion temperature of 32.5 °C, the agitation at 105 rpm, and pH at 5.5 (Mannan et al.
2007).
Several fungal strains belonging to the genera Cephalosporium, Paecilomyces,
Penicillium, Aspergillus, Trichoderma, and Mucor have been used in gas-phase bio-
filters for the treatment of waste gases (Liu et al. 2013; Kennes and Veiga 2004).
They are able to remove malodorous sulphur compounds, which are the main cause
of odour pollution, and may be used together with bacteria in biotrickling filters
(Liu et al. 2013).
Fungal cells are also able to bioremediate contaminated soil with, e.g., polycy-
clic aromatic hydrocarbons (Potin et al. 2004), heavy metals (Leyval et al. 1997),
and even radioactive material (Gray 1998). In fact, expensive fungal growth was
observed on the walls of the inner part of the Shelter of the damaged fourth Unit of
the Chernobyl Nuclear Power Plant (Zhdanova et al. 2000). Of the fungi isolated,
about 80 % were melanin-containing and pigmented micromycetes.
Final Remarks
Fungi are promising producers of commercially interesting compounds. These may

be secondary metabolites or the result of enzymatic bioconversion. However, to
achieve the best yields, environmental conditions must be adjusted and bioreactors
should be chosen to allow the best productivities and activities. Fungal behaviour is
difficult to model and predict. Nevertheless, several research groups are trying to
provide further understanding on these complex microorganisms.
Acknowledgements The author acknowledges Fundação para a Ciência e a Tecnologia, I.P.

(FCT), Portugal, for the exploratory project and financial support under program “Investigador
FCT 2013” (IF/01203/2013/CP1163/CT0002).
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Chapter 22
Microfungi in Biofuel and Bioenergy Research
Richa Raghuwanshi, Shalini Singh, Mohd. Aamir, Amrita Saxena,

Vijai Kumar Gupta, and R.S. Upadhyay
Introduction
The exigency for crude oil and the existing ebb of the petroleum reserve of the
planet have necessitated the use of alternative sources of energy for fuel production.
This can be inferred from the statistical data stated by the United Nations (UN),
according to which the world primary energy consumption will increase twofold
(24 billion tons coal equivalent per year) to the existing energy consumption calcu-
lated by World Energy Council (WEC) in 2009 due to the population increase to
about 10 billion by 2050 (Dashtban et al. 2009). Among the existing renewable
resources along with the close homology to the natural resource, biofuels have
gained greater attention these days. Biofuels refer to the type of fuel whose energy
is derived from biological carbon fixation. On this basis, this class includes fuels
that are derived from biomass conversion or solid biomass, liquid fuels, and also
various biogases.
On the basis of the mode of their production and depending on the starting mate-
rial for their production, up until now, four generations of biofuels have been under
consideration. First-generation biofuel refers to the biofuel derived from plant
materials like cereals, sugar crops, and oil seeds. These types include biodiesel,
green diesel, bioalcohols, bioethers, and biogas (Sims et al. 2010). Owing to the
R. Raghuwanshi, Ph.D.
Department of Botany, Mahila Mahavidyalaya, Banaras Hindu University,
Varanasi 221005, India
S. Singh, Ph.D. • M. Aamir, Ph.D. • A. Saxena, Ph.D. • R.S. Upadhyay (*)
Department of Botany, Banaras Hindu University, Varanasi 221005, India
e-mail: upadhyay_bhu@yahoo.co.uk
V.K. Gupta, Ph.D.
Molecular Glyco-biotechnology Group, Discipline of Biochemistry, School of Natural
Sciences, National University of Ireland Galway, Galway, Ireland
e-mail: vijaifzd@gmail.com; vijai.gupta@nuigalway.ie

DOI 10.1007/978-3-319-29137-6_22
544 R. Raghuwanshi et al.
decreasing availability of the plant materials, focus was shifted to the waste products
for biofuel production that constituted the second-generation biofuels comprising
mainly of cellulosic ethanol and biogas from municipal wastes, etc. The next step
toward the development of biofuel owing to the high-cost conversion of lignocel-
lulosic materials and decreased profit was the employment of microbes for the
production of biofuel starting the third-generation biofuels. Numerous classes of
bacteria and fungi were employed for successful conversion of raw materials into
economic biofuel for commercial use. Lignin-degrading fungi, cellulose-
decomposing bacteria, lipid-containing fungi, etc., were considered for effective
and low-cost production of biofuels. The present concern has been shifted to the use
of genetically developed photosynthetic cells for the conversion in biofuels, which
brings the fourth generation.
Biofuels produced using lignocellulosic biomass provide several benefits to soci-
ety such as (1) being renewable and sustainable, (2) indirectly helping a greenhouse
gas (GHG) that is responsible for global warming for fixation, (3) facilitating local
economy development, (4) reducing air pollution from burning of biomass, (5) bring-
ing energy security for countries dependent on imported oil, and (6) creating jobs for
engineers, fermentation specialists, process engineers, and scientists. Fungal biomass
biofuel production represents a pivotal approach to face high energy prices and con-
tribute to a net reduction of total greenhouse gas emissions. These microorganisms
have the capability to accumulate significant quantities of storage triacylglycerols
(TAGs), an attractive source of oil suitable for biodiesel production (Economou et al.
2011). The major reasons for the popularity and success in employing oleaginous
fungi for biofuel production may be summarized in the following points:
• High growth rate
• Extensive enzymatic system for efficient lipid production and accumulation
• Ability to utilize cheap waste materials as substrates
• Growth and cultivation independent of seasonal variations
• Easy alteration in lipid production by manipulating nutrient conditions
• Probability of using mutation of specific enzymes at specific steps to increase
lipid yield
• Act as potential hosts for cloning foreign genes related with lipid and PUFA
production.
Enzymes Involved in Biofuel Production
Though lignocellulosic biomass is a potential low-cost source of mixed sugars for

fermentation to produce fuel ethanol, but it is a naturally recalcitrant material com-
posed of cell walls having intricate network of celluloses, hemicelluloses (including
xyloglucans, arabinoxylans, and glucomannans), pectins (like homogalacturo-
nans, rhamnogalacturonan, and xylogalacturonans), lignins, and proteoglycans that
creates technical barriers for the cost-effective transformation of biomass to fer-
mentable sugars. Moderate yields and the resulting complex composition of sugars
22 Microfungi in Biofuel and Bioenergy Research 545
and inhibitory compounds lead to high processing costs. Cell walls in lignocellulosic
biomass can be converted to mixed-sugar solutions with lignin-rich solid residues
by sequential use of a range of thermochemical pretreatments and enzymatic sac-
charification. A wide variety of extracellular enzymes obtained from fungal biomass
are implicated in lignocellulosic biomass decomposition, a potential low-cost
source of mixed sugars for fermentation to fuel ethanol (Table 22.1).
Cellulases
Cellulases are an O-glycoside hydrolase group of enzymes used to hydrolyze the

cellulosic plant biomass to simple sugars that can be transformed (fermented) by
microbes for biofuel, primarily ethanol generation. Cellulases hydrolyze the β-1,4
glucan linkage in cellulose and produce glucose, cellobiose, and cello-
oligosaccharides. The cellulase enzymatic system consists of three enzymes acting
synergistically to hydrolyze the crystalline cellulosic biomass into the small sugars
Cellobiohydrolase (CBH), Endo β-1,4 glucanases (EG), and β- glucosidase (BGL).
Endoglucanase hydrolyzes the middle of a low crystalline cellulose and starts cel-
lulose breakdown by randomly attacking the interior of the amorphous regions of
the cellulose fiber, thus releasing oligosaccharides occupying different degrees of
polymerization and exposing its reducing and non-reducing ends (Sanchez et al.
2004). Cellobiohydrolases act processively on reducing and non-reducing chain
ends to release mainly cellobiose. β-Glucosidases (BGLs) hydrolyze the β-1,4 gly-
cosidic bond of cellobiose and cellodextrins to release glucose units. β-glucosidase
is competitively inhibited by glucose (Dashtban et al. 2009). Enzymatic hydrolysis
by cellulases depends on the physical properties of cellulose molecules such as
crystallinity, degree of polymerization (DP), and accessible surface area, as these
are the major factors responsible for controlling hydrolysis rate due to their effect
on enzyme binding and accessibility of substrates to the cellulase enzymes.
Crystallinity is a key factor affecting the hydrolysis of cellulose as the glycosidic
bonds in crystalline regions are difficult to hydrolyze compared to those in the
amorphous regions (Ahola et al. 2008). Several other novel enzymes assist in cel-
lulose degradation by acting in synergy with the exo- and endoglucanases (Leggio
et al. 2012) including copper-requiring polysaccharide monooxygenases. Some
proteins have elastin-like properties including Swollenin and other cellulase-
enhancing proteins, which contribute to its hydrolysis by increasing access of the
cellulase enzymes to the cellulose chain ends (Nakatani et al. 2013).
The majority of cellulases have a characteristic two-domain structure with a
catalytic domain and Cellulose Binding Domain (CBD). One of the major things
that restricts or directs the degradation of native cellulose and thus regulates the
catalytic activity of cellulolytic enzymes, is their different levels of orientation and
different degrees of cooperation between individual units. Depolymerization of
both crystalline and amorphous cellulose molecules to fermentable sugars is due to
synergistic action of endoglucanases, exoglucanases, and β-glucosidases (Dyk and
Table 22.1 Enzymes, characteristic properties, and their fungal sources involved in lignocellulosic biodegradation
Enzymes Principal types Characteristic features and catalytic function Fungal sources
Cellulolytic Endoglucanases Hydrolyzes accessible intramolecular β-1,4- Aspergillus niger, Penicillium, Fusarium, Coriolus,
enzymes glycosidic bonds of cellulose chains randomly to Volvariella, Rhizopus oryzae, Talaromyces emersonii,
produce new chain ends, increases the accessible Chaetomium, Trichoderma, Phanerochaete, Schizophyllum,
surface areas, and prevents the formation of T. harzianum, Schizophyllum commune, Mucor, Pycnoporus,
inhibitory products Bjerkandera, Trichoderma reesei, Phanerochaete
Exoglucanases Cleaves cellulose chains at the reducing and chrysosporium, Sporotrichum pulverulentum, Penicillium
non-reducing to produce cellobiose or glucose pinophilum, Aureobasidium pullulans (yeast)
β-glucosidases Hydrolyze cellobiose and cellodextrins to glucose
to prevent cellobiose inhibition
Cellobiose Dephosphorylates the phosphorylated moiety and Anaerobic Fungi
phosphorylase thus favors the hydrolysis by other cellulolytic Orpinomyces, N. patriciarum, Caecomyces, Neocallimastix,
enzymes Orpinomyces, and Piromyces
Hemicelluloytic Endoxylanases Generates oligosaccharides from the cleavage of Polyporus sulfurous, Aspergillus niger, Pycnoporus
Enzymes Exoxylanases xylan oligosaccharides, producing xyloses. All sanguineus, Neocallimastix frontalis, Trichoderma reesei,
α- xylosidase these act synergistically to depolymerize B. adusta, Anaeromyces mucronatus, P. chrysosporium
hemicellulosic sugars to their corresponding
fermentable monomers to produce biofuels and
other valuable co-products
ɑ-glucuronidase Hydrolyzes the ɑ 1,2 linkages between
D-glucuronic acid and xylose
β-mannosidases Hydrolyzes the endomannase-generated oligomers
β -glycosidase β-1,4 bonds
α-L-arabinofuranosidase Hydrolyzes the terminal arabinose residues from
Acetyl xylan esterase side chain of Xylan De-acetylates O-acetyl group
from acetylated Xylan
Ferulic acid esterase Cleaves ester bond between arabinose side chains
and ferulic acids
Enzymes Principal types Characteristic features and catalytic function Fungal sources
Lignin-degrading Lignin peroxidases Oxidizes non-phenolic part of lignin and along Phanerochaete chrysosporium, Fomitopsis palustris,
or lignin- with Laccases and MnPs causes degradation and Orpinomyces (anaerobic), Phlebia, Physisporinus rivulosus,
modifying delignification of woody component Dichomitus squalens, Piptoporus betulinus, Laetiporus
enzymes (LMEs) Manganese peroxidases Extracellular glycoproteins; catalyzes the portentosus, Trametes versicolor, Fusarium solani,
peroxide-dependent oxidation of Mn(II) (as the Ceriporiopsis, Gloeophyllum trabeum, Pycnoporus
reducing substrate) to Mn(II) and increases coccineus, Pycnoporus sanguineus, Cyathus, Coniophora
accessible surface for lignin degradation along puteana, Magnaporthe grisea, Myrothecium verrucaria,
with LiP Neurospora crassa
Laccases Blue multicopper oxidoreductases; uses molecular
O2 to oxidize organic compounds with the
coupling of electron reduction of dioxygen to
water and the oxidation of a vast variety of
substrates like phenols, lignin, amines,
arylamines.
Versatile peroxidases Dual oxidative ability and oxidizes various
substrates of other lignin peroxidases
Pletschke 2012). All the commercially available glycoside hydrolases are principally
isolated from fungi and it was estimated that in 2012, cellulases accounted for 20 %
of the total enzyme market which was approximately 6 billion dollars (Mathew
et al. 2008).
Cellulase System of Fungi
Filamentous fungi are a potent source of cellulases and secrete non-complexed cel-
lulases (not bounded to cell wall) into their extracellular environment. A wide vari-
ety of fungi, such as Aspergillus spp., Trichoderma reesei, Penicillium pinophilum
(Korotkova et al. 2009), Trichoderma viride (Gusakov et al. 2007), Phanerochaete
chrysosporium (Tsukada et al. 2006), Fomitopsis pinicola (Joo et al. 2010),
Fomitopsis palustris (Yoon et al. 2008), Talaromyces emersonii (Murray et al.
1984), Neocallimastix, Orpinomyces, and Piromyces (Steenbakkers et al. 2002) have
been reported with extensive ability of secreting extracellular cellualse enzymes.
Filamentous fungi are the major producer of cellulase and hemicellulase enzymes,
but the enzymatic component and their corresponding activity may differ in differ-
ent species and are also affected by various parameters like product inhibition.
Hypocrea jecorina (Trichoderma reesei) is the most extensively studied cellulolytic
fungus that secretes an array of cellulases (Kubicek 2013) and is the main industrial
source of cellulases. Aspergillus produces the enzymatic component of cellulase
and has strong hydrolytic activity, but it mainly produces BGLs compared to T.
reesei, as T. reesei is subject to product inhibition. When there is need for biomass
saccharification with T. reesei, it is often supplemented with Aspergillus BGLs. A
complex array of cellulases, hemicellulases, and ligninases are produced by
Phanerochaete chrysosporium (Broda et al. 1996). High titre of cellulase produc-
tion is also reported from Penicillium species like P. brasilianum (Jorgensen et al.
2007). For the efficient hydrolysis of lignocellulosic biomass, the major factor that
determines the lignocellulose saccharification is the catalytic efficiency of an indi-
vidual enzyme and its percentage composition in cocktail when a multienzyme
catalyst is used for conversion. The ideal cellulase complex must be highly active in
intended biomass feedstock, must be able to completely hydrolyze the feedstock,
withstand mild acidic pH, and finally must be cost-effective.
Approaches to Increase Cellulase Production
There are a wide variety of fungal species capable of cellulase production, but the
enzymatic yield and level of individual cellulase components are not satisfactory
for complete lignocellulosic biomass saccharification, so there is a need to enhance
the production of cellulase enzyme. One of the keys for developing cellulase is to
construct them either by assembly of enzymes to form cocktail or to engineer cel-
lulase producers to express the desired combination of cellulases. For instance, an
enzymatic cocktail has been produced by mixing T. reesei cellulases with other
enzymes like xylanases, pectinases, and BGLs and this cocktail is tested for ligno-
cellulosic biomass saccharification from different feedstocks. Solid-state fermenta-
tion (SSF) is one of the most important cost-effective technologies for biodegradation
of lignocellulosic biomass employing cellulolytic microbes. Other approaches to
enhance cellulase production include increasing the copy number of the BGL gene
and the amount of BGL in cellulase enzymes secreted by T. reesei (Fowler and
Brown 1992) or altering the cellulose mixture profile of T. reesei by introducing
glucose-tolerant BGL gene into the fungus (White et al. 2000). Site-directed muta-
genesis, expression cassettes, and antisense technology are some recent techniques
used frequently to modulate the fungal biosystem for enhanced cellulase production
(Gincy and Sasikumar 2007). Potent cellulase genes from different fungi can be
isolated, cloned, and expressed in their fungal hosts. Enhanced production of cel-
lulases can also be achieved by the use of promoters that are insensitive to glucose
repression (Watanabe and Tokuda 2001).
Hemicellulases
Hemicelluloses are highly branched and mostly noncrystalline heteropolysaccha-

rides. Sugar units that are generally found in hemicellulose are pentoses (D-xylose,
L-arabinose), hexoses (D-galactose, L-galactose, D-mannose, L-rhamnose, L-fucose),
and uronic acids (D-glucuronic acid) (Glazer and Nikaido 2007). Hemicellulose
generally comprises 15–35 % of plant biomass. Xylan and glucomannan are
amongst the most relevant hemicellulose units. Xylan is a major structural hetero-
polysaccharide in plant cells composed of homopolymeric backbone chains of 1,4
linked B-D-Xylopyranose units (Koukiekolo et al. 2005). An enzyme or a group of
enzymes that hydrolyzes the Xylan and glucomannan units to simple sugars are
called hemicellulases. Xylan is found at the interface between lignin and cellulose
and is believed to be accountable for fiber cohesion and overall plant cell-wall sta-
bility (Collins et al. 2005). More complex structures of hemicellulose require syn-
ergistic action of various types of hemicellulases for efficient degradation. Xylanases
like cellulases can also be divided into endo-acting xylanases (E.C.3.2.1.8 available
in GH5,8,10,11,43), exo-acting xylanases (E.C.3.2.1.156) found in GH8 acting
from the reducing end, and often complemented with xylosidases (E.C.3.1.2.37),
for example, GHI 3,39,52,54,116,120 acting from non-reducing ends. Use of other
hemicellulosic degrading enzymes is classified under glycoside hydrolase and car-
bohydrate esterase (CE) families for degrading cellulose and hemicellulose moi-
eties. For instance, mannanases (E.C.3.2.1.78) catalyze different mannan-containing
hemicelluloses and classified under the category (GH 26,113), Mannosidases
(E.C.3.2.1.25) (GHI2,5), galactosidases (E.C.3.2.1.23) (GH2,3,35,42), and others
including Arabinofuranosidases (E.C.3.2.1.55) (GH3,43,51,54,62) together with
other enzymes degrade hemicelluloses to monomeric sugars (Table 22.2, Fig. 22.1).
Fungal xylanases are generally active at mesophilic temperature (40–60 °C) with a
Table 22.2 Glyoside Enzyme Enzyme families

hydrolase and carbohydrate
Endoxylanase GH-5,8,10, 11,43
esterase family for cellulosic
degradation Beta-xylosidase GH-3,39,43,52,54
Alpha-L-arabinofuranosidase GH-3,43,41,54,62
Alpha glucuronidase GH-4,67
Alplia-galactosidase GH-4,36
Acetyl xylan esterase CE 1,2,34,5.6,7
Feruloyl esterase CE1
Fig. 22.1 Composition of

lignocellulosic biomass
Lignin
Hemicellulose (phenolics)
(xylose)
26%
30%
Cellulose
(glucose)
44%
slightly acidic pH; however, xylanases have also been reported to be active in
extreme environments. Psychrophilic fungi, such as Penicillium sp., Alternaria
alternata, and Phoma sp., have been isolated from the Antarctic environment.
Endo-1, 4-β-xylanase (1, 4-β-D-xylan xylanohydrolase; EC 3.2.1.8) hydrolyzes
the glycosidic bonds in the xylan backbone to reduce its degree of polymerization
and thus increasing accessibility of cellulose to enzymatic hydrolysis releasing
xylo-oligomers which can further produce other small sugars (Polizeli et al. 2005).
Exo-1, 4-β-D-xylosidase (1,4-β-D-xylan xylohydrolase; EC 3.2.1.37) hydrolyzes
xylobiose and short-chain xylooligosaccharides generated by the action of endox-
ylanases, releasing D-xylose residues from the non-reducing end. β-xylosidases
hydrolyze only xylobiose and their affinity for xylooligosaccharides decreases with
the increasing degree of polymerization. Xylose-utilizing species including Candida
sp., Geotrichum sp., Sporopachydermis sp., Trichosporon sp., Pichia sp., and
Sugiyamaella sp. have been isolated from buffalo feces (Wanlapa et al. 2013) or
from soil (Zhang et al. 2014). Other enzymes involved in hemicellulosic degrada-
tion includes α-L-arabinofuranosidases (EC 3.2.1.55) hydrolyzing the terminal
arabinose residues from the side chains of xylan and other arabinose containing
polysaccharides (Saha 2003). α-D-glucuronidases (EC 3.2.1.139) hydrolyze the
α-1,2 linkages between the 4-O-methylglucuronic/D-glucuronic acid and xylose
residues in glucuronoxylan. The hydrolysis of the stable α-(1, 2)-linkage is the bot-
tleneck in the enzymatic hydrolysis of xylan. Acetyl xylan esterase (EC 3.1.1.6)
removes the O-acetyl groups from acetylated xylan. This enzyme plays an impor-
tant role in the hydrolysis of xylan, since the acetyl groups can interfere in the action
of enzymes that cleave the xylan backbone, and so their removal facilitates the
action of xylanases (Polizeli et al. 2005).
Lignin-Modifying Enzymes (LMEs)
Lignin is the second most abundant constituent of the plant cell wall, where it pro-
tects cellulose against hydrolytic attack by saprobic and pathogenic microbes.
Lignin degradation plays a major role in carbon recycling in the ecosystem as well
as converting plant biomass for second-generation biofuel (ethanol) production.
The fungal lignin degradation process is oxidative and nonspecific, which decreases
methoxy, phenoxy, and aliphatic content of lignin, cleaves aromatic rings, and
forms new carbonyl groups by the action of enzymes, namely, laccase, lignin per-
oxidase, and manganese peroxidase, etc. These changes in the lignin molecule
result in depolymerization and carbon dioxide production (Timothy et al. 2011).
White rot fungi are reported to be efficient in secreting LMEs with some examples
like Auricularia polytricha, Flammulina velutipes, Ganoderma lucidum, G. applana-
tum, G. australe, G. capense, G. carnosum, G. fornicatum, G. gibbasum, G. resina-
ceum, G. stipitatum, G. trabeum, Irpex lacteus, Phanerochaete chrysosporium,
Pleurotus sajor-caju, Pleurotus ostreatus, Pleurotus dryinus, Pleurotus tuberregium,
Lentinula edodes, Trametes hirsuta, and Trametes versicolor (syn. Coriolus versicolor)
(Kannan et al. 1990; Fang et al. 1997; Elissetche et al. 2006; Arboleda et al. 2008;
Elisashvili et al. 2008, 2009; Dinis et al. 2009; Erden et al. 2009; Asgher et al. 2012;
Pinto et al. 2012; Manavalan et al. 2013; Salvachua et al. 2013; Shevchenko et al. 2013).
Lignin Peroxidases (LiPs)
Lignin peroxidases are heme containing glycoproteins also known as Heme peroxi-
dases that catalyze the H2O2-dependent oxidative depolymerization of a vast variety
of non-phenolic lignin compounds (Wong 2009). LiPs oxidize the substrates in
multistep electron transfers and have high reduction potential in comparison to
other heme peroxidases and also do not require any chemical mediator for its reac-
tion. Among fungal genera mostly White Rot Fungi are the major producer of
Lignin peroxidases or ligninases. Phanerochaete chrysosporium (Pointing et al.
2005) and Trametes versicolor (syn. Coriolus versicolor) are major fungal lignin
degraders. P. chrysosporium is a potent source of extracellular ligninases with some
peroxide generating enzyme glyoxal oxidase (GLOX) along with other ligninases.
Manganese Peroxidases (MnPs)
MnPs are extracellular glycoproteins and also classified with Heme Peroxidases,
secreted in multiple isoforms, and contain one molecule of Heme as Protoporphyrin
(IX). MnP catalyzes the peroxide-dependent oxidation of Mn (II) (as the reducing
substrate) to Mn (III) released from the enzyme surface by forming complexes with
oxalate or other metal chelators. This chelated Mn (III) complex then acts as a reac-
tive low-molecular-weight, diffusible redox mediator of phenolic substrates
including simple phenols, amines, dyes, and phenolic lignin substructures. Fungal
genera that secrete MnPs include P. chrysosporium, Panus tigrinus (Lisov et al.
2003), Lenzites betulina (also reported as Lenzites betulinus) (Hoshino et al. 2002),
Phanerochaete flavido-alba (de la Rubia et al. 2002), Agaricus bisporus (Lankinen
et al. 2001), and Bjerkandera sp. (Palma et al. 2000).
Versatile Peroxidases (VPs)
Versatile Peroxidases are glycoproteins with hybrid properties capable of oxidizing

substrates of other peroxidases like LiPs and MnPs. VPs form an attractive lignino-
lytic enzyme group due to their dual oxidative ability to oxidize Mn (II) and other
phenolic and non-phenolic aromatic compounds. VPs can oxidize substrates of both
high and low redox potentials. The major fungal genera secreting VPs include
Pleurotus eryngii, P. ostreatus (Cohen et al. 2001), Bjerkandera adusta (Wang et al.
2003), Bjerkandera fumosa, and Pleurotus pulmonarius; basidiomycetous fungal
genera secreting VPs include P. tigrinus (Lisov et al. 2007). VPs have a wide variety
of biotechnological applications due to their catalytic versatility in catalyzing those
reactions where other peroxidases fail to catalyze the same.
Phenol Oxidases (Laccases)
Laccases are glycosylated blue multicopper oxidoreductases using molecular oxy-

gen to oxidize various aromatic and non-aromatic compounds by coupling the elec-
tron reduction of dioxygen into water with the simultaneous oxidation of a vast
variety of substrates, such as phenols, arylamines, anilines, thiols, and lignins.
Laccases are produced and secreted by a wide range of fungal genera including
white rot fungi Lentinus tigrinus (Farroni et al. 2007), T. versicolor (Necochea et al.
2005), and Cyathus bulleri (Mishra and Bisaria 2006). Several brown rot fungi are
also potent producers of laccases including Coniophora puteana. Many ascomyce-
tous genera are also reported as potent producer of laccase Melanocarpus albomy-
ces (Hakulinen et al. 2006), Chaetomium thermophile (Ishigami and Yamada 1986),
Magnaporthe grisea (Iyer and Chattoo 2003), Myrothecium verrucaria 24G-4
(Sulistyaningdyah et al. 2004), and Neurospora crassa (Schilling et al. 1992).
Potential Strains of Oleaginous Microfungi Employed

in Biofuel Production
An emerging potential alternative for biodiesel production is represented by micro-

bial lipids single-cell oils (SCOs), which oleaginous microorganisms can accumulate
up to 70 % or more of their biomass of their dry weight. Some of the oleaginous spe-
cies show the ability to metabolize pentoses, demonstrating the potential to produce
triacylglycerol (TAG) from lignocellulosic biomass. Microbial TAGs may be a

prospective alternative feedstock and proven to be a pivotal for a sustainable bio-
diesel industry. Nowadays, the possibility to track lipid production in real time can be
achieved by Fourier transform infrared spectroscopy, which is essential for a viable
and successful development of an industrial production process (Ami et al. 2014).
Important oleaginous fungi employed for biodiesel production are as follows:
Rhodosporidium toruloides
The red yeast Rhodosporidium toruloides is an oleaginous mesophilic species of

class Ustilaginomycetes which can accumulate lipids to above 70 % of its dry cell
weight from a wide variety of carbon sources and can transform carbohydrates from
lignocellulosic hydrolysate into long-chain fatty acids that contribute to biodiesel
production. This species is able to carry out diverse biochemical reactions such as
biodegradation of epoxides, biphenyls, and oxiranes, biosynthesis of carotenoids,
and other types of biotransformations (Yu et al. 2010). Yang et al. (2014) studied the
two-stage process of Rhodosporidium toruloides Y4 for the conversion of crude
glycerol into lipid. This yeast strain is capable of accumulating lipids up to 76 % of
cell dry weight and provides an attractive route to integrate biodiesel production
with microbial lipid technology for better resource utilization efficiency and eco-
nomical viability (Fig. 22.1).
Novel biochemical approaches remain to be developed to improve microbial
lipid technology (Yang et al. 2014). Sulfate limitation technology was found to be
effective to promote accumulating substantial amounts of intracellular lipid in
Rhodosporidium toruloides Y4. The sulfate-limitation approach to control lipid bio-
synthesis should be valuable to explore nitrogen-rich raw materials as the feedstock
for lipid production (Wu et al. 2010). Gas chromatography analysis revealed that
lipids from R. toruloides Y4 contained mainly long-chain fatty acids, such as oleic
acid, palmitic acid, stearic acid, and linoleic acid, and act as a potential alternative
oil resource for biodiesel production (Li et al. 2007). On the other hand, Kumar
et al. (Kumar et al. 2014) reported the 20.05-Mb draft genome of the red yeast
Rhodosporidium toruloides MTCC 457, predicted to encode 5993 proteins, 4
rRNAs, and 125 tRNAs and found to be valuable for molecular genetic analysis and
manipulation of lipid accumulation. Fourier Transform Infrared Spectroscopy
(FTIR) can be proposed as a powerful tool for the development of a viable biodiesel
production in oleaginous yeasts, such as Cryptococcus curvatus and Rhodosporidium
toruloides, and of non-oleaginous yeast Saccharomyces cerevisiae.
Yarrowia lipolytica
Yarrowia lipolytica is tropical marine yeast and can undergo extensive modifica-
tions for converting a wide range of hydrophobic and hydrophilic biomass to fatty
acid-based products and enhance its potential in the sustainable production of
biodiesel, functional dietary lipid compounds, and other value-added oleochemical

compounds. Y. lipolytica could be used as a potential feedstock for biodiesel pro-
duction when grown on glucose and inexpensive wastes (Katre et al. 2012). All of
these features make Y. lipolytica suitable in industrial applications of lipid-modifying
enzymes and highlight its potential for use in sustainable production of second-
generation biofuels. Simple and renewable substrates, such as molasses,
N-acetylglucosamine, sewage sludge, palm oil mill, olive oil mill wastewater, whey,
municipal wastewater, industrial fats, etc., have been demonstrated to increase the
economy of the bioconversion process by Y. lipolytica (Liang and Jiang 2013). It is
also known to convert glycerol to SCO and hence can play dual roles in the upstream
and downstream of the biodiesel industry. The nitrogen-limited cultures of Y. lipo-
lytica demonstrated increased production of organic acids with enhanced accumula-
tion of lipids (Papanikolaou et al. 2002). Use of genetic engineering under a
nitrogen-limiting condition has proved efficient in augmenting the biomass and cel-
lulosic lipid yields following the co-fermentation strategies (Sestric et al. 2014).
Two-stage fed-batch system for conversion of volatile FAs to the SCO has been
proved useful to obtain high biomass using Y. lipolytica (Cherry and Fidantsef
2003). Abghari and Chen (2014) demonstrated the use of Y. lipolytica to produce
nanoproducts and lipid-based bioproducts that provide an effective tool for bridging
between biotechnology and nanotechnology through development of novel biocata-
lysts and nano-oils (Fig. 22.2)
Cryptococcus Species
The oleaginous yeast Cryptococcus curvatus (Diddens & Lodder) Golubev is a

promising feedstock for biofuel production. These species are used to hydrolyze
biomass that has been pretreated using dilute acid and produce lipids that may be
used as feedstock for producing biofuels (Yu et al. 2010). In Cryptococcus curvatus
volatile fatty acids (VFA) from dark fermentation hydrogen production were tested
as carbon sources for the culture of oleaginous yeast, which is a promising feed-
stock for biofuel production. On the other hand, Cryptococcus laurentii 11 biomass
when supplemented with sugarcane molasses indicated a predominant (86 %) pres-
ence of neutral lipids with high content of 16- and 18-carbon-chain saturated and
monosaturated fatty acids and thus can be considered suitable for the production of
biodiesel (Castanha et al. 2014). High cell density fed-batch cultivation on low-cost
Fig. 22.2 Microbial lipid production based on conventional (path A) and two-stage (path B) pro-
cess (Yang et al. 2014)
substrate, viz. crude glycerol, indicated a high oleic acid content followed by pal-
mitic acid, stearic acid, and linoleic acid, and the oil was transesterified to biodiesel
(Thiru et al. 2011). Lipid from C. curvatus was found to be a quality-sufficient
source of oil as a transportation fuel in terms of cetane, iodine values, and oxidation
stability (Ryu et al. 2013). Dairy wastes were also used as substrates and achieved
production of high concentrations of sophorolipids using a two-stage cultivation
process for the yeast Cryptococcus curvatus ATCC 20509 (Johny 2013). C. curva-
tus lignocellulosic materials, such as corn fiber and sweet sorghum, are prepro-
cessed and can be used to produce liquid biofuels (Liang et al. 2014). C. terricola
used for fuel production through consolidated bioprocessing produced high propor-
tions of C16:0 and C18 fatty acids when grown on starch and proved to be a promis-
ing alternative source for biodiesel production (Tanimura et al. 2014).
Cunninghamella echinulata and Mortierella isabellina
These strains are considered potential producers of single-cell oil (SCO) containing
γ-linolenic acid, a polyunsaturated fatty acid. Mortierella isabellina (current name:
Umbelopsis isabellina (Oudem.) W. Gams) can be considered as a promising pro-
ducer of SCO that can be subsequently converted into second-generation biodiesel
(Chatzifragko et al. 2011). The growth of Cunninghamella echinulata on various
nitrogen containing raw materials (corn gluten, corn steep, whey, yeast extract, and
tomato waste hydrolysate) yielded various quantities of γ-linolenic acid (GLA)-rich
cellular lipids. Growth on tomato waste hydrolysate yielded 17.6 g/L of biomass
containing 39.6 % oil and 800 mg/L GLA. On xylose containing media M. isabel-
lina accumulated 65.5 % and C. echinulata 57.7 % of lipid and produced 6.7gL−1 of
single-cell oil and 1119 mgL−1 of γ-linolenic acid. On the side, M. isabellina pro-
duced GLA-rich SCO from pear pomace, an agro-industrial waste accumulating in
large amounts in several Mediterranean countries (Fakas et al. 2009). C. echinulata
when grown on tomato waste hydrolysate medium rapidly, took up glucose and
produced large amounts of lipids that contain GLA-rich triacylglycerols (TAG) and
hence may be of commercial interest. Actually, C. echinulata has been successfully
used by several researchers for GLA production (Papanikolaou et al. 2008), but
cultivation of this mold on media containing organic nitrogen has been rarely
reported. Certik and Shimizu (1999) compared the effects of various organic and
inorganic nitrogen sources on lipogenesis in a strain of C. echinulata and found that
the use of organic nitrogen increased lipid accumulation.
Aspergillus spp. and Penicillium spp.
Penicillium spp. and Aspergillus spp. strains have been revealed as appropriate
lipase producers, but the studies dealing with the production of microbial lipids by
Penicillium spp. and Aspergillus spp. are insufficient. Studies dealing with the
production of fat by Aspergillus nidulans, Aspergillus flavus, Aspergillus fumigatus,

Aspergillus sydowii, or Aspergillus niger have been reported (Hui et al. 2010).
Waste cooking olive oil was an adequate substrate for the growth of Aspergillus sp.
and Penicillium expansum producing remarkable quantities of lipid-rich biomass
(Papanikolaou et al. 2011). Studies were done on the use of potato processing
wastewater for microbial lipid production by Aspergillus oryzae with the purpose of
recycling potato processing wastewater for biodiesel production (Muniraja et al.
2011). In Aspergillus oryzae whole-cell biocatalyst, coexpression of Fusarium het-
erosporum lipase (FHL) and mono- and di-acylglycerol lipase B (mdlB) has been
developed to improve biodiesel production. For enzymatic biodiesel production
from plant oil hydrolysates, an Aspergillus oryzae whole-cell biocatalyst expresses
the Candida antarctica lipase B (r-CALB) with high esterification activity which
resulted in the biodiesel production (Adachi et al. 2013). In Aspergillus oryzae,
highly efficient biodiesel production was achieved by using the lipase activity of the
whole-cell biocatalyst. In Aspergillus awamori, Taguchi orthogonal array (OA) is
useful for optimizing the number of functional factors at a time which were involved
in the lipid production and the pH individually showed significant influence on the
lipid synthesis (Prakasham et al. 2007). Aspergillus awamori enhanced the lipid
production by 31 % and the lipids with fatty acid ester linkages and free fatty acids
can produce fatty acid methyl esters (FAME) after transesterification that can be
used as biodiesel. Also, the mutant strain of A. niger NMG12/4 showed maximum
lipase activity of 15.5 U cm−3 at 96 h, which is prospective for the development of
industrial biotechnology for production of extracellular lipase (Toscano et al. 2015).
In Penicillium decumbens, production of cellulases effectively degrades the ligno-
cellulose for the second-generation biofuel production (Liu et al. 2013).
Rhodotorula glutinis
Rhodotorula glutinis is an oleaginous yeast that produces copious quantities of lip-

ids in the form of triacylglycerols (TAG) and can be used to make biodiesel via a
transesterification process. The ester bonds in the TAG are broken leaving behind
two products, i.e., fatty acid methyl esters and glycerol, which provide an inexpen-
sive carbon source (Easterling et al. 2009). In R. glutinis, fatty acids are activated in
an ATP-dependent manner and an enzyme acyl–acyl carrier protein (ACP) plays a
role in activating fatty acids for triacylglycerol biosynthesis. There is plenty of evi-
dence to suggest that this organism has the potential to be a source of fatty acids for
the production of biodiesel. R. glutinis have the ability to grow and accumulate
lipids when grown on glycerol. Over 70 % of biodiesel production costs are due to
the expense of feedstocks such as soybean and rapeseed oil. Also, the lipid produc-
tion potential of the red yeast Rhodotorula glutinis grown on non-detoxified hydro-
lysates from wheat straw and miscanthus as carbon sources showed the lipid
composition of C 16:0, C 18:1, and C 18:2 indicating a good level of suitability for
biodiesel production (Mast et al. 2014).
Trichoderma reesei
The enzyme producer T. reesei stands out among industrially applied microorgan-
isms because it can degrade cellulose at the rates sufficient for industrial use and a
wide range of mutants have been developed for T. reesei. T. reesei serves today as a
model organism for the regulation and biochemistry of (hemi) cellulose degradation
(Kubicek et al. 2009). The microfungus Trichoderma reesei is a well-known pro-
ducer of lignocellulolytic enzymes that are used for depolymerization of plant lig-
nocellulosic biomass (Martinez et al. 2008). T. reesei is already the main industrial
source for cellulases and hemicellulases, but despite the fact that industrial strains
produce more than 100 g/l of cellulases (Cherry and Fidantsef 2003), efforts are
needed to reduce costs and maximize yield and efficiency of the produced enzyme
mixtures. Furthermore, T. reesei has the ability to utilize all the lignocellulose sug-
ars for producing ethanol (Xu et al. 2009). Huang et al. (2014) demonstrated direct
ethanol production from lignocellulosic sugars and sugarcane bagasse by a recom-
binant Trichoderma reesei strain HJ48. In T. reesei, cost-effective lignocellulolytic
enzyme is produced when supplemented on a cane molasses medium (He et al.
2014). On the other side, Javanovic et al. (Jovanović et al. 2014) focused on the
potential of producing erythritol in T. reesei from lignocellulosic biomass. As such
a strong producer of cellulases and hemicellulases revealed for T. reesei, 10 cellu-
loytic and 16 xylanolytic enzyme-encoding genes, it is likely that T. reesei is able to
grow on cheap biowaste material like wheat straw as the sole carbon source
(Dashtban et al. 2013). The high lipid containing biomass in T. reesei can be used to
extract oil and the contents can be termed as bio-oil (or biodiesel or myco-diesel
after transesterification). The resulting bio-oil production from wastewater treat-
ment by T. reesei reactors was found to be 74.1 mg/L, whereas biomass containing
bio-oil contents (%w/w) was 9.82 % in 96 h. (Bhanja et al. 2014). This study sug-
gests that wastewater can be used as a potential feedstock for bio-oil production
with the use of oleaginous fungal strains and which could be a possible route of
waste to energy.
Novel Technologies to Meet the Challenges of Biofuel

Research
Metabolic Engineering
Metabolic engineering is a novel approach that has developed microbial cell facto-
ries for converting renewable carbon sources into biofuels, widely useful for indus-
trial application (Jang et al. 2012). Major use of metabolic engineering in biofuel
synthesis is to engineer obese microbes for overproduction of fat and oils, improv-
ing tolerance of microbes to biofuels (ethanol) and lastly high-throughput screens
for various extracellular compounds. Improving tolerance of microbes to biofuel
generated and enhancing robust growth and production under adverse industrial
conditions are still one of the major challenges for metabolic engineering, as adverse
and hostile conditions may elicit multigenic responses coordinated at the transcrip-
tomics and proteomics level. Microbial metabolism is quite complex and the bio-
synthetic pathway to produce biofuels requires a complex array of multiple
enzymatic reactions. Current molecular biology techniques can effectively alter
enzyme levels to increase the flux toward biofuel synthesis. Metabolic engineering
allows fine-tuning and modulation of both expression level and target activity of
proteins/enzymes with the help of some traditional techniques that enable engineer-
ing and de novo synthesis of promoters, ribosome binding sites, and entire coding
regions or by regulating the choice of plasmids and their copy numbers, promoter
engineering, codon optimization, synthetic scaffolds, directed evolution or modifi-
cation of key enzymes, and knockout/knockdown of competitive pathways
(Nowroozi et al. 2014). Some new tools that enable genome engineering and
system-wide identification of genes that confer target traits are very useful. These
emerging techniques include multiscale analysis of library enrichment (SCALEs)
that provide quantification of the effects of expression of specific genes, trackable
multiplex engineering (TRMR) (Warner et al. 2010), coexisting/coexpressing
genomic libraries (CoGeL) (Nicolaou et al. 2011), genome-scale analysis of library
sorting (GALibSo) (Stadlmayr et al. 2010), multiplex automated genome engineer-
ing (MAGE) (Wang and Chen 2009), conjugative assembly genome engineering
(CAGE) (Isaacs et al. 2011), and global transcription machinery engineering
(gTME) (Alper et al. 2006). New genetic techniques, such as RNA Interference,
CRISPRs, or TALENs, offer new capabilities to edit microbial metabolisms (Sun
and Zhao 2013).
Ethanol fermentation by yeast is the most developed biofuel process, but low
combustion energy and high purification costs prevent the wide use of ethanol as an
economical fuel. Therefore, researchers have engineered microbes to produce new
fuels. Advanced biofuel examples include higher alcohols via the keto-acid and the
Ehrlich pathway, terpene-based fuels (e.g., isopentenol) from the mevalonate path-
way, and fatty acid ethyl esters and alkanes from fatty acid biosynthesis pathways.
Despite the development of these diverse biofuel producers, it is still challenging to
commercialize biofuel processes due to the poor microbial productivity in large
bioreactors and the low profit margins of biofuels (Lamonica 2014).
Global Transcription Machinery Engineering (gTME)
Global transcription machinery engineering (gTME) is an approach that alters the

essential proteins regulating the transcriptomes and introduces transcriptional-level
modifications that are transferable between strains. Mutagenesis of the basal tran-
scription factors leads into global reprogramming of transcription of genes and
results into new phenotypes which can be isolated by various screening methods.
Since gTME is independent of preliminary information like enzymes and their
kinetic parameters, biochemical routes, and pathways, multigenic or polygenic
traits such as tolerance toward different types of stresses can also be addressed. The
genetic change that causes the optimal global reprogramming can then be trans-
ferred to other strains (Alper and Stephanopoulos 2007). In contrast to other evolu-
tionary engineering approaches where random mutations accumulate in the entire
genome, the gTME approach allows for genotype–phenotype correlations traceable
to a single mutant protein. Due to the lack of regulation of the transcription factor,
the gTME strategy is able to change the metabolic strength and direction. gTME has
been shown as an efficient solution to improve substrate utilization, product toler-
ance, and production in yeast (Çakar et al. 2012). gTME enables the creation and
isolation of polygenic mutants under several conditions, thus facilitating pheno-
types that would be difficult to obtain with conventional gene modification tech-
niques. gTME approach has been successful in improving the tolerance of yeasts to
high concentration of sugar and ethanol (Tyo et al. 2007).
Enzyme Engineering
One of the major limitations of manufacturing biofuels from plant biomass is the
presence of highly refractive lignocellulosic components causing major technical
hurdles in the biomass saccharification and hence production of biofuels. Cellulases
and hemicellulases used in biomass saccharification have optimum temperature and
pH range with low hydrolytic efficiency. Biomass saccharification at high tempera-
tures decreases their activity and catalytic efficiency. For complete and efficient
bioconversion of lignocellulosic biomass to biofuels, there is need to develop an
ideal cellulase system that must be highly active on desired biomass feedstock,
completely hydrolyze biomass, must be active at mildly acidic pH, withstand pro-
cess stress, and most importantly to be cost-effective. Another approach has been to
prepare “Enzymatic cocktail.” Enzyme cocktails have been developed by mixing T.
reesei cellulases with other enzymes like pectinases, xylanases, and BGLs, and
these cocktails were employed to hydrolyze various feedstocks (Berlin et al. 2007).
Biomass degradation enzymes from thermophilic fungi demonstrate higher
hydrolytic capacity despite the fact that extracellular enzyme titres are typically
lower than more conventionally used species (Berka et al. 2011). Several approaches
have been pursued, including directed evolution using error-prone Polymerase
Chain Reaction-based mutagenesis of cellulase genes, adaptive evolution using
natural selection to specific environmental conditions, or rational protein design to
improve the enzymatic activity of cellulases or to expand the physiological condi-
tions at which the enzymes are active. “Rational Design” approach of engineering
cellulases involves site-directed mutagenesis of conserved residues (identified by
sequence comparison between homologues from different species). Site-directed
mutagenesis of non-active site residues to amide carboxylate pairs enhanced cata-
lytic activity and increased pH susceptibility. Similarly in Directed evolution
approach catalytic efficiency of cellulases is improved by random mutations along
the length of given DNA sequences. “DNA shuffling” approach selects DNA
sequences that are randomly fragmented using DNaseI and then recombined by
annealing and extension of DNA strands using self-primed PCR, followed by selec-
tion of clones with desired properties from a library of recombined fragments
(Patten et al. 1997).
System-Level Approaches and Biofuel Production
Computational tools like genomics, transcriptomics, proteomics, glycomics and lig-

nomics, and fluxomics can be helpful in understanding core system biology. The
system-level approach to understanding complex metabolic and regulatory net-
works enables the design of more efficient microorganisms for the production of
valuable molecules, including biofuels. Sequencing industrialized strains and then
comparing it with their progenitors may give vital information in designing new
microbial systems that increase bioconversion efficiency and lower biofuel cost.
Significant progress in the development of functional genomic tools in recent years
has powered the elucidation of complex phenotypes and the engineering of new
ones for the development of promising industrial strains (Zhang et al. 2006).
Transcriptomics focuses on measuring expression of RNA under specified condi-
tions and determines the full and coordinated set of molecular response which helps
to elucidate the regulatory network and evaluate the models of cellular response.
The use of transcriptional profiling tools, such as DNA microarrays, allows the
study of gene expression by identifying differentially expressed genes under differ-
ent experimental conditions or resulting from certain genetic perturbations.
Currently, microarray technology is widely employed for analyzing the gene
expression profiles from heterogeneous system critical to biomass conversion. A
proteomic study reveals the identification and quantification of protein complexes
present in both plant and microbial systems. Proteomics can be used to explore a
microbe’s protein-expression profile under various environmental conditions as the
basis for identifying protein function and understanding the complex network of
processes facilitated by multiprotein molecular machines (Delaunois et al. 2014).
Metabolomics studies target fuel production, including methods to isolate,
extract, and analyze labile metabolites such as those involved in cellular energy
metabolism (e.g., ATP, GTP, NADP, and NADPH). Metabolomics makes use of
high-throughput analyses like Nuclear Magnetic Resonance (NMR) and Gas or
Liquid Chromatography coupled to Mass Spectrometry (GC-MS and LC-MS) for
the quantification of intra- and extracellular small-molecular-weight metabolites.
Metabolic studies also serve as a screening tool to investigate the unknown effect of
a specific genetic modification on the phenotype of the organism, and as a means to
describe metabolism kinetics by performing pulse experiments (Buchholz et al.
2002). Glycomics (profiling materials related to structural polysaccharides) and lig-
nomics (profiling materials related to lignin) have essential capabilities and infor-
mation which will help us understand native and modified pathways for the synthesis
of cell-wall polymers by tracking precursor consumption, generating and utilizing
intermediate structures, and exploring their connection to plant cell-wall chemical
composition and physical-chemical structure and also elucidate substrate modification
by tracking structural changes and concentration fluxes in saccharification products

that may be linked to harsh pretreatments. Fluxomics studies analyze the fluxes of
diverse substrates through complex networks of metabolic pathways. Quantifying
metabolic fluxes in microorganisms allows identification of rate-limiting steps in a
biosynthetic pathway that could be improved by genetic manipulation or by altera-
tions in cultivation conditions (Tang et al. 2009).
Genomics and Metagenomics
Next generation DNA sequencing approach has explored the widespread possibili-
ties of degradation of lignocellulolytic biomass by comprehensive analysis of their
genomes, transcriptomes, proteomes, and interactomes. This technique is nowadays
used to determine the whole-genome sequence related to lignocellulosic biomass
bioconversion. For example, the genome of white rot fungus P. chrysosporium and
brown rot fungus Postia placenta has been sequenced to explore the presence of a
wide array of genes related to biomass degradation and conversion. Genome
sequencing tools have revealed that T. reesei (syn. Hypocrea jecorina) is a powerful
degrader of agricultural crop residues encoding fewer plant cell-wall polysaccha-
ride degrading cellulases and hemicellulases than any other sequenced fungus
(P. chrysosporium and brown rot fungus Postia placenta) (Martinez et al. 2009).
Metagenomics provides a culture-independent genome analysis of entire micro-
bial communities of a particular environmental niche. Metagenomic studies for lig-
nocellulosic biomass degradation can explore the potential and novel bioagents for
effective hemicellulose and lignin conversion to improve biomass utilization for
cost-effective biofuel production from communal or unculturable populations and
also play a key role in sequencing new genomes harboring cellulolytic components
acquired by lateral gene transfer (Fig. 22.3). The outcomes of these functional and
comparative studies will include a repertoire of new enzymes and proteins available
for engineering approaches (e.g., designer cellulosomes or free cellulase systems).
Biomass conversion to sugars and biofuels requires optimizing microbial break-
down of structural sugars and fermentation of complex sugar mixtures. A number
of microbial communities have evolved over millions of years to maximize and
coordinate these capabilities, with several of the better-studied ones associated with
ruminants and the hindgut of termites, and hence a reasonable number of model
fermentative communities that can degrade lignocelluloses are critical targets for
metagenomic analysis (Mhuatong et al. 2015; DeAngelis et al. 2010).
Biotechnology in Fungal Biofuel Production
One of the major barriers in the efficient use of biomass-derived sugars is the lack of
microbial biocatalysts that can grow and function optimally in highly stressed and
harsh environments created by both biomass hydrolysis and cellular metabolism.
Fig. 22.3 Schematic representation of the fatty acid-derived bioproducts production through
engineered lipid pathway using Y. lipolytica cell factory (Abghari and Chen 2014)
Environmental Samples
Traditional Screening of Metagenomics Metatranscriptomics

mirobes
Extraction of Metagenomic
Enrichment culture to DNA Extraction of Extraction of
induce protein expression RNA proteins
Construction of metagenomic
library Analysis of Analysis of peptide
RNA(mRNA) MS/MS
Function driven Sequence driven
mRNA Isolation.cDNA
analysis analysis cDNA library and sequence
synthesis cloning in
suitable systems and assembly using next generation
making cDNA library sequencing(NGS)
Substrate specific Genome sequence
Screening analysis
Gene transcript Protein hits

Gene Identification Gene Identification
hits
using bioinformatics
Gene identification identification of

using probes Gene Synthesis/Gene cloning and gene
expression studies in heterologous systems Products
Fig. 22.4 Metagenomic approaches for discovery of novel enzymes (modified from Sebastian
et al. 2013)
Genetic engineering plays a key role in the transformation of microbes into the
desired cell factories with high efficiency of bioenergy generation. Genetic engi-
neering has improved the microbial biocatalysts to enhance the extent of biomass
saccharification. A wide variety of examples are available for gene transfer tech-
nologies for the expression of genes from different microbes to increase the catalytic
properties of Glycosyl hydrolases. One of the most current usages of gene transfer
technology is the development of ideal microbes for consolidated bioprocessing
(CBP). CBP has been shown to offer large cost benefits relative to other process
configurations in both near-term and futuristic contexts (Lynd et al. 2005). Selection
of suitable and efficient cellulolytic enzymes is a key factor in the process of engi-
neering non-cellulolytic organisms with high product yields. CBP where the ligno-
cellulosic biomass utilization and product formation properties are sequestered in
one single microorganism is widely considered an ultimate low-cost configuration
for cellulose hydrolysis and fermentation. One major disadvantage using mycelial
fungi for ethanol production is their slow bioconversion rate compared to yeasts and
high tolerance to bioethanol and currently we do not have any natural microbe avail-
able for CBP at a desired efficiency for industrial bioethanol production. The main
two strategies can be employed to develop CBP organisms: (1) engineering natural
cellulolytic microorganisms to improve product-related properties, i.e., so-called
native host strategy, which engineers organisms with their native ability to use cel-
lulose or pentose sugars to improve product-related properties, and (2) engineering
non-cellulolytic organisms by the use of gene transfer technology to develop recom-
binant microbes. Ethanol yields in A. niger can be increased by expression of a
pyruvate decarboxylase gene from Zymomonas mobilis. However, the major focus
for producing CBP microbe is the heterologous expression of the cellulase gene in
natural ethanologens and the most commonly used host for heterologous expression
of cellulase genes for CBP is the baker’s yeast, S. cerevisiae (Nakatani et al. 2013).
All three classes of cellulase genes have been expressed in S. pastorianus (Fitzpatrick
et al. 2014). Similarly, the key point of CBP for biodiesel production is the engineer-
ing of a microorganism that can efficiently depolymerize biomass polysaccharides
to fermentable sugars and efficiently convert this mixed-sugar hydrolysate into
FAEEs (Lin et al. 2013). Another approach of genetic engineering for enhancing
cellulase activity is to increase the gene copy number. Episomal plasmids have been
extensively used to express cellulase genes. Genetic engineering also offers to
develop chimeric cellulosomes for efficient degradation of biomass substrate either
by incorporating bacterial or fungal cellulases.
Biomass conversion to bioenergy and biofuels requires an array of enzymatic
system. The catalytic efficiency and efficacy of enzymes decrease with reaction
time. The nanotechnology approach in biofuel production mainly focuses on the
development and application of reusable nanocatalysts in bioconversions of bio-
mass to biofuels. Designing nanocatalysts for biomass degradation and biofuel gen-
eration is one of the most important fast-growing and unexplored research areas of
Nanotechnology. The approach utilizes the assessment of the activity of designed
hydrolytic nanocatalyst by adsorbing them or ligating them through chemical means
on carbon nanotubes (CNT). Optimization of such chemical ligation will be very
important because it would offer the advantage of performing repeated uses of the
same CNT–enzyme conjugate after their isolation from processed biomasses
(Wabeke et al. 2014; Neto and De Andrade 2013).
Challenges in Fungal Biofuel Production
Microbial biofuels are a good alternative to petroleum-based fuels. They offer sev-
eral benefits to society and the environment. Countries in the world have set their
own targets to replace petroleum fuel by biofuels. Today, the contribution of enzyme
costs to the economics of lignocellulosic biofuel production continues to be a much-
debated topic. Some authors argue that the cost of enzymes is a major barrier for
biofuel production (Brijwani et al. 2010), while others assume that it is not, as it will
decrease with technological innovation or other advances (Aden and Foust 2009).
The cost of enzymes for biofuel applications seriously hampers robust techno-
economic analysis of biofuel production processes. The contribution can be low-
ered by shifting to lower cost feedstocks, reducing the fermentation times, and
reducing the complexity of the process to drive down capital costs. Producing as
many co-products as possible in a biorefinery will help to reduce the cost of biofuel
production. It is important that a biorefinery should be established in an appropriate
location that has good water resources, access to feedstocks, and energy that is
needed to process the feedstock.
Future Prospects
Keeping in view the fast depleting conventional resources of energy from the
planet along with the hazardous impact of their use on the environment, the abilities
of microbes to produce biofuels appear as an eco-friendly alternative. Conversion
of plant biomass using microbial systems provides an excellent alternative for pro-
duction of biofuel on a commercial basis. Recommended aims of future efforts to
upgrade the fungal biofuel production include appropriate genetic and metabolic
manipulation of the strains, applying protein engineering strategies, and achieving
high productivity and product recovery at a larger scale. An effort to increase bio-
fuel production has led scientists to discover genes in microfungi that improve their
tolerance to ethanol, allowing them to produce more ethanol from the same amount
of nutrients. Genetic engineering may be used to manipulate the lipid-associated
metabolic pathway. Further exploration on regulatory and transport mechanisms,
transcriptional machinery, and signal transduction pathways involved in lipid accu-
mulation and degradation will pave the way to better understanding and utilization
of this platform.
Conclusion
Microfungi provide a promising platform to produce a wide range of lipid-based

bioproducts. Microfungal lipids would be the best solution to produce biodiesel, to
overcome the conflict between food and fuel. They could compete with conven-
tional fuels only by improving the technology and increasing both the biomass and
the lipid yields. Consequently, the development of oleaginous microorganism
strains offering high lipid content for biodiesel production is of critical importance
for an effective industrial application.
Acknowledgment The authors wish to thank the Department of Botany, Banaras Hindu
University (BHU), Varanasi-221005, India, and DST-INSPIRE fellowship scheme for the finan-
cial assistance.
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Chapter 23
Interactions of Microfungi and Plant-Parasitic
Nematodes
James LaMondia and Patricia Timper
Fungal- and Plant-Feeding Nematodes
Fungal-feeding and plant-parasitic nematodes use a specialized mouthpart, the

needle-like stylet, along with modifications of the pharynx to pierce either fungal
hyphal cells or plant cells and ingest the cell contents (Hussey et al. 2002). Many
different stylet-bearing nematodes have been shown to feed on fungi. In most cases,
ecologists consider that nematodes with stylets which have not been shown to feed
on plants should be classified as fungal feeders. There is considerable overlap, as
some fungal feeders are also known plant parasites and others are associated with
and suspected to feed on plants. The genera Aphelenchus, Aphelenchoides,
Ditylenchus, and Tylenchus are among the most common plant- and fungal-feeding
nematodes in this category (Freckman and Caswell 1985). Fungivorous nematodes
commonly exist in soil in association with small populations of many different fun-
gal species, so it is not surprising that most fungal-feeding nematodes can feed on
and complete a life cycle by feeding on a wide variety of fungi (Ruess and Dighton
1996; Ruess et al. 2000; Townshend 1964) with varying degrees of success. It has
been demonstrated that not all fungi result in the same level of nematode population
increase and reproductive success, so it may not be surprising that nematodes may
prefer to feed on multiple fungi, especially as the suitability of individual fungi as a
food source may change over time depending on whether (or when) compounds
J. LaMondia, Ph.D. (*)

e-mail: James.Lamondia@ct.gov
P. Timper, Ph.D.
USDA ARS, Tifton, GA 31793, USA
e-mail: Patricia.Timper@ars.usda.gov

DOI 10.1007/978-3-319-29137-6_23
574 J. LaMondia and P. Timper
toxic to nematodes may be produced (Mankau 1969; Ciancio 1995; Hasna et al.
2007). Ditylenchus or Aphelenchus nematodes had different preferences of fungi for
feeding and development (Pillai and Taylor 1967). For example, Fusarium solani
and Alternaria solani were excellent hosts for both nematodes, but Phytophthora
cactorum only supported Ditylenchus whereas Rhizoctonia solani resulted in large
populations of Aphelenchus. Hasna et al. (2007) studied the attraction of Aphelenchus
avenae and Aphelenchoides sp. nematodes to different fungal species as food
sources and subsequently measured population increases on those fungi. They
determined that both nematode species were differentially attracted to all seven
fungi that they tested; however, nematode fecundity was not necessarily correlated
to nematode attraction to the fungus as a food source. Ikonen (2001) observed simi-
lar results with six different fungi, not all fungi supported equal reproduction, or
even resulted in nematode reproduction. In fact, nematodes were also attracted to
fungi which did not serve as a feeding host and did not result in nematode reproduc-
tion (Townshend 1964).
Most nematode species in the genus Aphelenchoides are fungivores (perhaps
more than >100 different species). While most nematodes do not feed on pure cul-
tures of fungi in nature, Aphelenchoides (and also Ditylenchus species) can feed
directly on commercial mushroom beds and consume hyphal cell contents, resulting
in the destruction of mycelium and the loss of the mushroom crop (Hesling 1966).
A few species of foliar bud and leaf nematodes such as Aphelenchoides fragariae,
A. besseyi, and A. ritzemabosi are well known as important plant pathogens and
result in serious plant diseases. Some fungal-feeding nematodes can indirectly
reduce plant growth and vigor by feeding on and damaging the mycorrhizal fungi
responsible for increased nutrient uptake by plants (Ingham 1988; Riffle 1967;
1971; Ruess et al. 2000; Sutherland and Fortin 1968). Aphelenchoides compostic-
ola, Aphelenchus avenae, and Ditylenchus myceliophagus fed on fungi in soil,
including mycorrhizal fungi, and damaged mycelia, but the effects observed on
plant growth were small (Giannakis and Sanders 1989). Aphelenchoides bicaudatus
was able to parasitize endomycorrhizal fungi and restrict fungal growth in a manner
similar to ectomycorrhizal fungi (Shafer et al. 1981). The arbuscular mycorrhizal
fungi Gigaspora margarita and Glomus coronatum supported population increases
of Aphelenchus avenae in pots that did not occur when these fungi were not present
on plants. The nematodes reduced subsequent root colonization by the fungi and G.
coronatum spores were visibly damaged by nematode feeding, while Gi. margarita
was not (Bakhtiar et al. 2001). Ingham (1988) documented that plant-parasitic nem-
atodes and mycorrhizal fungi are usually mutually inhibitory and nematodes feed-
ing on fungi typically reduced fungal populations while high levels of mycelium
associated with roots reduced nematode feeding and infection. In some cases, better
root growth resulting from mycorrhizae increased nematode populations; in other
instances, nematode feeding and associated wounding increased the incidence of
root infection by mycorrhizal fungi.
There are few morphological differences to separate Aphelenchoides species, so
Rybarczyk-Mydłowska et al. (2012) utilized small subunit rDNA sequences to dis-
tinguish species. Two plant-parasitic nematodes, Aphelenchoides besseyi and
23 Interactions of Microfungi and Plant-Parasitic Nematodes 575
A. ritzemabosi, grouped together in a cluster phylogenetically isolated from

A. fragariae, A. subtenuis, and other fungal-feeding (or fungal- and plant-feeding)
species. Even so, A. besseyi, which causes leaf and bud crimp disease on strawberry,
was able to complete its life cycle on a pure culture of Alternaria citri (Wang et al.
1993). Aphelenchoides besseyi can survive in the environment in the absence of
strawberry in a number of vegetable crops, in ornamental and weed species, or in
strawberry residue, straw, or even in soil alone. When viable roots or root hairs are
not present, these obligate parasitic nematodes are assumed to feed on the fungi liv-
ing on incorporated organic matter in soil.
Fungal-feeding nematodes in soil are often assumed not to be parasites or to be
only minor plant parasites or pathogens. Sutherland (1967) did not observe
Aphelenchus avenae feeding or invasion of roots of seven species of pine and spruce
seedlings, concluding that the nematodes survived by feeding on the fungi associ-
ated with the plant rhizospheres. Other researchers have observed that A. avenae
may be associated with necrotic roots, as the nematode was observed moving in and
laying eggs in the cortex of roots, and was a “facultative plant parasitic with domi-
nant necrobiotic tendencies” (Steiner 1936). Christie and Arndt (1936) observed
both Aphelenchus and Aphelenchoides spp. feeding and reproducing on fungi in
culture and on plant cells in the early stages of necrosis as well as adjacent healthy
plant cells. They concluded that these nematodes were more than just saprobes.
Additional researchers have shown that A. avenae could feed and reproduce on
sterile plant tissue cultures in the total absence of fungi (Barker and Darling 1965),
and in addition to feeding on fungi, could feed on root hairs, and enter and feed on
healthy root tissues (Chin and Estey 1966).
Karim et al. (2009) identified more than 5000 expressed sequence tags represent-
ing more than 2200 genes from A. avenae. These ESTs included genes encoding
cell wall-degrading enzymes that were similar to those previously found in other
plant-parasitic nematodes, supporting the observed dual role of this nematode as
both a fungal feeder and a plant parasite. They postulated that plant parasitism by
nematodes evolved from fungal feeding and that A. avenae may be an important
animal for future studies of the evolution of plant parasitism in nematodes.
To further support this hypothesis, the A. avenae-expressed sequence tags were
more similar to the pinewood nematode Bursaphelenchus xylophilus than to the
potato cyst nematode Globodera pallida or the root-knot nematode Meloidogyne
arenaria. Bursaphelenchus xylophilus is also known to be a mycophagous nema-
tode that feeds on fungi such as the blue stain Ceratocystis fungus symbiotic with
bark beetles. This nematode likely also has evolved from a fungal feeder to be an
important plant parasite. The pinewood nematode was originally described as
Aphelenchoides xylophilus, a fungal-feeding nematode associated with bark bee-
tles. The beetles transmitted both the nematode and blue stain fungi (Steiner and
Buhrer 1934) to dead or dying pines. Pinewood nematodes were later shown to be
a very serious pathogen of pines in Japan (Mamiya 1972), feeding on living plant
tissue to cause tree death (Kiyohara and Suzuki 1978). There are a number of
Bursaphelenchus species, most of which feed on fungi in dead and dying trees.
Bursaphelenchus fungivorous can reproduce on at least 54 different fungal species
(Townshend 1964). Apparently, the pinewood nematode B. xylophilus evolved

from a fungal-feeding ancestor to feed on plant tissue, but in the absence of living
plant cells it too can have a completely mycophagous life cycle. For example, if it
is transmitted to dead or dying pines by beetle oviposition it can complete its life
cycle without living plant tissues (Graham 1967). The B. xylophilus nematode can
utilize wood-inhabiting fungi such as Ceratocystis blue stain fungi to increase pop-
ulations in dead trees, and the presence of the fungus also increased spread of the
nematode by beetle vectors (Maehara and Futai 1997). The presence of additional
wood-inhabiting fungi such as Trichoderma virens (J.H. Mill. et al.) Arx (reported
as Gliocladium virens) enhanced the survival of Bursaphelenchus xylophilus in dry-
ing pine tissues and also served as a food source for the nematode, increasing popu-
lations and nematode recovery from pine seedlings (McGawley et al. 1985).
Alternatively, transmission of pinewood nematodes to living trees during matu-
ration feeding would require plant feeding to complete the life cycle. Cellulases
produced by B. xylophilus associated with plant feeding were likely acquired from
fungi through horizontal gene transfer (Jones et al. 2005). Researchers used
expressed sequence tags to study the proteins nematodes use to parasitize plants and
demonstrated that genes encoding for cell wall-degrading enzymes appear in plant-
parasitic nematodes but not in other nematodes or in other animals studied. A num-
ber of genes have been identified; some are similar to bacterial sequences and others
are similar to known fungal sequences. Both are likely due to independent horizon-
tal gene transfer. In B. xylophilus, a cellulase similar to fungi and associated with
the ability to parasitize living plants was likely the result of horizontal gene transfer
and, like with the Aphelenchoides example cited above, associated with the evolu-
tion of plant parasitism (Kikuchi et al. 2004). In addition, a glucanase present in a
number of Bursaphelenchus species may be the result of horizontal gene transfer
from bacteria. As β-1,3-glucanases are constituents of fungal cell walls, these pro-
teins are likely associated with the fungal-feeding lifestyle of the genus (Kikuchi
et al. 2005).
The genus Ditylenchus is another example of a stylet-bearing nematode con-
taining both plant-parasitic and fungal-feeding species. Ditylenchus dipsaci and
D. destructor (stem and bulb nematodes) are economically important pathogens of
many plants (Faulkner and Darling 1961). Ditylenchus dipsaci was able to repro-
duce on Musicillium theobromae (Turconi) Zare & W. Gams (reported as Verticillium
theobromae) and Cladosporium spp. (Viglierchio 1971). Ditylenchus destructor
was also shown to feed and reproduce on at least 64 species of fungi representing 40
genera, 8 orders, and all classes (Anderson 1964; Faulkner and Darling 1961).
Undoubtedly, the ability to feed on many different fungi in plants, decomposing
plant tissues, or soil plays an important role in the survival of many plant-parasitic
nematodes over time when living host plants are not present (Hooper and Southey
1978). This is certainly an under-investigated area of research and may be a mecha-
nism responsible for survival of plant-parasitic nematodes despite management
with non-host rotation crops.
Nematodes as Biocontrols of Plant Pathogenic Fungi
Because fungal-feeding nematodes can be attracted to and actively feed on plant

pathogenic fungi, they may have some abilities to reduce pathogen biomass and act
as biological controls of plant disease. Rhoades and Linford (1959) were likely the
first to demonstrate that Aphelenchus avenae could control Pythium root rot in corn.
A number of mycophagous nematodes were shown to feed and reproduce on root
rotting and vascular wilt fungi (Nickle and McIntosh 1968). Aphelenchus avenae
was shown to control other root rot fungi such as Rhizoctonia solani and Fusarium
solani in pot experiments with alfalfa seedlings (Barnes et al. 1981) and R. solani,
F. solani, and F. oxysporum in beans or peas (Hong and Estey 1985; Klink and
Barker 1968), and in growth chambers at a range of soil moisture conditions when
added to soils at realistic population levels observed under field conditions (Lootsma
and Scholte 1997). Aphelenchus avenae could control R. solani on beans although
different isolates of the fungus differed in ability to support nematode populations
(Barker 1964). The level of R. solani control was not correlated with ability of the
isolate to serve as a nematode host. Potato stem canker caused by Rhizoctonia solani
inoculated to soil as sclerotia was significantly suppressed by A. avenae feeding and
nematode populations increased over time. Klink and Barker (1968) determined
that early feeding by A. avenae could inhibit the formation of sclerotia by R. solani.
Both A. avenae and Aphelenchoides spp. suppressed Rhizoctonia solani and reduced
damping off of cauliflower seedlings. In a series of experiments, Lagerlof et al.
(2011) concluded that A. avenae resulted in better disease control than the
Aphelenchoides spp. tested and that there was no interaction with the disease-
suppressive activity of various composts.
Ditylenchus spp. also fed on and inhibited the development of Fusarium wilt of
carnation (Schindler and Stewart 1956). Jun and Kim (2004) investigated combina-
tions of different Trichoderma species with Aphelenchus avenae for control of
Pythium and damping off of radish seedlings. They found that T. harzianum plus
nematodes resulted in the best disease control, while other Trichoderma species
either had no impact or resulted in reduced disease control. Their conclusion was
that the T. harzianum was a good food source that increased nematode populations
which also fed on Pythium, where the other Trichoderma species were less favor-
able or even antagonistic to the nematodes.
Cryphonectria parasitica, the fungal pathogen which causes chestnut blight, can
be partially controlled using hypovirulent strains of the fungus as a biological con-
trol (Hogan and Griffin 2008). These hypovirulent strains are infected with a virus
that reduces virulence and results in nonlethal superficial cankers on the trees
(Elliston 1985). Both virulent and hypovirulent strains of C. parasitica are good
hosts of the fungivorous nematode Aphelenchoides hylurgi (Griffin et al. 2009,
2012), and experiments demonstrated that the nematodes were able to spread propa-
gules of the hypovirulent strain. The direct effects of nematode feeding on the
hyphae on canker development were not determined, but perhaps more importantly,
the researchers postulated that Aphelenchoides hylurgi may be an effective means

of spreading hypovirulence to new cankers, increasing the incidence and efficacy of
biological control under natural conditions.
Inhibition of Fungal Biocontrol
Alternatively, fungal-feeding nematodes may feed on the biocontrol fungi which

themselves parasitize plant pathogenic fungi. Bae and Knudsen (2001) collected an
Aphelenchoides sp. from field soils that fed on the biocontrol fungus Trichoderma
harzianum. In culture, the nematode fed on T. harzianum hyphae, increased in num-
ber, and reduced radial growth of the fungus. In both untreated and heat-treated
soils, the Aphelenchoides sp. increased populations over time, reduced biological
control activity against Sclerotinia sclerotiorum, and reduced recovery of T. harzia-
num from soil, indicating that natural soil populations of fungivorous nematodes
may reduce the biocontrol activity of other soil fungi against plant pathogens.
Nematodes may also interact with endophytic fungi. Endophytic nonpathogenic
Fusarium oxysporum has been shown to negatively affect Meloidogyne incognita,
M. javanica, Pratylenchus goodeyi, and Radopholus similis in banana, tomato, and
rice (Dababat and Sikora 2007b; Mwaura et al. 2010; Sikora 2008). Endophytes
may produce metabolites toxic to nematodes or systemically alter root exudates to
reduce attraction or induce repellency (Vu 2005). Endophytes may also be mutual-
istic with nematodes. While not a plant parasite, the fungal-feeding nematode
Paraphelenchus acontioides appears to interact with a fungal endophyte, Fusarium
cf. torulosum, of brome grass Bromus tectorum, in a manner advantageous to both
the nematode and the fungus (Baynes et al. 2012). The presence of the nematode
increased the incidence of infection of the endophyte in the plant by some as yet
unknown mechanism, and the nematode preferentially grazed on this endophyte.
Nematode Interactions with Plant Pathogenic Fungi in Disease

Complexes
In a Presidential paper presented at the 1930 meeting of the American

Phytopathological Society, H. S. Fawcett stated the oft-cited quote “Nature does not
work with pure cultures alone but most frequently with associations.” He also stated
that “… a number of diseases may require an association of organisms for their
occurrence and cannot be produced by infection of one organism alone” (Fawcett
1931). Since that time, many diseases of complex etiology have been discovered.
While none of the examples of “synergism” that were cited in his 1930 address
included nematodes, nematodes certainly interact with fungi in complex diseases.
In the process of feeding on plants, plant-parasitic nematodes necessarily cause
wounds and can also result in numerous changes in host physiology. The damage to
and effects on plants can differ substantially based on how different nematodes
feed. Ectoparasites, nematodes which feed on plants while remaining for the most
part outside the root, typically feed on plant cells by insertion of the stylet, a sclero-
tized mouthpart, into cells. Repeated insertion into the same or different cells causes
microinjection wounds which occur in conjunction with salivation and pharyngeal
gland secretions while cutting into or through cell walls, and the stylet is used to
ingest cell contents (Wyss 2002). Endoparasitic nematodes enter the roots and can
be migratory, moving into plant tissues while feeding on cell contents in a manner
similar to ectoparasites, or sedentary. Sedentary endoparasites enter roots and settle
into one location for feeding and development. These nematodes can secrete effec-
tor proteins to actively suppress plant defense responses and can change cellular
identity and metabolism to induce the development of specialized feeding cells
which concentrate nutrients for developing nematodes (Ali et al. 2015; Mitchum
et al. 2013).
Nematode-induced root wounding, cell senescence and death, local or systemic
host physiological changes, and nematode-induced suppression of plant defense
responses may each act to increase the incidence or severity of diseases that are
primarily caused by fungal pathogens. Nematode–fungal interactions in complex
disease can be additive in effect or synergistic when disease is significantly greater
than the sum of disease caused by each pathogen individually. Many examples of
complex diseases caused by the interaction of more than one pathogen and includ-
ing nematodes have been proposed. Sikora and Carter (1987) proposed limiting the
definition of complex diseases involving plant-parasitic nematodes to those based
on field observations of disease where both nematodes and additional pathogens can
be accounted for, environmental factors and population densities of both nematodes
and fungal propagules that are realistic and in a range based on actual observed data,
timing of inoculations that are realistic and determined by environmental conditions
and phenology of the crop/pathogen(s) interaction, and where experiments were
conducted with appropriate factorial/multifactorial designs used to produce statisti-
cal evidence of a synergistic complex interaction.
What is likely the very first example of a nematode–fungal disease complex was
described by Atkinson in 1892 and involved an increased severity of Fusarium wilt
of cotton caused by Fusarium oxysporum f. sp. vasinfectum when plants were also
infected with root-knot nematodes (Meloidogyne spp.). Subsequently, most cases of
synergistic interactions between nematodes and fungi involve the sedentary endo-
parasitic root-knot and cyst nematodes increasing disease caused by Fusarium or
Verticillium wilt fungi. Meloidogyne spp. have been shown to interact with Fusarium
wilt in a number of crops, including alfalfa (Griffin 1986), bananas (Jonathan and
Rajendran 1998), beans (France and Abawi 1994), chickpeas (Uma Maheswari
et al. 1997), coffee (Bertrand et al. 2000), cotton (Atkinson 1892; De Vay et al.
1997; Abd-El-Alim et al. 1999), lentils (De et al. 2001), peas (Siddiqui and
Mahmood 1999), soybeans (Xing and Westphal 2013), tobacco (LaMondia 1992;
LaMondia and Taylor 1987), tomatoes (Abawi and Barker 1984; Suleman et al.
1997), and watermelon (Sumner and Johnson 1973). Cyst nematodes can act in a
similar manner to increase wilt diseases. The potato cyst nematodes Globodera
rostochiensis (Evans 1987) and G. pallida (Hide et al. 1984; Storey and Evans
1987) interact with Verticillium spp. in potato plants to increase disease severity,
and Fusarium wilt of tobacco is most damaging in the presence of the tobacco cyst
nematode G. tabacum (LaMondia and Taylor 1987).
In addition to wilt diseases, a number of complex nematode–fungal root rot dis-
eases have been described. Meloidogyne incognita has been associated with black
root rot of cotton caused by Thielaviopsis basicola (Walker et al. 2000). Neither
pathogen causes significant disease individually; plant mortality only occurs when
both pathogens are present. Management of the nematode component alone can
reduce the severity of the fungal root rot disease (Fichtner et al. 2005). Meloidogyne
enterolobii (Gomes et al. 2013) and M. mayaguensis (Gomes et al. 2011) interact
with Fusarium solani to result in a decline of guava due to a complex root rot syn-
drome. Meloidogyne javanica can interact with Rhizoctonia solani to increase root
rot and pod disease in peanut (Abdel-Momen and Starr 1998), and M. incognita
increases the severity of root rot by R. solani in grapevines (Walker 1997). Early
infection with the potato cyst nematode Globodera pallida increased root rot in
potato by R. solani (Bhattarai et al. 2010), and the sugar beet cyst nematode
Heterodera schachtii increased infection and disease severity caused by R. solani in
beets (Polychronopoulos et al. 1969). The soybean cyst nematode Heterodera gly-
cines interacts with Fusarium virguliforme (=F. solani f. sp. glycines) to result in the
sudden death syndrome of soybean, significantly increasing root and crown rot,
chlorosis, defoliation, and pod abortion symptom development when both patho-
gens are present (McLean and Lawrence 1993; Xing and Westphal 2013).
Nematodes may influence the development of fungal diseases in several ways.
Root wounding occurs as infective juveniles enter and migrate through the root
cortex to suitable feeding locations behind the root tip. Nematodes migrating
through root tissue may also act to disperse fungal inoculum (Neher 2010). Inagaki
and Powell (1969) suggested that wounding alone was the primary mechanism for
the migratory endoparasite Pratylenchus brachyurus-incited increases in infection
by Phytophthora parasitica in tobacco. They investigated artificial wounding and
timing of the interaction and concluded that nematode infection 1 week earlier but
not 2–3 weeks increased infection by P. parasitica was consistent with mechanical
damage due to invasion. However, additional effects of artificial wounding and
nematodes could also occur. A stronger argument for wounding as the primary rea-
son for increasing fungal infection was presented by observation of Rhizoctonia
solani growing in those areas of the root damaged by cyst nematode (Heterodera
schachtii) invasion in sugar beet roots (Polychronopoulos et al. 1969). Likewise,
Verticillium infected via Globodera pallida juvenile invasion routes in potato
(Storey and Evans 1987). Infection by the fungus was most likely if nematode
wounds were fresh and infection was reduced over time after roots responded to
damage. In addition to nematode movement through tissues, root wounding may
also be caused by physical damage to the cortex as the developing female inside the
root expands greatly in size as she develops to maturity and produces eggs. Cortical
cells can be damaged as they are compressed, split, or separated from adjacent cells.
R. solani was shown to infect areas of the root damaged by the development of the
northern root-knot nematode M. hapla inside root galls (Fagbenle and Inskeep
1987). Heterodera daverti interacts synergistically with Fusarium oxysporum or F.
avenaceum to decrease subterranean clover plant dry weight, but only if the fungal
pathogens were inoculated 1–2 weeks prior to the nematode (Nordmeyer and Sikora
1983a). Rather than the nematode increasing infection by the fungal pathogen, the
fungi may make roots more attractive and likely to be infected by the nematode. In
fact, in vitro research (Nordmeyer and Sikora 1983b) showed that nematodes moved
in greater numbers toward culture filtrate treated roots and diffusates from infected
roots than control roots. Fungal factors such as cell wall-degrading enzymes may
increase chemical orientation factors or make roots more easily penetrated.
Migratory endoparasitic nematodes such as lesion nematodes, Pratylenchus spp.,
have been widely studied as important factors increasing potato early dying disease
in combination with Verticillium dahlia or V. albo-atrum (Martin et al. 1982; Wheeler
et al. 1992). In this disease complex, nematode infection acts to increase the impact
of relatively low populations of Verticillium spp. to result in significantly increased
early plant senescence and tuber yield losses that would not otherwise occur in the
absence of the nematode. Root wounding is often cited as the likely mechanism of
this interaction as Pratylenchus juveniles and adults infect, feed, and migrate inter-
cellularly through the root cortex. However, certain species of Pratylenchus, includ-
ing P. penetrans and P. scribneri, can increase potato early dying, whereas P.
crenatus, which causes similar wounds due to cortical invasion, does not (Riedel
et al. 1985). In fact, further research has shown that geographically isolated distin-
guishable populations of P. neglectus can differ in ability to interact with Verticillium
spp. to incite early dying (Hafez et al. 1999). The fungal pathogen can also differ in
ability to interact with a nematode partner in a disease complex. Pratylenchus pen-
etrans synergistically increased Verticillium wilt of peppermint and spearmint by V.
dahlia VCG 2B but not with VCG 4A (Johnson and Santo 2001). Faulkner et al.
(1970) utilized split-root systems in peppermint to demonstrate that Pratylenchus
brachyurus could systemically increase the incidence and severity of Verticillium
wilt on different root systems of the same plant. Clearly, migratory endoparasites can
do more than just wound roots to impact subsequent disease by Verticillium.
Pratylenchus penetrans has been associated with significantly increased root rot
in perennial strawberry beds caused by Rhizoctonia fragariae, often leading to
removal of fields from production. Lesion nematode feeding and movement in
strawberry roots directly results in cell damage and death. The indirect effects of
lesion nematode infection are discoloration of the endodermis and early polyderm
formation, followed by localized areas of secondary growth and cortical cell senes-
cence or death. Senescing root tissue or dying cells resulting from the direct and
indirect effects of P. penetrans were more susceptible to R. fragariae, leading to
increased infection and cortical root rot (LaMondia 2004). Unlike the Pratylenchus-
Verticillium system (Faulkner et al. 1970), inoculation of lesion nematodes and R.
fragariae on separate root systems of the same plant did not result in increased
disease, indicating that local rather than systemic effects of P. penetrans were most
important in the interaction of these pathogens in strawberry black root rot
(LaMondia 2003).
The stem and bulb nematode Ditylenchus dipsaci can feed on different plant tissues
than the nematodes discussed to this point, but Ditylenchus spp. can also interact
with fungi to cause or increase plant disease. Fusarium basal rot of onion was shown
to be significantly greater when both D. dipsaci and Fusarium spp. were present
(Trifonova and Koleva 2002). Synergistic interactions were observed when nema-
todes were inoculated concomitantly or when nematodes followed inoculation with
the fungus. Crown and root rot of sugar beet caused by Rhizoctonia solani was also
synergistically increased by coinfection with D. dipsaci (Hillnhutter et al. 2011).
The authors concluded that the fungus gained access to the plant through wounds
caused by the nematode based on fungal infection at those sites. Foliage blight of
Phlox subulata resulting from dual infection of D. dipsaci and Botrytis cinerea
resulted in severe dieback and the failure of Botrytis gray mold control with appro-
priate fungicides (LaMondia unpublished). This lack of disease control was assumed
to be due to fungicide resistance in the Botrytis pathogen, but was determined
instead to be due to ongoing damage by the stem nematode that resulted in second-
ary infection of dead and dying leaves and stems by Botrytis cinerea. Fusarium wilt
of alfalfa by F. oxysporum f. sp. medicaginis was increased by infection with D.
dipsaci in both field and greenhouse studies (Griffin 1992). Vrain (1987) deter-
mined that D. dipsaci and not Pratylenchus penetrans interacted with V. albo-atrum
to increase the severity of wilt symptoms and affect forage yields.
Root-knot and cyst nematode-induced giant cells and syncytia increase meta-
bolic activity and effectively concentrate nutrients as a food source for the station-
ary developing nematodes (Jones 1981; Jones and Northcote 1972). Many
researchers have reported that fungi such as F. oxysporum, F. solani, Pythium spp.,
and R. solani colonize nematode-infected tissues in a manner not observed in the
absence of nematode infection (Abdel-Momen and Starr 1998; McLean and
Lawrence 1993; Meléndez and Powell 1970; Negrón and Acosta 1989). In addition
to physical cell damage, leakage of cell contents from feeding cells may also influ-
ence infection. Golden and Van Gundy (1972) determined that R. solani and
T. basicola responded to M. incognita-induced galls and increased leakage of elec-
trolytes and organic compounds by growing more vigorously and actively coloniz-
ing nematode-infected roots, especially as females expanded and matured. In fact,
perhaps because damaged tissues are such good sources of nutrition, it has been
reported that root-knot nematode infection can result in root disease by fungi usu-
ally considered to be nonpathogenic or weakly pathogenic, such as Curvularia,
Penicillium, and Trichoderma (Combettes 1983).
Fungal Antagonism of Nematodes
Antagonistic interactions between microfungi and plant-parasitic nematodes are as

numerous as they are varied. While some facultative plant-parasitic nematodes,
such as Aphelenchoides fragariae and Bursaphelenchus xylophilus, feed on fungi
as well as plants, the majority of fungal–nematode interactions are detrimental to
the nematode. Fungi kill or disrupt the life cycle of nematodes by parasitism, by
production of toxins or extracellular enzymes, and by inducing resistance in plants.
Many reviews have been written about the interaction between microfungi and
plant-parasitic nematodes (Liu et al. 2009; Stirling 2014; Davies and Spiegel 2011;
Dong and Zhang 2006; Morton et al. 2004; Lopez-Llorca et al. 2008). Hence, this
section is not intended to be a comprehensive literature review, but to provide the
reader with a detailed overview of the ecological, cellular, and molecular interac-
tions between fungi and their host nematodes.
Parasites of Motile Nematode Stages
Except for the eggs, most nematode life stages are actively moving in their environ-
ment, which presents a challenge for relatively slow-growing fungal parasites.
Fungi have adapted to parasitize mobile stages of nematodes by employing trapping
structures or adhesive conidia to immobilize or make firm contact with nematodes
that touch them. These fungi have a broad host range, parasitizing microbivorous
nematodes as well as plant- and animal-parasitic nematodes.
Fungi Producing Traps
Fungi that produce trapping structures are often referred to as predatory or carnivo-
rous because they trap, wound, or paralyze nematodes before consuming them in a
manner reminiscent of carnivorous plants. Most trapping fungi are true pathogens
that infect a living host; however, some kill the host prior to infecting. Trapping
fungi are found primarily in the Orbiliaceae (Ascomycota) and in several families
in the Basidiomycota (Table 23.1). The taxonomy of the trapping fungi in the
Orbiliaceae has undergone numerous changes in the last 20 years. Previously, the
genera were classified based on morphology of their conidia (Drechsler 1937;
Rubner 1996); however, current classification of these fungi is based on morphol-
ogy of the trapping structures and molecular similarity (Ahren et al. 1998; Scholler
et al. 1999; Li et al. 2005). Scholler et al. (1999) accepted 82 species of trapping
fungi in the Orbiliaceae. Dactylellina spp. produce stalked adhesive knobs with
some species also producing nonconstricting rings or adhesive branches.
Arthrobotrys spp. produce adhesive networks, while Dreschlerella spp. produce
constricting rings that dramatically clamp around the nematode body. Traps are
typically produced on hyphae; however, under conditions of low nutrition or
intense competition, traps are produced directly from germinating conidia
(Persmark and Nordbring-Hertz 1997). Additionally, the traps of Dactylellina and
Dreschlerella are continuously produced regardless of the environment, whereas the
traps of Arthrobotrys are produced in response to a combination of low nutrition
and the presence of nematodes (Nordbring-Hertz 1977; Scholler and Rubner 1994).
Table 23.1 Genera of nematode-trapping fungi

Genus Order (family) Trap type Citation
Dactylellina Helotiales (Orbiliaceae) Stalked adhesive knobs; Li et al. (2005)
nonconstricting rings
Arthrobotrys Helotiales (Orbiliaceae) Adhesive networks Li et al. (2005)
Dreschlerella Helotiales (Orbiliaceae) Constricting rings Li et al. (2005)
Stylopage Zoopagales (Zoopagaceae) Adhesive hyphae Barron (1977)
Resupinatus Agaricales Hourglass-shaped Thorn and
(Tricholamataceae) adhesive cells Barron (1984)
Pleurotus Agaricales (Pleurotaceae) Paralytic droplets Barron and
Thorn (1987)
Hohenbuehelia Agaricales (Pleurotaceae) Hourglass-shaped Thorn and
adhesive cells Barron (1984)
Coprinus Agaricales (Agaricaceae) Spiny balls Luo et al. (2007)
Stropharia Agaricales (Strophariaceae) Spiny acanthocytes Luo et al. (2006)
Hyphoderma Polyporales (Meruliaceae) Adhesive stephanocysts Tzean and Liou
(1993)
Recent evidence suggests that nematode pheromones known as ascarosides are

involved in triggering trap production in Arthrobotrys (Hsueh et al. 2013). Some
genera in the Basidiomycota and Zygomycotous fungi also produce adhesive
hyphae or knobs to secure nematodes prior to infection, though these trapping
structures are morphologically distinct from the Orbiliaceae (Table 23.1).
Basidiomycetes also form unique structures which kill or immobilize nematodes
prior to consuming them. Pleurotus ostreatus produces droplets of toxin on hyphal
stalks which instantly paralyze nematodes that come in contact with the toxin
(Barron and Thorn 1987). Recently, spiny acanthocytes of Stropharia spp. and
spiny balls of Coprinus comatus have been shown to mortally wound nematodes
with their sharp projections before rapidly colonizing their prey (Luo et al. 2004,
2006, 2007; Zouhar et al. 2013). Additional descriptions and images of trapping
structures can be found in (Barron 1977; Stirling 1991; Nordbring-Hertz et al.
2001; Tunlid and Ahren 2011).
The mechanism of trapping and infection has primarily been studied in
Arthrobotrys oligospora and related Orbiliaceae (see Tunlid and Ahren 2011 for
review). Early studies indicated that adhesion of A. oligospora to the nematode
cuticle involved lectins on the traps binding with carbohydrates on the nematode
surface (Nordbring-Hertz and Mattiasson 1979; Premachandran and Pramer 1984).
An N-acetylgalactosamine-binding lectin (AOL) was subsequently isolated from
A. oligospora; however, AOL-deletion mutants showed no loss of nematode para-
sitism (Balogh et al. 2003). Moreover, none of the seven genes encoding for lectins
that were identified in A. oligospora by Yang et al. (2011) were upregulated during
trap formation. The mechanism of adhesion is still unresolved. Following capture,
the fungus penetrates the nematode cuticle by means of mechanical force and
extracellular proteases (Veenhuis et al. 1985). The nematode is paralyzed during

the initial penetration of the cuticle by the fungus; evidence indicates that a serine
protease is involved in the paralysis (Ahman et al. 2002). At the point of entry, an
infection bulb is formed from which trophic hyphae develop. Nematode death and
colonization of the cadaver is rapid (48–60 h) and vegetative hyphae soon emerge
from the degraded cadaver (Dijksterhuis et al. 1994).
Despite the broad host range of trapping fungi, some nematodes are resistant to
capture. Gaspard and Mankau (1987) evaluated 16 species of nematodes and found
that nematodes in the class Secernentea were trapped by Dactylellina ellipsospora
(Monacrosporium ellipsosporum), whereas nematodes in Adenophorea were not
trapped. Even within Secernentea, nematodes can exhibit resistance to trapping
fungi. Cyst nematodes (Globodera and Heterodera spp.), for example, are able to
evade capture by fungi with adhesive traps (Wimble and Young 1983; Den Belder
and Jansen 1994; Jaffee 1998), but not those with constricting rings (Jaffee 1998).
Nematodes that are resistant to trapping fungi may possess surface components that
mask receptors for fungal attachment on the nematode cuticle. Mendoza de Gives
et al. (1999) demonstrated that three mutants of Caenorhabditis elegans, which had
altered surface coats, were more efficiently captured than the wild type by
Arthrobotrys (Duddingtonia) flagrans. The authors hypothesized that the mutants
had lost a component of their surface coat or cuticle that in the wild type provides a
barrier to recognition by the fungus.
Plant-parasitic nematodes, for the most part, are found in and around living
plants, while several of the nematode-trapping basidiomycetes (Hohenbuehelia,
Hyphoderma, Pleurotus, and Resupinatus) are found in decaying wood, compost,
and barnyard soil (Thorn and Barron 1984, 1986; Tzean and Liou 1993). Although
these fungi can trap and consume plant-parasitic nematodes, they typically do not
co-occur in the same habitat. The exception is Hohenbuehelia (syn. Nematoctonus),
which has been isolated in low frequencies from plant rhizospheres (Persmark and
Jansson 1997). It is unclear how commonly the spiny traps of Coprinus or
Stropharia are found in association with plant-parasitic nematodes because, until
recently, they had not been recognized as nematode-killing structures. Luo et al.
(2006) demonstrated that Stropharia rugosoannulata readily colonized woodland
soil where it produced abundant acanthocytes. The trapping fungi in Orbiliaceae
and Stylopage are also found in decaying wood, dung, and humus (Duddington
1951); however, they have been regularly isolated from agricultural soils including
the rhizosphere and endosphere of plant roots where they could potentially interact
with plant-parasitic nematodes. The abundance of these fungi is generally greater
in the rhizosphere than in the bulk soil (Persmark and Jansson 1997; Peterson and
Katznelson 1965; Gaspard and Mankau 1986). This is not surprising given that the
rhizosphere is more densely populated with host nematodes (Persmark and Jansson
1997), both plant parasites and microbivorous, than the bulk soil and some of the
trapping fungi may obtain nutrition from the root exudates. Interestingly, A. oligos-
pora was found to penetrate barley and tomato roots and colonize the cortex but
not the vascular tissue (Bordallo et al. 2002). The fungus demonstrated directed
growth towards roots and penetrated the epidermis via appressoria. Two other trap-
ping fungi, A. musiformis and A. psychrophila, did not show directed growth
towards roots.
Addition of organic matter to soil often increases the abundance of trapping
fungi (Cooke 1962a, b; Wang et al. 2003a; Jaffee 2002, 2004). The organic matter
can serve as a source of nutrition for the fungi but also increases numbers of micro-
bivorous nematodes which serve as hosts (Linford et al. 1938). Moreover, in agri-
cultural soils, abundance of trapping fungi has been found to be greatest in the top
10 cm where organic matter content is highest (Persmark et al. 1996; Stirling et al.
2011). Abundant trapping fungi, however, does not always lead to suppression of
nematode populations. These fungi show varying degrees of dependence on nema-
todes for nutrition, both within and between genera. Species of Arthrobotrys tend to
be competitive saprobes and consume nematodes as an alternative food source dur-
ing periods of intense competition (Jansson and Nordbring-Hertz 1979; Cooke
1963). Their abundance may increase in response to a carbon source without pro-
ducing traps (Jaffee 2002, 2004). Conversely, species of Dactylellina and
Dreschlerella tend to be weak saprobes and are more dependent on nematodes for
nutrition. These predominantly parasitic species of trapping fungi increase in
response to nematode density, whereas more saprobic species do not (Jaffee et al.
1993; Persmark et al. 1996).
Although trapping fungi in the Orbiliaceae are frequently encountered in agricul-
tural soils and in close association with plant roots, they have not proven to be reli-
able in suppressing populations of plant-parasitic nematodes (Stirling 1991). Many
of the early attempts to control these nematodes with trapping fungi focused on
species of Arthrobotrys, which are not principally parasites. Galper et al. (1995)
found that in soil, Dactylellina spp. and Dreschlerella spp. were more effective in
trapping the root-knot nematode, Meloidogyne javanica, than were Arthrobotrys
spp. In other studies, Dactylellina cionopaga and Dreschlerella dactyloides, applied
in alginate pellets, reduced root penetration and galling by root-knot nematodes
(Stirling and Smith 1998; Jaffee and Muldoon 1997). However, Persson and Jansson
(1999) were unable to demonstrate suppression of root-knot nematodes by D. dac-
tyloides or Dactylellina spp. formulated in alginate pellets even though several of
the fungi, particularly D. ellipsospora, abundantly colonized the tomato rhizo-
sphere. Attempts to determine whether native trapping fungi are suppressing popu-
lations of plant-parasitic nematodes have also yielded inconsistent results. Amending
soil with sunn hemp residue increased the abundance of parasitic trapping fungi, but
improved suppression of the reniform nematode, Rotylenchulus reniformis, was not
observed (Wang et al. 2003b). Yet, in soils cultivated with sugarcane, both the
diversity and the abundance of trapping fungi were correlated to suppression of
Radopholus similis (Stirling et al. 2011). The latter study utilized a bioassay to mea-
sure nematode suppression which reduced the variability associated with determin-
ing suppression based on resident nematode populations.
Fungi Producing Adhesive Conidia
Fungi that attach to nematodes via adhesive conidia are often referred to as “endo-
parasites”; however, this term is misleading because the trapping fungi are also
endoparasites of nematodes. Compared to the trapping fungi, there are only a few
nematophagous fungi producing adhesive conidia. Two species in the
Entomophthorales and Kickxellomycotina, Meristacrum asterospermum and
Zygnemomyces echinulatus, and one species in the Agaricales, Nematoctonus
leissporus (Pleurotaceae), produce adhesive conidia (Barron 1977; Saikawa et al.
1997); however, very little is known about their ecology or impact on nematode
populations. Most of the research has focused on a handful of fungi in the
Hypocreales that produce adhesive conidia, Drechmeria coniospora, Drechmeria
(Verticillium) balanoides, and Hirsutella spp.; both genera are in the
Ophiocordycipitaceae, which contains other parasites of invertebrates (Quandt
et al. 2014). The new genus and species Esteya vermicola, described by Liou et al.
(1999) from infected pinewood nematode (Bursaphelenchus xylophilus), also pro-
duces adhesive lunate conidia. This fungus is a member of the Ophiostomataceae,
which are typically found growing saprophytically in living trees in association
with bark beetles.
Drechmeria coniospora is an obligate pathogen of nematodes that is readily iso-
lated from different soils (Glockling and Holbrook 2003). The fungus produces
clusters of club-shaped conidia on a conidiophore. These conidia are not immedi-
ately infective and must undergo a maturation process to produce an adhesive knob
on the narrow end of the conidium (Van den Boogert et al. 1992). Knob formation
is not influenced by the presence of nematodes and occurs only after the conidia are
released from the conidiophore and dispersed in the environment; aggregated
conidia are inhibited from maturation. Conidia of D. coniospora primarily adhere to
the head region of adults and juveniles, and additionally to the tails of male nema-
todes; however, in some nematode species, the conidia adhere over the entire cuticle
(Jansson et al. 1985). The mechanism of adhesion involves proteins in the adhesive
material binding to proteins excreted from nematode sensory organs and does not
involve a lectin–carbohydrate interaction as previously hypothesized (Jansson
1993). Conidial attachment to a particular nematode species does not always lead to
infection; the recognition signal for infection by D. coniospora appears to be more
specific than the recognition signal for adhesion (Jansson et al. 1985, 1987). On a
susceptible host, the conidium will form an appressorium that presses firmly against
the nematode cuticle, possibly with the aid of an adhesive, before forming a penetra-
tion tube into the pseudocoel (Dijksterhuis et al. 1990). The nematode pseudocoel is
then colonized by trophic hyphae with a characteristic wavy appearance. Nematode
death occurs within 48 h of initial infection. Interestingly, the fungus does not fully
colonize the nematode cadaver before the outgrowth of conidiophores and sporula-
tion (Dijksterhuis et al. 1991). Approximately, 5000–10,000 conidia are produced
per nematode. Because D. coniospora infects only a few species of plant-parasitic
nematodes, it has not been frequently evaluated as a biological control organism.
Drechmeria balanoides has acorn-shaped conidia and is an obligate pathogen of

nematodes. The adhesive material on the conidia is formed shortly after their release
from the phialide (Sjollema et al. 1993). Infection is initiated by formation of an
appressorium followed by a penetration tube, but unlike D. coniospora, an infection
bulb forms after penetration. Conidiophores emerge from the cadaver about 60 h
after initial infection. Atkinson and Dürschner-Pelz (1995) determined that cadav-
ers of Globodera rostochiensis and Ditylenchus dipsaci yielded 12,000–16,000
conidia per cadaver (Atkinson and Dürschner-Pelz 1995). Little is known of the
host range of D. balanoides. Durschnerpelz and Atkinson (1988) indicated that this
fungus had a broader host range among plant-parasitic nematodes than did D.
coniospora. However, few studies have evaluated the biological control potential of
this fungus except against the stem nematode D. dipsaci. The fungus was found
infecting D. dipsaci within the foliage of white clover; moreover, inoculation of
white clover seed with D. coniospora reduced nematode numbers in the foliage
compared to plants without fungal inoculation (Hay and Regnault 1995; Hay and
Bateson 1997).
Three species of Hirsutella are obligate parasites of nematodes: H. rhossiliensis
and H. minnesotensis have broad host ranges including numerous plant-parasitic
nematodes, while H. vermicola is primarily a pathogen of bacterivorous nematodes
and will not be discussed further (Xiang et al. 2007). Hirsutella rhossiliensis has
been found worldwide parasitizing a variety of nematode species, while H. minne-
sotensis has been found predominantly parasitizing the soybean cyst nematode
(Heterodera glycines) in China and the USA (Liu and Chen 2000; Xiang et al. 2007;
Costa et al. 2012). The conidia of these two species are produced singly on phialides
and are unable to attach to nematodes once they are dislodged from the phialides
(McInnis and Jaffee 1989). Attachment of conidia and infection can occur any-
where on the nematode cuticle. Little is known of the mechanism of infection. Two
serine proteases have been isolated from H. rhossiliensis and presumably are
involved in the infection process (Wang et al. 2007, 2009). Following infection, the
trophic hyphae colonize the body of the nematode killing it in a few days. When the
cadaver is completely colonized by the fungus, hyphae containing sparsely spaced
phialides and conidia radiate out from the cadaver. Conidia production by H. rhos-
siliensis is determined by the size of the nematode; 112 and 700 conidia/cadaver
were produced from parasitized sugar beet cyst nematode (Heterodera schachtii)
and ring nematode (Mesocriconema xenoplax), respectively (Jaffee and Zehr 1983;
Jaffee et al. 1990). Although both H. rhossiliensis and H. minnesotensis can
parasitize a broad range of nematodes, there is some host specialization among
isolates of these fungi (Tedford et al. 1994; Liu and Chen 2001; Xiang et al. 2007).
It is noteworthy that cyst nematodes in the genus Heterodera tend to be more sus-
ceptible to these two Hirsutella spp. than root-knot nematodes (Meloidogyne spp.),
the reverse of that observed for trapping fungi (Xiang et al. 2007).
Hirsutella rhossiliensis and H. minnesotensis have been the subject of more stud-
ies to determine their potential for biological control of plant-parasitic nematodes
than any other nematophagous fungi producing adhesive conidia. Environmental
factors can have large effects on rates of nematode parasitism; soil texture, moisture,
and temperature influence both fungal growth and nematode movement. Acquisition
of H. rhossiliensis conidia by H. schachtii was greater in silty clay and loamy sand
than in coarse sand (Jaffee et al. 1990). Similarly, parasitism of H. glycines by H.
minnesotensis declined with increasing sand content (Xiang et al. 2010). The large
pore sizes in the sand both limit the movement of H. schachtii, and thus contact with
the adhesive conidia, and also allow the nematode to move through the pore without
touching conidia. Rates of nematode parasitism by Hirsutella spp. are reduced at
both high and low soil water contents. Nematodes require water films to move in
order to contact conidia; however, when soil pores are filled with water, nematode
movement is limited and the fungus does not produce conidia (Tedford et al. 1992;
Xiang et al. 2010). Warmer soil temperatures should increase fungal growth and
nematode activity leading to greater acquisition of conidia. However, Xiang et al.
(2010) found that persistence of H. minnesotensis was greatest at cooler soil tem-
peratures (5–10 °C). Rates of parasitism by both H. rhossiliensis and H. minnesoten-
sis are greater in acidic soil than in neutral and basic soils (Jaffee and Zasoski 2001;
Liu and Chen 2009). Jaffee and Zasoski (2001) provided convincing evidence that
pH has an indirect effect on H. rhossiliensis by influencing antagonists of the fun-
gus; pH had no influence on nematode parasitism in soil that had been heated to kill
most organisms. Nevertheless, other research indicates that pH may also have a
direct effect on these fungi (Jaffee and Zehr 1983; Liu and Chen 2009). Soil fauna
and flora can substantially reduce the activity of H. rhossiliensis. Sporulation of the
fungus from alginate pellets was inhibited by both micro- and macro-organisms
(Jaffee 2000; 1999; Jaffee et al. 1997b). Enchytraeids, in particular, frequently con-
tributed to the decline of pelletized H. rhossiliensis in soil (Jaffee et al. 1997a). The
fungus appears to be more sensitive to biotic inhibition when it is formulated in
alginate pellets than when it is growing from infected nematodes (Jaffee 2000).
Hirsutella spp. are frequently found in agricultural fields, sometimes parasitizing
large numbers of cyst (Heterodera spp.) and ring (Mesocriconema spp.) nematodes
(Muller 1985; Jaffee et al. 1988, 1989; Liu and Chen 2000). Moreover, soils con-
taining high densities of H. rhossiliensis collected from the field suppressed pene-
tration of cabbage roots by H. schachtii and egg production by H. glycines (Jaffee
and Muldoon 1989; Chen 2007). Despite the promising results in naturally infested
soil, applications of H. rhossiliensis as pelletized hyphae to microplot or field soil
failed to suppress populations of plant-parasitic nematodes (Tedford et al. 1993;
Jaffee et al. 1996) due, in part, to biotic inhibition of the formulated fungus.
Unlike the other fungi producing adhesive conidia, Esteya vermicola appears
to be a facultative parasite of nematodes (Wang et al. 2011b). The fungus has been
found on three continents (Asia, Europe, and South America) and isolated from
wood, pinewood nematodes, and nematodes in pine forest soil (Wang et al. 2014).
Esteya vermicola produces two types of conidia: one type is lunate with a central
structure that resembles an endospore and a second type is cylindrical to bacil-
loid; only the lunate conidia are adhesive to nematodes (Liou et al. 1999). The
adhesive material is formed on the concave side of the conidia. Similar to
Hirsutella spp., the conidia are unable to attach to nematodes if they are dislodged
from the conidiophore (Wang et al. 2008). Following attachment to the nematode
cuticle, E. vermicola forms an infection peg which then expands into a bulb. The
fungus grows endoparastically before emerging from the cadaver to produce only
lunate conidia. The cylindrical conidia are formed on nutrient-rich media and do
not infect nematodes (Liou et al. 1999). Although the host range of E. vermicola
has not been fully characterized, it was shown to be a virulent pathogen of both
the pine wilt nematode and microbivorous nematodes (Wang et al. 2008). Of the
eight plant-parasitic nematodes tested by Wang et al. (2014), the fungus infected
B. xylophilus, B. mucronatus, Aphelenchoides besseyi, and Ditylenchus destruc-
tor, but not Meloidogyne incognita, Heterodera avenae, or Pratylenchus pene-
trans. However, the rate of infection by E. vermicola was greater for B. xylophilus
than for any of the other nematode hosts suggesting some host specialization. The
fungus shows promise as a biological control agent of the pinewood nematode.
Injecting logs with a conidial suspension of E. vermicola reduced numbers of
pine wilt nematode by 49–79 % after 2 months. Moreover, spraying pine seed-
lings with a suspension of the fungus 1 month prior to inoculating with pinewood
nematode reduced wilting and increased tree survival (Wang et al. 2011a).
Attraction of Nematodes to Nematophagous Fungi
In addition to traps and adhesive conidia, some nematophagous fungi also utilize
another tactic to increase encounters with their hosts: they produce substances that
attract nematodes to them. The attraction intensity is related to the dependency of
the fungus on nematodes for nutrition (Jansson and Nordbring-Hertz 1979, 1980;
Jansson 1982a). Facultative saprobes such as Arthrobotrys spp. are generally less
attractive than obligate parasites such as Drechmeria spp.; Dactylellina spp. show
an intermediate level of attractiveness. The mycelia of nematophagous fungi attract
nematodes, but the presence of either traps or adhesive conidia increases the level
of attraction (Jansson 1982a, b). In more recent studies, attraction of pinewood
nematodes was greatest for E. vermicola, intermediate for Dreschlerella (=
Dactylaria) brochopaga, and least for Botrytis cinerea (Wang et al. 2010a).
Interestingly, E. vermicola is able to mimic the scent of pine trees by emitting vola-
tile compounds (two monoterpenes and a terpenoid) which are among the com-
pounds that attract pinewood nematodes to their host trees (Lin et al. 2013).
Parasites of Sedentary Nematode Stages
Root-knot nematodes (Meloidogyne spp.) and cyst nematodes (Heterodera and

Globodera spp.) are among the most economically important plant-parasitic nema-
todes worldwide. After penetrating the roots of a host plant and establishing a feed-
ing site, these nematodes become sedentary. Both root-knot and cyst nematodes lay
their eggs in a cluster surrounded by a gelatinous matrix; some eggs are also retained
within the body of female cyst nematodes. These tight clusters of eggs are particu-
larly vulnerable to fungal parasitism. The gelatinous matrix provides the eggs some
protection from microorganisms (Orion et al. 2001); however, specialized patho-
gens are able to overcome this protection. There is also circumstantial evidence that
some bacteria found within egg masses may also protect the eggs from nematopha-
gous fungi (Kok et al. 2001).
Fungi that are specialized for parasitizing sedentary stages of nematodes (eggs,
sedentary juveniles, and females) are from different lineages within the Hypocreales
including Pochonia spp. (Clavicipitaceae), Purpureocillium lilacinum (syn.
Paecilomyces lilacinus; Ophiocordycipitaceae), and Trichoderma spp.
(Hypocreaceae). Brachyphoris (syn. Dactylella) oviparasitica (Orbiliaceae), which
is related to trapping fungi but does not form traps, also parasitizes sedentary stages.
All of these fungi are facultative parasites of sedentary nematode stages; their abil-
ity to grow saprobically depends on the isolate, with some isolates exhibiting greater
levels of saprobic growth, often at the expense of parasitic activity (Siddiqui et al.
2009). The fungi listed above are commonly found in agricultural soils, often asso-
ciated with root-knot and cyst nematodes (Stirling 2014).
There are several species of Pochonia known to parasitize nematodes (Zare et al.
2001); however, most of the research has focused on P. chlamydosporia. Members
of the genus produce conidia on verticillate phialides as well as stalked, multicel-
lular chlamydospores (dictyochlamydospores). The two subspecies of P. chlamydo-
sporia are differentiated by whether conidia are produced in heads (var.
chlamydosporia) or in chains (var. catenulate), and both subspecies are parasites of
nematodes.
In the absence of nematodes, P. chlamydosporia is able to grow saprobically in
the rhizosphere of plants. Presumably, the fungus obtains nutrition from root exu-
dates and is often found in greater abundance in the rhizosphere of plants than in
bulk soil (De Leij et al. 1993; Mauchline et al. 2002). Colonization of the rhizo-
sphere by P. chlamydosporia varies among plant species (Manzanilla-Lopez et al.
2011; Bourne et al. 1996). For example, Bourne et al. (1996) observed greater sap-
robic colonization on brassicas (kale and cabbage) than on solanaceous plants
(tomato and eggplant). The presence of nematodes within the root system also
increases the abundance of P. chlamydosporia, particularly after egg deposition
begins; this increase may be due to parasitic growth or additional nutrient leakage
from galls (Bourne et al. 1996). The fungus has also been observed to grow endo-
phytically within the roots of both monocots and dicots (Bordallo et al. 2002;
Manzanilla-Lopez et al. 2011; Escudero and Lopez-Llorca 2012).
Upon contact with nematode eggs, P. chlamydosporia colonizes the surface of
the egg forming numerous appressoria and penetration pegs (Escudero and Lopez-
Llorca 2012; Segers et al. 1996). An alkaline serine protease designated VCP1 is
involved in infection of nematode eggs by P. chlamydospora (Morton et al. 2003a).
Polymorphisms within the gene encoding this enzyme are related to host preference
for either cyst or root-knot nematodes among fungal isolates. Other extracellular
enzymes (chitinases, lipases, and esterases) are also produced by P. chlamydospo-
ria, but their role in pathogenicity is not known (Esteves et al. 2009). Although it is
possible for isolates from root-knot nematode to infect cyst nematodes and vice
versa, the infection efficiency is greater when fungal isolates are obtained from the
same nematode genus (Siddiqui et al. 2009). In addition to differences in the VCP1
gene, isolates of P. chlamydosporia from root-knot nematodes can also be distin-
guished from those isolated from cyst nematodes with DNA fingerprinting (Morton
et al. 2003b).
Pochonia chlamydosporia has been a primary contributor in soils suppressive to
the cereal cyst nematode (Heterodera avenae) in England and Europe (Kerry et al.
1982; Kerry and Crump 1998), as well as soils suppressive to the southern root-knot
nematode (Meloidogyne incognita) in California (Bent et al. 2008). The fungus has
also been evaluated in numerous greenhouse and field studies for suppression of
plant-parasitic nematodes (see reviews by Siddiqui and Mahmood 1996; Timper
2011). The effectiveness of P. chlamydosporia in suppressing nematode popula-
tions depends on several factors in addition to the fungal isolate. The fungus is more
effective in suppressing nematode populations on moderate to poor host plants for
the nematode than on good host plants because of lower reproductive rates of the
nematode and, in the case of root-knot nematodes, smaller galls (Bourne and Kerry
1999). Bourne et al. (1996) observed greater parasitism of M. incognita eggs on
potato, which produced smaller galls with more exposed egg masses, than on
tomato, which produced large galls with more egg masses embedded in the gall tis-
sue. Similarly, P. chlamydosporia is less effective in reducing nematode popula-
tions in soils with heavy nematode infestations (De Leij et al. 1992). The presence
of the fungus in the rhizosphere early in the growing season is also critical.
Suppression of the cyst nematode H. schachtii by P. chlamydosporia was correlated
with the number of pre-gravid females infected rather than later infections of eggs
(Kerry 1988). Atkins et al. (2003) took advantage of the ability of P. chlamydospo-
ria to colonize roots in the absence of nematodes by applying the fungus to non-host
crops of M. incognita grown in rotation with tomato. The non-host crops reduced
populations of the nematode while supporting growth of the fungus leading to
greater suppression of M. incognita by P. chlamydosporia in the tomato crop.
Purpureocillium lilacinum has been isolated worldwide from soil, decaying veg-
etation, insects, nematodes, and mammals (Luangsa-Ard et al. 2011) and is com-
monly associated with plant-parasitic nematodes in agricultural fields (Kilama et al.
2007; Gaspard et al. 1990; Stirling and West 1991; Bernard et al. 1996). Given the
diverse substrates on which P. lilacinum is found, it is not surprising that there is
considerable genetic variability among isolates (Tigano-Milani et al. 1995b).
Isolates of the fungus also vary in their pathogenicity to nematode eggs; however,
there is no relationship between genetic clustering and pathogenicity (Tigano-
Milani et al. 1995a; Gunasekera et al. 2000). Unlike P. chlamydosporia or B. ovi-
parasitica, there is no indication of host specificity among isolates of P. lilacinum
for particular nematode genera. Encounters with nematode eggs and other sedentary
stages appear to be random, as no directed growth of P. lilacinum toward eggs has
been observed (Holland et al. 1999). When hyphae of the fungus grow over nema-
tode eggs, the hyphae become appressed to the egg surface and appressoria form at
the site of penetration. Although P. lilacinum is able to penetrate the nematode cuti-
cle and infect all sedentary stages, appressoria were only observed forming on eggs
(Holland et al. 1999; Khan et al. 2006). The fungus is believed to utilize both
mechanical pressure and enzymes to penetrate the nematode cuticle and egg shell
(Holland et al. 1999).
There is good evidence that some of the chitinases and proteases produced by P.
lilacinum are involved in pathogenicity. A serine protease from the Samson strain
of the fungus degraded the vitelline layer of M. hapla eggs and killed the embryos
(Bonants et al. 1995). Overexpression of the serine protease gene increased parasit-
ism of M. incognita eggs by 20 % (Wang et al. 2010b). Khan et al. (2004) observed
similar effects of a serine protease from strain 251 on nematode eggs, but also
observed greater structural damage to the eggs when both the protease and chitin-
ases from the fungus were combined than when the enzymes were applied sepa-
rately. Moreover, among isolates of P. lilacinum and P. chlamydosporia, efficacy
against M. incognita was strongly correlated with in vitro protease and chitinase
production (Wei et al. 2009). In some cases, P. lilacinum may kill nematodes with
toxins prior to infecting them. Both acetic acid and leucinostatins have been impli-
cated as the primary toxic metabolite in culture filtrates of P. lilacinum (Djian et al.
1991; Park et al. 2004); however, it is not clear whether these toxins are involved in
nematode mortality in the rhizosphere. Isolates of P. lilacinum varied in leuci-
nostatin production, but those isolates producing the toxin were only weakly patho-
genic to nematodes eggs (Park et al. 2004). Isolates may vary in their mode of action
with some being primarily pathogenic, while others are primarily toxic to nema-
todes. Similarly, different modes of action may be required for consuming different
hosts. For example, strain 251 readily infected eggs of Meloidogyne spp. but not
eggs of R. similis. Nevertheless, the eggs of R. similis appeared abnormal and other
stages of the nematode were immobilized when in contact with P. lilacinum hyphae;
these stages were eventually colonized by the fungus (Khan et al. 2006).
There have been conflicting reports about whether P. lilacinum colonizes the
rhizosphere or endosphere of plant roots. Some of these discrepancies may be due
to differences among isolates of the fungus; however, inconsistencies in root colo-
nization have also been observed for a single isolate. In two studies, rhizosphere
colonization of several host plants was not observed for P. lilacinum strain 251 and
persistence of the strain in soil was not increased in the presence of different crop
plants compared to fallow soil (Rumbos and Kiewnick 2006; Kiewnick and Gullino
2009). In a third study, however, abundance of strain 251 was greater in the rhizo-
sphere of sugar beet and rape than in non-rhizosphere soil (Manzanilla-Lopez et al.
2011). Rhizosphere colonization by strain 251 may depend on crop species or
cultivar or environmental factors such as soil type. For example, Manzanilla-Lopez
et al (2011) reported that strain 251 preferentially colonized the rhizosphere of rape
and sugar beet, but not the rhizosphere of potato or wheat. Cabanillas et al. (1988)
observed endophytic colonization of excised tomato roots by a Peruvian strain of P.
lilacinum. In a more extensive study with 10 crop species, P. lilacinum strain 251
was found endophytically at low levels in some, but not all crop species, and at high
levels in barley (Rumbos and Kiewnick 2006). These results were in contrast to
Holland et al. (2003) who did not observe endophytic growth of strain 251 in eight
crop species, including tomato and barley.
Although P. lilacinum is commonly found parasitizing nematode eggs in agricul-
tural soils, it has never been associated with soils that are suppressive to nematodes.
Abundance of P. lilacinum strain 251 in soil was not influenced by the presence of
host nematodes, suggesting that the fungus is not strongly dependent on nematodes
as a food source (Rumbos et al. 2008). The importance of soil organic matter as an
alternative food source of the fungus is unclear. Increasing the organic matter in soil
enhanced persistence of the P. lilacinum, while increasing the sand content dimin-
ished persistence (Rumbos et al. 2008). However, persistence may have also been
influenced by leaching of conidia, which would have been reduced by the organic
matter and increased by the sand content. Application of organic matter in the form
of cow manure had no influence on either the percentage suppression of nematodes
by P. lilacinum or re-isolation of the fungus from M. incognita females and egg
masses (Siddiqui and Futai 2009).
Purpureocillium lilacinum is commercially available in several countries for
control of plant-parasitic nematodes and numerous studies have evaluated its effi-
cacy under greenhouse and field conditions (see Stirling 1991; Siddiqui and
Mahmood 1996; Stirling 2014 for reviews). In general, the fungus shows greater
efficacy in suppressing nematode populations when it is applied 1–2 weeks before
planting to allow longer exposure of nematode eggs to infection (Anastasiadis et al.
2008; Mendoza et al. 2007). As mentioned earlier, P. lilacinum has low persistence
in soil; therefore, multiple applications early in the season have shown greater
reduction in nematode numbers than single applications (Cabanillas and Barker
1989; Mendoza et al. 2007; Anastasiadis et al. 2008; Udo et al. 2013).
Brachyphoris oviparasitica and the sterile fungus designated Arkansas Fungus
(ARF) are closely related based on phylogenic analysis of rRNA genes (Yang et al.
2012) and will be discussed together as related Brachyphoris species. Both B. ovi-
parasitica and ARF parasitize eggs of cyst and root-knot nematodes (Kim and
Riggs 1991; Stirling et al. 1979). Nematodes that are embedded in the root are pro-
tected from infection; however, juveniles and females of cyst nematodes break
through the epidermis of the root as they develop and are readily parasitized by
these fungi (Kim and Riggs 1991; Timper et al. 1999; Becker et al. 2013; Stirling
et al. 1979). Additionally, ARF parasitizes eggs and sedentary females of the reni-
form nematode, R. reniformis (Wang et al. 2004b). Though not well studied, there
appears to be some host specificity among isolates of ARF. For example, isolates
from the soybean cyst nematode (H. glycines) infected the eggs of several other
species of Heterodera and M. incognita, but did not infect eggs of the reniform
nematode or the tobacco cyst nematode (Globodera tabacum) (Kim and Riggs
1991; Wang et al. 2004b). The mechanism of infection by B. oviparasitica and ARF
is unknown. When infecting juveniles and females of cyst nematodes, both fungi
produce dense mats of mycelium on the nematode cuticle (Timper et al. 1999;
Becker et al. 2013). Penetration holes are formed under these mats in a manner sug-
gestive of enzymatic activity (Kim et al. 1992).
The fungus ARF is able to colonize and decompose organic matter in bulk soil
(Wang et al. 2004a). After application of mycelium to soil without nematodes, ARF
formed abundant mycelial mats primarily in the bulk soil with only a few mats
attached to roots (Timper et al. 1999). Similarly, B. oviparasitica did not show tro-
pism towards roots (Becker et al. 2013), suggesting that, unlike P. chlamydosporia,
these fungi do not preferentially colonize the rhizosphere. Stirling et al. (1979),
however, observed greater abundance of B. oviparasitica in rhizosphere soil from
peach than in non-rhizosphere soil, possibly due to the presence of parasitized egg
masses on the root. More research is needed to determine whether B. oviparasitica
and ARF colonize roots in the absence of nematodes.
Brachyphoris oviparasitica was first identified in peach soils that were suppres-
sive to root-knot nematodes in California (Stirling and Mankau 1978). The fungus
was later shown to be the primary organism responsible for the natural suppression
of root-knot nematodes in peach and the suppression of the sugar beet cyst nema-
tode (H. schachtii) in an experimental field site in California (Stirling et al. 1979;
Westphal and Becker 2001; Yin et al. 2003). The fungus ARF has been associated
with natural suppression of the soybean cyst nematode (H. glycines) in Arkansas
(Kim and Riggs 1991). Additionally, ARF has been isolated from H. glycines from
several locations in the Mid-South region of the USA. (Kim et al. 1998). Although
B. oviparasitica and ARF have shown promise as biological control agents of cyst
and reniform nematodes in greenhouse and microplot experiments, no studies have
evaluated these fungi under field conditions (Olatinwo et al. 2006a, b, c; Wang et al.
2004b; Timper and Riggs 1998). As with P. chlamydosporia, the host plant contrib-
utes to the level of nematode suppression by B. oviparasitica. A greater percentage
of M. incognita eggs were parasitized by B. oviparasitica on peach than on tomato
(96 % vs. 57 %), presumably because the fungus was more efficient at colonizing
the smaller egg masses on peach than the larger egg masses on tomato (Stirling
et al. 1979).
The genus Trichoderma is well known for containing widely distributed free-
living soil fungi, but species and strains in this genus are also associated with other
habitats, as plant symbionts, and as fungal parasites. The Trichoderma anamorph
has been linked with the teleomorph Hypocrea. The taxonomy of these fungi is very
difficult due to limited and variable morphological characters and is becoming
increasingly reliant on molecular analyses for species identification (Samuels 2006).
Trichoderma species are widely adapted to interact with plants and plant patho-
gens over a wide range of environments to increase plant growth and reduce plant
disease and have many attributes which allow them to be successful in different
environments. These fungi can grow saprobically, endophytically, or in the rhizo-
sphere and phyllosphere transition zones; they produce large numbers of propagules
and antibiotic compounds that result in competitive advantages against many other
microorganisms, and they can be successful mycoparasites (Howell et al. 2000;
Howell 2006; Woo et al. 2006). As a result, Trichoderma strains have been devel-
oped as successful biological controls of fungal plant pathogens (Harman 2006).
Trichoderma spp. antagonize nematode by multiple mechanisms. Many of the
attributes which allow Trichoderma to be successful biological control agents of
fungi may also result in control of plant-parasitic nematodes (Sharon et al. 2011).
Certain Trichoderma spp. produce proteases and chitinases which may play a role
in suppression of nematode populations (Sharon et al. 2011). Trichoderma strains
that can grow in the gelatinous matrix of egg masses previously thought to protect
eggs from microbes may directly parasitize root-knot nematode eggs (Sharon et al.
2001). Howell (2002) reported that Trichoderma can metabolically break down
root exudates as they diffuse into the rhizosphere or spermosphere before they can
stimulate the germination of pathogen propagules such as oospores, effectively
masking plant roots and preventing infection. This same process may be utilized to
reduce egg hatch and reduce or delay root infection by nematodes which hatch in
response to host root exudates. In addition, T. atroviride produces nematicidal com-
pounds that reduce hatch of immature eggs, but increase hatch of mature eggs
(Sharon et al. 2007).
Antagonistic Plant Symbionts
Several fungi which form symbiotic associations with plants resulting in improved
plant growth also suppress populations of plant-parasitic nematodes; they are often
endophytic within plant roots. Unlike the fungi discussed previously, these fungi
are not specialized for parasitizing nematodes, but instead antagonize nematodes by
producing toxins, altering root exudates, or inducing a resistance response in plants.
Toxins and Extracellular Enzymes
While numerous fungi produce metabolites in liquid media that are toxic to nema-
todes, it is not clear if these metabolites are produced in sufficient quantities in roots
to affect nematode viability or behavior. Therefore, only studies demonstrating
activity of metabolites against nematodes at concentrations found in root tissue or
root exudates will be discussed below.
Neotyphodium spp. are asexual forms of Epichloë (Clavicipitaceae) which are
mutualistically associated with grasses in the family Poaceae. Neotyphodium spp.
are exclusively endophytic and seed transmitted. After the seed germinates, the
mycelium grows intercellularly in the stem and leaf sheath, but does not grow into
the leaf blade or roots. The most well studied of these grass/endophyte associations
has been tall fescue (Schedonorus arundinaceus) and its endophyte N. coenophialum,
and perennial ryegrass (Lolium perenne) and its endophyte N. lolii. These endo-
phytes confer resistance to many abiotic and biotic stresses, including herbivory
from mammals, insects, and nematodes. Neotyphodium spp. produce four groups
of alkaloids which are involved in either toxicity or feeding deterrence to verte-
brates and invertebrates that feed on grasses containing them (Schardl et al. 2004).
The endophyte in tall fescue is able to suppress populations of plant-parasitic nem-
atode, notably Pratylenchus scribneri and Meloidogyne marylandi, even though

the fungus is not present in the roots (West et al. 1988; Kimmons et al. 1990; Timper
et al. 2005). Although the mechanism by which the fungus confers nematode resis-
tance to the plant is unknown, it is believed that one or more of the biologically
active alkaloids are involved in the suppression. Root extracts of endophyte-
infected tall fescue paralyzed P. scribneri and reduced egg hatch and viability of M.
incognita J2 (Bacetty et al. 2009; Meyer et al. 2013). Root exudates of endophytic
tall fescue also reduced egg hatch and J2 viability, suggesting that compounds both
within the root and exuded from the root are involved in nematode suppression.
Except for peramine, most of the other alkaloids (ergovaline, lolitrem B, and the
lolines) are found in low concentrations in roots (Siegel and Bush 1996). Knockout
mutants of Neotyphodium sp. lacking ergot alkaloids, including ergovaline, still
conferred resistance to P. scribneri in annual ryegrass, indicating that the ergot
alkaloids are not essential for nematode suppression (Panaccione et al. 2006).
Lolitrem B is not produced in the tall fescue/N. coenophialum association and is,
therefore, not a likely source of resistance to nematodes. Of the bioactive alkaloids
produced by Neotyphodium spp., the lolines have the greatest potential to affect
nematodes. These alkaloids are found in both root tissue and exudates of endophyte
infected tall fescue at concentrations that are toxic to P. scribneri (Bush et al. 1993;
Bacetty et al. 2009).
Alteration of Root Exudates
Fusarium spp. are cosmopolitan, ecologically diverse fungi in the family Nectriaceae
(Hypocreales). Although Fusarium spp. are important plant pathogens, many spe-
cies and strains are nonpathogenic to plants. Fusarium spp. are commonly found
growing endophytically within the roots of a number of plant species with F. oxys-
porum being the most common species (Macia-Vicente et al. 2008). Hallmann and
Sikora (2011) have reviewed research evaluating the efficacy and mode of action of
Fusarium endophytes for suppression of plant-parasitic nematodes. One of the
mechanisms of nematode suppression appears to be altered root exudates. Fewer M.
incognita and Radopholus similis were attracted to and infected roots containing F.
oxysporum strain 162 than control plants (Dabatat and Sikora 2007b; Hallmann and
Sikora 2011). Moreover, in the absence of plants, root exudates from tomato con-
taining F. oxysporum attracted fewer M. incognita than exudates from control
plants. It is not clear whether the exudates contain a toxin which repels the nema-
todes or contains components that are less attractive to the nematodes. The effect of
F. oxysporum in reducing attraction and penetration of roots may be dependent on
the strain of the fungus or the plant species. In banana, there was no difference in
attraction or penetration by R. similis between roots without endophytes and roots
containing one of three different strains of F. oxysporum (Athman et al. 2006, 2007).
Arbuscular mycorrhizal fungi (AMF) form obligate mutualistic relationships
with plants. These fungi live within the cortical tissue of roots and enhance plant
growth through nutrient uptake and suppression of pathogens. There are several
genera of AMF, all in the order Glomerales (Morton and Benny 1990), including
Gigaspora, Scutellospora (Gigasporaceae), Glomus, Sclerocystis (Glomaceae),
Acaulospora, and Entrophospora (Acaulosporaceae). Numerous studies have inves-
tigated the interaction between AMF and plant-parasitic nematodes. Recent reviews
of the literature indicate that in most studies, plants colonized by AMF have lower
nematode populations than plants without AMF (Hol and Cook 2005; Hallmann
and Sikora 2011; Veresoglou and Rillig 2012). Whether or not AMF confer resis-
tance to plant-parasitic nematodes depends on the genus of AMF, AMF-plant speci-
ficity, genus of nematode, and the timing of AMF inoculation. The mechanism by
which AMF influences nematode populations is also likely dependent on the genus
of AMF and plant. In both tomato and banana, AMF colonization of roots appears
to alter the root exudates leading to fewer nematodes penetrating AMF compared to
non-AMF roots (Vos et al. 2012a, b). On agar plates, root exudates from AMF
plants repelled R. similis, whereas exudates from non-AMF plants were either neu-
tral or attracted the nematode.
Piriformospora indica (Basidiomycota: Sebacinaceae) is a plant growth-
promoting fungus that endophytically colonizes roots of a wide range of plants.
This fungus has been shown to increase plant tolerance to stress, induce disease
resistance, and increase plant yields (Waller et al. 2005). In two greenhouse studies,
P. indica reduced populations of H. schachtii in Arabidopsis and H. glycines in
soybean (Daneshkhah et al. 2013; Bajaj et al. 2015). One mechanism of suppression
appears to be altered root exudates; exudates of colonized soybean roots were less
attractive to infective juveniles of H. glycines than exudates from control roots
(Daneshkhah et al. 2013).
Induced Resistance
In the last 20 years, it has become increasingly clear that many nonpathogenic
microorganisms living in the rhizosphere or endosphere of plant roots trigger a
heightened immune response in the plant (Pieterse et al. 2014). This phenomenon is
referred to as induced systemic resistance (ISR). In ISR, the microbes prime the
plant defense system leading to a faster or stronger resistance response to pathogen
infection. Using a split-root system to physically separate the inducing organism
from plant-parasitic nematodes, several studies have demonstrated that AMF-
colonized plants elicit a systemic resistance response in grapevine, tomato, and
banana against nematodes (Elsen et al. 2008; Hao et al. 2012; Vos et al. 2012c). In
both tomato and grapevine, expression of defense-related genes was greater in AMF
plants challenged with nematodes than in AMF alone or nematode alone plants,
suggesting that AMF prime the plant defenses for subsequent nematode infection
(Li et al. 2006; Hao et al. 2012; Vos et al. 2013).
Other endophytic antagonists of nematodes are also capable of inducing systemic
resistance in plants. Numerous studies with Trichoderma spp. have demonstrated
ISR responses against a wide range of plant pathogens (Harman 2006; Woo et al.
2006). The ability to colonize plant tissue as an endophyte may be key to induction
of plant defense responses (Howell 2006). Trichoderma harzianum (strain T10) was
shown to induce systemic resistance to the root-knot nematode M. javanica in a
split-root tomato system, alone or in combination with salicylic acid or jasmonic
acid (Selim et al. 2014). Similarly, endophytic, nonpathogenic strains of F. oxyspo-
rum also induce resistance to plant-parasitic nematodes in banana and in tomato (Vu
et al. 2006; Dababat and Sikora 2007a; Martinuz et al. 2012). Paparu et al. (2013)
further showed that the F. oxysporum primed banana for greater expression of
defense-related genes when infected by R. similis. Although not demonstrated with
plant-parasitic nematodes, Pedrotti et al. (2013) used a split-root hydroponic system
to show that root infection by P. indica triggers a priming of defense responses in
Arabidopsis. It is likely that plants colonized by P. indica exhibit ISR responses
against nematodes given the pervasive association between endophytes and ISR
responses.
Interestingly, trapping fungi and parasites of sedentary stages of nematodes, par-
ticularly endophytic strains of these fungi, are also capable of inducing systemic
resistance to nematodes. Colonization of roots by both endophytic and rhizospheric
strains of A. oligospora reduced nematode numbers and increased defense-related
enzymes in tomato compared to plants inoculated only with M. incognita or without
nematode and fungus (Singh et al., 2013). The endophytic strain was superior to the
rhizospheric strain both in terms of suppressing nematode numbers and enhancing
the immune response of the plant. An endophytic strain of P. chlamydosporia caused
a moderate induction of genes involved in ISR in barley (Hordeum vulgare); how-
ever, the plants were not challenged with plant-parasitic nematodes to conclusively
demonstrate priming for resistance to nematodes (Larriba et al. 2015).
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Chapter 24
Pathogenic Microfungi Associated
with Spartina in Salt Marshes
Wade H. Elmer
Introduction
Diebacks and/or disease outbreaks caused by fungal pathogens in natural ecosys-

tems, such as salt marshes, are not well studied, in part, due to the rarity of these
events, and their perceived economic insignificance when compared to agricultural
systems. However, it may be surprising to many that salt marshes are one of the
most valuable ecosystems and outrank tropical rain forests and coral reefs for pro-
ductivity. Carbon sequestration can approach more 3 kg dry matter/m2/year which
outperforms coral reefs by 40 % (Bertness 2007). Furthermore, their value in
absorbing excess nitrogen and phosphorus, detoxifying pollutants, and providing
habitat for marine animals elevates the salt marsh ecosystem as the most valuable
natural ecosystem on the planet in terms of productivity.
What is also surprising is the low number of dieback events or disease outbreaks
that have been documented in salt marshes. This may be, in part, due to confusion
in recognizing a disease outbreak versus the usual death and recovery observed dur-
ing accretion and subsidence that is common to a salt marsh. It is interesting that salt
marshes are exclusively dominated by only a few foundation plant species. In tem-
perate climates, species in the genera Spartina and Juncus compose the majority of
acreage, whereas in tropical regions, salt marshes yield to the species of mangroves
(Rhizophora spp.). Most of the low marshes in northern and southern temperate
climes are dominated by species of Spartina. In the northwestern Atlantic down
through the Gulf, and spotted locales in Southwestern Atlantic marshes, Spartina
alterniflora Loisel dominates the low marsh habitat. Northeastern Pacific marshes
are dominated by S. foliosa whereas tidal marshes on the west and east coast of the
W.H. Elmer, Ph.D. (*)

Department of Plant Pathology and Ecology, The Connecticut Agricultural Experiment
Station, P. O. Box 1106, New Haven, CT 06504, USA
e-mail: Wade.Elmer@ct.gov

DOI 10.1007/978-3-319-29137-6_24
616 W.H. Elmer
South American continent are colonized by S. densiflora. In the northeastern

Atlantic marshes in Europe, S. maritima dominates the low marsh.
However, over the past century, S. alterniflora has invaded many foreign marshes
and hybridized with native species to generate fertile hybrids (Ainouche et al. 2009;
Sloop et al. 2009). This has resulted in a wide genetic assortment of genotypes that
confer both selective advantages (resistance) and disadvantages (susceptibilities) to
fungi and stressors. For example, the first recorded account of a dieback was
observed on a Spartina hybrid in the 1950s along the marshes in the Hampshire
basin in England (Goodman 1959; Goodman et al. 1959; Goodman and Williams
1961). A hybridized Spartina species called S. anglica, an allopolyploid hybrid
derived from the chromosome doubling of S. x townsendii [a cross between S. alter-
niflora and S. maritima (Ayers and Strong 2001)], had dominated southern England
marshes. Over 200 ha of marshes declined and died. Since the attempt by Goodman
et al. (1959) to transmit the dieback from unhealthy plants to healthy plants failed,
they concluded a parasite was not involved. This conclusion was further supported
by the observation that, of over 20 fungal species isolated from dead rhizomes onto
nonselective media, none was demonstrated to be pathogenic. Although many
potential pathogens like Fusarium spp. were not reported, these species might eas-
ily have been missed in their study, since dead, not declining, plants were sampled,
and since selective agars were not used.
However, even when a fungal pathogen is identified, the actual contributions of
the disease to the dieback of the marsh are often indirect or not clear. Examples
below will depict different cases. Sometimes a pathogen is clearly present, yet eco-
logical costs are marginal whereas in other examples, a combination of factors, such
as genetic shifts in the host species, herbivores, and/or other plant stressors like
drought, rising sea levels, excessive salinity, nitrogen, and phosphorus, may interact
with a fungal pathogen and lead to a major dieback. Deciphering the actual contri-
butions of the fungal pathogen becomes a daunting task in light of climate change
stressors. The following chapter highlights four examples where microfungi were
associated with dieback/disease events in salt marshes. When possible the life
cycles, genetic relationships, and ecological implications are presented.
Fusarium palustre (W. H. Elmer & R. E. Marra) and Other

Fusarium spp.
Distribution
Fusarium palustre is an endophytic fungus that is closely associated with S. alterni-

flora in Northern hemispheres (Elmer and LaMondia 2011; Elmer and Marra 2011;
Elmer et al. 2013) (Fig. 24.1). It was recently described and found exclusively in
salt marshes extending from the Gulf of Mexico to New England. It has also been
isolated from the invasive S. alterniflora plantings that have invaded salt marshes in
24 Pathogenic Microfungi Associated with Spartina in Salt Marshes 617
Fig. 24.1 Macroconidia of

Fusarium palustre
Brittany, France (unpublished data). A survey of salt marshes in Argentina did not
find F. palustre on S. alterniflora or S. densiflora, indicating the fungus may have
geographical limits (unpublished data).
On Dongtong wetland on Chongming Island in Shanghai, China, the invasive S.
alterniflora was deliberately introduced and spread into areas where other native
species began to die back. One such species was the native common reed grass,
Phragmites australis. It was revealed that F. palustre had spread from S. alterniflora
onto Phr. australis and was associated with a major dieback of that plant (Li et al.
2014). The dieback was only observed in marshes where S. alterniflora had invaded
and was never isolated in pure stands of Phr. australis. Aside from S. alterniflora,
Phr. australis is the only other host reported for F. palustre.
Along with F. palustre are many species of Fusarium that are found colonizing
S. alterniflora that appear to be avirulent or only slightly pathogenic (Elmer and
LaMondia 2011; Elmer and Marra 2011; Elmer et al. 2013). These species fall into
the F. incarnatum-equiseti species complex (O’Donnell et al. 2009). There have
been other reports where Fusarium spp. associated with salt marsh plants were
reported in the literature. The first report was a brief description of a Fusarium sp.,
called F. spartinae Ellis & Everh., observed on leaves of Spartina stricta Roth (syn
S. maritima) (Ellis and Everhart 1902). However, the species description, made
in vivo, was far too generic to be considered synonymous with F. palustre and no
isolates were saved. Other surveys have mentioned Fusarium sp. (Gessner and
Goos 1973; Gessner and Kohlmeyer 1976), but the isolates were not identified and
are no longer available. Therefore, it is not certain if these previous reports had been
on F. palustre.
There was another interesting association between a Fusarium species and the
ergot fungus (Claviceps purpurea) that was found colonizing S. anglica in England
(Preece et al. 1994). F. heterosporum Nees & T. Nees was found associated with the
618 W.H. Elmer
ergots, but no pathology between the F. heterosporum and C. purpurea or S. anglica

was documented (Preece et al. 1994; Raybould et al. 1998).
Description
When DNA sequence queries of the translation elongation factor (tef1) of F. palus-
tre isolates did not closely match any known species of Fusarium, a phylogenetic
analysis was performed (Elmer and Marra 2011). Combined partial sequences of
three genes, b-tubulin, calmodulin, and tef1, were aligned for 20 F. palustre isolates
along with other Fusarium species. Strong bootstrap support provided evidence that
the F. palustre isolates along the Atlantic coast were closely related and represented
a new species (Elmer and Marra 2011). Its closest known relative based on these
sequences was F. sporotrichioides and F. langsethiae.
The fungus produces macro- and meso-conidia in monophialides in vitro, but the
fungus has not been observed sporulating on the host. This may be due to the fre-
quent washing and removal of spores from tidal action. It is not clear what role
spores function in infection or dispersal. The fungus has been isolated from marsh
water, but the propagule was not identified [unpublished data]. Chlamydospores are
produced intercalary in mycelium and these propagules may function more in dis-
persal on pieces of tissue than the other conidia. Chlamydospores and thick-walled
hyphae may also function during periods of stress as survival propagules. During
low tides when drought conditions prevail, saline conditions can be excessive on
and around S. alterniflora plants. F. palustre is more saline tolerant than most
Fusarium spp. Elmer and LaMondia (2014) found that hyphae of F. palustre were
uninhibited on NaCl-amended agar at levels of 0.27 M NaCl (equivalent to marsh
water) whereas genetically similar terrestrial species, F. sporotrichioides, showed
immediate inhibition at 0.14 M NaCl. F. palustre has also been found to produce
T-2 toxins (personal communication with Dr. Susan McCormick, ARS, Peoria, IL),
but the role of these toxins on pathogenicity or survival is not clear.
Pathology and Life Cycle
In the Western Atlantic, F. palustre has been implicated in Sudden Vegetation

Dieback (SVD) (Fig. 24.2). Of over 10 species of Fusarium found colonizing
Spartina only F. palustre isolates were capable of inciting stem rot and plant stunt-
ing (Elmer and LaMondia 2011; Elmer and Marra 2011; Elmer et al. 2013).
However, in other studies, isolates of Fusarium in the Gibberella fujikuroi species
complex (O'Donnell et al. 1998) could also incite disease, but these isolates may be
more restricted to southern locales (Elmer et al. 2013).
SVD affects primarily S. alterniflora and is characterized as a rapid decline that
begins with thinning and/or browning of the aboveground foliage of S. alterniflora
Fig. 24.2 Sudden vegetation dieback of Spartina alterniflora along intertidal creek bank in
Branford, CT
followed by death of the rhizomes (Alber et al. 2008; Elmer et al. 2013; Smith and
Carullo 2007). Once plants die, barren areas of remnant peat remain indefinitely.
The defining signature of SVD is death of the rhizomes and a very slow recovery
that can take from 1 to more than 10 years. It was originally called Brown marsh
(McKee et al. 2004). Surveys have reported up to 10 Fusarium species have been
isolated from S. alterniflora in SVD sites, but 3 out of 4 colonies isolated from S.
alterniflora give rise to F. palustre (Elmer et al. 2013). The fungus was consistently
recovered from plants in SVD sites, but can still be found in low densities in marshes
where no SVD occurs. The fungus has been isolated from roots, crowns, stems, and
seeds, but the incidence is usually greatest in the basal stem sections (Elmer and
LaMondia 2011). Isolation from marsh soil is relatively rare, so F. palustre may not
persist as a typical soilborne pathogen.
Inoculation of healthy plants with F. palustre rarely results in death. Stem lesions
do result from stab inoculations and generally stunting is observed following conid-
ial drenches of roots (Elmer 2014; Elmer and Marra 2011; Elmer et al. 2013) (Fig.
24.3). Infected plants usually have less vigor than healthy plants and symptoms are
always greater when plants are stressed by drought or poor nutrition [(Elmer 2014;
Elmer and LaMondia 2011); unpublished data]. Therefore, F. palustre may operate
more as a component of a multilayered ecosystem under multiple stressors. Recent
studies have shown that fungal infection can render plants more vulnerable to
620 W.H. Elmer
Fig. 24.3 Stem lesions on Spartina alterniflora following pathogenicity tests with different iso-
lates of Fusarium palustre
herbivory by the purple marsh crab (Sesarma reticulatum) (Elmer 2014). Intense
grazing by the purple marsh crab was strongly correlated with SVD sites (Altieri
et al. 2012; Holdredge et al. 2009) and controlled studies found that the purple
marsh crab grazed more on disease-stressed S. alterniflora plants than on healthy
plants (Elmer 2014). One possible mechanism that could explain the increased
attraction is chemotaxis where stressed plants may emit volatiles that attract crabs.
No such attractants have been identified. However, S. alterniflora is unique in that
it contains dimethylsulfoniopropionate (DMSP), a naturally occurring putative
osmolyte, which is oxidized to dimethylsulfoxide (DMSO) during periods of stress
(Husband et al. 2012). Studies in Georgia found that the DMSO:DMSP ratio was a
sensitive indicator of presymptomatic stress in S. alterniflora and consistently
greater in leaves and stems of plants in dieback sites (McFarlin and Alber 2013). In
one preliminary trial, we have found the healthy S. alterniflora transplants sprayed
with DMSO at 2.5 μmoles/ml and set in mecocosms with purple marsh crabs were
grazed significantly more in the first 24 h than untreated control plants (unpublished
data, P > 0.001). The role of volatile compounds as a chemoattractant in S. alterni-
flora is still not clear.
Fig. 24.4 Ergot of

Spartina alterniflora
caused by Claviceps
purpurea var. spartinae
(Courtesy of Court
Stevenson and Lorie
Staver, University of
Maryland)
Claviceps purpurea var. spartinae R.A. Duncan & J.F. White
Distribution
Ergot, caused by the ascomycetous fungal pathogen, Claviceps purpurea, was first
discovered along the Gulf of Mexico in 1895 on S. alterniflora (Tracy and Earle
1895) (Fig. 24.4). C. purpurea appeared to be a resident fungus in most US and
European marshes causing disease on S. alterniflora, S. foliosa, and Spartina
hybrids, but incidence can be very low to over 96 % depending on host and environ-
mental conditions (Eleuterious 1970; Eleuterius and Meyers 1974; Ellis and
Everhart 1902; Fisher et al. 2005a). Eleuterius and Meyer (1974) reported that
Distichlis spicata, Spartina patens, and Spartina cynosuroides could also serve as
hosts of C. purpurea.
622 W.H. Elmer
Description
Duncan et al. (2002) proposed listing the pathogen on Spartina as a separate variety
of C. purpurea and named it C. purpurea var. spartinae. They found that sequences
of the ITS regions placed the Spartina pathogen into the clade of C. purpurea, but
distinguished it based on morphological differences in the sclerotia and unique
alkaloid profiles. It is interesting that the sclerotia from isolates of C. purpurea var.
spartinae were able to float in saline water whereas sclerotia from isolates from
other hosts sank. This adaption to the marsh environment provides a selective
advantage and aids in dispersal. Pažoutová et al. (2002) further classified the C.
purpurea population colonizing S. anglica in Britain and S. alterniflora in the
USA. In addition to floating sclerotia, they noted unusually long cylindrical conidia.
Molecular assays utilizing RAPDs, AFLPs, and sequences from rDNA compared
isolates from other hosts and concluded that the Spartina isolates were a genetically
distinct, homogeneous population of C. purpurea. The same morphological and
genetic markers were found also in S. alterniflora isolates from Spartina from the
USA. All Spartina isolates belonged to a fungal chemotype that produces the alka-
loids ergocristine and ergocryptine (Pažoutová et al. 2002). Given the similarity
between the Spartina isolates, it was speculated that a common origin was likely
and that the British stands of S. anglica were likely colonized by isolates introduced
from America on S. alterniflora.
Pathology and Life Cycle
Infection in the UK was greatest on S. alterniflora florets when the plant had recently
recolonized manmade beaches and sites where only barren peat persisted (Preece
et al. 1994). They also noted greater incidence on plants closer to the water than
more inland, but offered the observation that this may be due more to conditions
that affect flowering than environmental conditions that affect ergot infection and
development. However, these observations led the authors to suggest that plant
stress might increase infection and to be mindful of how marsh disturbances like
canals and other ecological modifications might respond to ergot infection. In gen-
eral, outbreaks are considered relatively rare (Fisher et al. 2005b). However, the
ergot disease of Spartina can rapidly spread when cool, wet conditions prevail and
when a more susceptible homogeneous germplasm dominates the marsh.
Not much information is available on the actual infection cycle on Spartina, but
it likely follows the same patterns known for most terrestrial plants (Tudzynski and
Scheffer 2004). Gray et al. (1990) studied ergot on S. anglica in England and stated
that windblown ascospores derived from flask-shaped perithecia on overwintering
sclerotia land on grass florets at anthesis in the spring to germinate. Once the cuticle
has been invaded, the hyphae colonize the ovarian tissue, grow down toward the
base of the ovary, and colonize the vascular tissue. The pathogen develops a
Fig. 24.5 Lesions of Spartina rust caused by Puccinia sparganioidis in initial stages (Courtesy of
Carrie Knott, University of Kentucky)
mycelial stroma, called a sphacelium, in the ovary and produces masses of conidia
that are exuded into a sugar-rich fluid derived from phloem sap. These conidia pro-
vide the summer inoculum that initiates the spread of the disease during the growing
season. The ergot fungus is homothallic and the perithecia are produced on the
sclerotia which drop from the plant as the plant senesces. Sclerotia float with the
tides into rack lines and barren mud flats to overwinter (Gray et al. 1990). It is
unclear how long the sclerotia would remain viable in the saline water.
In England and on the west and east coast of the USA, widespread ergot epidem-
ics have been recorded in salt marshes (Fisher et al. 2005b; Gray et al. 1990;
Pažoutová et al. 2002; Raybould et al. 1998; Van Dyke and Amerson 1976). From
1983 to 1995, a detailed survey was conducted in England on the incidence of ergot
on the hybridized S. anglica (Raybould et al. 1998). Raybold et al. (1998) reported
that the disease caused no overall differences between the number of seed set on
infected compared to uninfected inflorescences. However, when the rate of infec-
tion was considered, heavily infected inflorescences had less seed set whereas inci-
dences less than 10 % produced more seed per inflorescence. Each year infection by
ergot was relatively uniform on S. anglica so no wide diversity in susceptibility was
thought to exist in the host population (Raybould et al. 1998).
Conversely, in the San Francisco Estuary, outbreaks of ergot regularly occurred
on the native S. foliosa, but the hybrids that formed between the introduced S. alter-
niflora and the native S. foliosa were more resistant to ergot and sustained lower
levels of infection (Fisher et al. 2005a, b). Since these hybrids were more robust and
competitively superior to S. foliosa, they have spread extensively throughout these
marshes. In marshes where the native and hybrid coexist, the higher rates of
624 W.H. Elmer
Fig. 24.6 Lesions of Spartina rust caused by Puccinia sparganioidis in later stages (Courtesy of
Carrie Knott, University of Kentucky)
infection on the native S. foliosa reduce plant fecundity more than on the hybrid,
which in turn speed the displacement of the native S. foliosa in these ecosystems.
Puccinia sparganioidis Ellis & Barthol
Distribution
Puccinia sparganioidis (often reported as Puccinia sparganioidis Ellis & Tracy) is

a macrocyclic rust disease of Spartina spp. that shares three of five spore stages on
its alternate host, ash (Fraxinus spp.) (Figs. 24.5 and 24.6). It was first observed by
W. G. Farlow in 1883 in Massachusetts and labeled as Uromyces spartinae Farl.
(Arthur 1902). His report coincided with a major outbreak of the fungus on white
ash (Fraxinus americanus) (Anonymous 1916; Arthur 1902). At present, the known
distribution of Pu. sparganioidis includes the USA and Canada east of the Rocky
Mountains, Mexico, and Brazil (Anonymous 1916; Arthur 1902; Gray et al. 1990).
The disease has previously been reported on S. alterniflora in Connecticut,
Delaware, Florida, Louisiana, Maine, Maryland, Massachusetts, Mississippi, North
Carolina, New Hampshire, Rhode Island, Vermont, and Virginia (Davelos et al.
1996). There are no reports of it occurring in Asia, Europe, or South America on
Spartina spp. or Fraxinus spp.
Description, Pathology, and Life cycle
As an obligate parasite, Pu. sparganioidis, is only found in association with its hosts
(Fraxinus spp. and Spartina spp.). It is a heteroecious macrocyclic rust possessing
five spore stages in succession, two of which must occur on a Spartina spp. and the
other three on ash (Fraxinus spp.) (Arthur 1902) In the spring, overwintering telio-
spores germinate on Spartina residues and the basidiospores are released where
they infect the current-year tissues of ash, causing spermogonia. Aecia develop in
these lesions on ash and release aeciospores that must infect a species of Spartina.
Once infection has occurred, uredinia develop in early summer releasing uredinio-
spores that repeatedly infect Spartina spp. causing numerous orange, long, hypo-
phyllous lesions. The uredinial lesions become erumpent giving rise to new colonies
of urediniospores. Uredinia eventually develop into brownish-black telia in the fall.
However, Kaur et al. (2010) did not observe telia on S. alterniflora in Louisiana.
Basidiospores then infect the ash the following spring if weather conditions are
favorable for infection.
The disease has been noted in the salt marsh on S. alterniflora, S. cynosuroides,
and S. patens. There are few reports stating it developed on Distichlis spicata. The
fungus is also reported throughout the Midwest where it completes its life cycle on
prairie cord grass (Spartina pectintata). On ash, it is reported on several (Fraxinus)
species including white, green, and occasionally, black ash. Although outbreaks are
relatively common, there is no evidence, thus far, that Spartina rust is limiting or has
major ecological costs to Spartina. Van Dyke and Amerson (1976) found more rust
infection of S. alterniflora on plants grown with higher soil water salinity and sug-
gested surface salts may be inhibitory to aeciospores and urediniospores.
However, the damage caused to Fraxinus can be aesthetically limiting causing
disfigurement and premature defoliation. Heavy infections over several years could
weaken the tree. It is likely that the same scenarios could result in weakening
Spartina spp. in the marsh if infections were heavy and prolonged over many
seasons.
Studies to determine whether clonal selection in the host could occur and result
in more virulent pathotypes found no pattern between clones of S. pectinata
(Davelos et al. 1996). Phylogenetic analyses based on 5.8S rDNA, ITS found that
Pu. sparganioidis belonged to a highly supported clade (Group 1) within the family
Pucciniaceae. Its closest relative was Puccinia physalidis (Dixon et al. 2010).
626 W.H. Elmer
Fig. 24.7 The salt marsh periwinkle snail Littoraria irrorata grazing on stems of Spartina alter-
niflora colonized by Phaeosphaeria spartinicola
Phaeosphaeria spartinicola Leuchtm
Distribution and Description
Phaeosphaeria spartinicola is an ascomycetous endophyte of Spartina spp. and

presumably found wherever Spartina is grown. It was first described in 1991
((Leuchtmann and Newell 1991) Kohlmeyer and Volkmann-Kihlmeter 2002).
Reports of Pha. spartinicola and many of its synonyms are listed as species of
Leptosphaeria and Pleospora and have been reported along the Pacific and Atlantic
Coast of the USA (Gessner and Goos 1973; Gessner and Kohlmeyer 1976; Jones
et al. 2002), England (Goodman 1959), and South America. Reports by Gessner and
Goos (1973) and Gessner and Kohlmeyer 1976) found that 75–100 % of the aboveg-
round biomass was colonized by Leptosphaeria spp. and Pleospora spp., which are
likely synonyms of Pha. spartinicola. Lyons (2007) found Pha. spartinicola was
ubiquitous on S. alterniflora in all Georgia marshes sampled. In addition, different
species of Mycosphaerella are often found as well on Spartina, but the identity and
taxonomic status of these species are not as clear (Kaur et al. 2010; Li et al. 2014).
Although Koch’s Postulates have never been satisfied with Pha. spartinicola on
healthy plants, the ubiquity of this species in salt marshes raises several important
questions regarding their ecological role.
Life cycle and ecology. Most discussions on Pha. spartinicola centered on its
primary role as a saprobe and its function as a secondary decomposer of S. alterni-
flora and other marsh grass species (McKee et al. 2004; Newell 1996, 2001a; Newell
and Barlocher 1993). Inclusion in this chapter (a chapter that is devoted to patho-
gens) is still warranted due to the important role this species plays in the dieback
events that occur in the southern USA marshes where a facultative mutualism exists
between herbivorous periwinkle snail (Littoraria irrorata) (Fig. 24.7). Pha. spar-
tinicola provides much of the dietary sustenance for the snail (Newell 2001b;
Raybould et al. 1998). During periods of drought, grazing by the snail was associ-
ated with major dieback of S. alterniflora in southern marshes (Silliman et al 2005;
Silliman and Newell 2003). Snails wound S. alterniflora leaves with their radula
and then deposit fungal-infested fecal matter into freshly grazed wounds. The snail
then returns to the plant after the fungus has sporulated in the wounds and selec-
tively consumes the fungal mycelium and spores. The fungus appears to colonize
the wound only as a necrotroph. As a result, the impact of this facultative mutualism
between snails and Pha. spartinicola led to major destruction of certain salt marsh
communities.
Other associations have been documented between Pha. spartinicola and herbi-
vores on S. densiflora in Argentina (Daleo et al. 2009). Crab grazing facilitated
colonization of necrotic tissues by Pha. spartinicola which in turn reduced produc-
tivity by interpreting the photosynthates production by more than 50 % (Daleo et al.
2009). Although a true disease condition has not been verified by Koch’s postulates,
the role of Pha. spartinicola in marsh ecology may extend beyond its primary role
in decomposition.
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Index
A Allergic bronchopulmonary aspergillosis

Aamir, M., 543–545, 548–561, 563–565 (ABPA), 437
Abarenkov, K., 47–62 Allergic bronchopulmonary mycosis (ABPM),
Abdel-Wahab, M.A., 275 437, 443
Abghari, A., 554 Allergy, 436, 437, 439–441
ABPA. See Allergic bronchopulmonary ACAAI/AAAAI Joint Taskforce (JTF)
aspergillosis (ABPA) Practice Parameter, 430
ABPM. See Allergic bronchopulmonary allergic and non-allergic fungi, 443, 444
mycosis (ABPM) allergic fungal sinusitis, 438–439
Acremonium species, 183 allergic rhinitis, 429
Acute lymphoblastic leukemia (ALL), 397 asthma-free room, 429
Adams, R.I., 386, 398 exposure and sensitization, 435
Adhikari, A., 378 fungal allergic proteins, 434
Aerobiology pathway, 316, 317 fungal protein cross-reactivity, 434–435
Aflatoxin fungi and cross-reactivity, 433
A. flavus and A. parasiticus fungi and innate immunity
prevalence, 477 CLRs, 439
adverse effects, 478 Dectin-1, 440
Aspergillus pseudotamarii, 476 Dectin-2, 440
atoxigenic strains, 477 definition, 439
carcinogenicity, 478 DNA sequencing technology, 441
corn/peanuts, A. flavus infection, 476 MBL, 440
cyclopiazonic acid, 478 mincle, 440
distribution, 477 molecular patterns, 439
and fumonisin, 476, 478 TLRs, 440
lymphocytes, phagocytes, mast cells and transitions and developmental stages, 439
basophils, 478 human exposure and fungal ecology
milk, 470 allergic rhinitis and asthma, 437
soil fungi, 477 ascospore and basidiospore levels, 436
Agaricomycetes, 269 carbon source, 436
Aigialus spp., 275 damp building exposure, 437
Aime, M.C., 153 facilitating factors, 436
Ajello, L., 460 Institution of Medicine publication Damp
Albaek, M.O., 529 Indoor Spaces and Health, 437
Alfenas, R.F., 206 mechanistic studies, 437

DOI 10.1007/978-3-319-29137-6
632 Index
Allergy (cont.) Ascomata, 220, 221, 224, 230, 232

outdoor airborne fungal spore Ascomycetes, 220, 231, 232, 269, 274
estimations, 436 Ascomycota, 268–270, 273, 275, 276
plaster and solid wood, 436 Aspergillus fumigatus, 183
weather conditions, 436 Aspergillus spp., 555–556
wind speed, 436 Ayers, T.T., 72
hyphae grow, 430 Azygosporogenesis, 141
medical use, 430
microfungi extracts, 430–432
recombinant allergens, 441–442 B
Rhodotorula, 433 Bahkali, A.H.A., 275
scalp and feet, 430 Bandoni, R.J., 335
sequencing allergenic proteins, 442–443 Barbeau, D.N., 384
spores, 430 Barcode. See DNA barcoding
testing and treatment, 433 Barghoorn, E.S., 278
Wallemia sebi, 433 Barker, K.R., 577
All-Fungal Tree of Life (AFTOL) project, 127 Bärlocher, F., 297, 302, 330, 334, 335
Amado, M., 429–444 Barnes, C., 429–444
Amend, A.S., 385, 399 Barron, G.L., 100
American Phytopathological Society, 578 Basal lineages, 65
American Society of Heating, Refrigerating Baschien, C., 285, 286, 288, 290, 294–305
and Air-Conditioning Engineers Basidiobolomycetes, 128–130
(ASHRAE), 374 Basidiobolus, 127–130, 133, 135, 136, 138,
Amerson, H.V., 625 139, 142
Amylocarpus encephaloides, 271 Basidiobolus meristosporus, 130
Anderson, J.L., 302 Basidiomata, 230, 232
Annellation, 171, 191 Basidiomycetes, 230–232, 268, 269
Annotation, names and taxonomic Basidiomycota, 269
re-annotation, 51–52 Baudoinia compniacensis,
Anomalizing selection, 177 415, 420
Antagonistic plant symbionts, 596–599 Bauer, R., 154
Anther smuts, 150, 151 Bayesian analysis, 66
Aphelenchoides, 574 Bayesian relaxed-clock method, 243
Aphelenchoides hylurgi, 578 Benjamin, C.R., 72, 76, 77
Aphelenchus avenae, 577 Benjamin, R.K., 72, 76, 77, 98
Aquatic fungi Benny,G.L., 65–111
adhesive mucilaginous material, 295 Bensch, K., 375
adhesive phase, 294 Ben-Ze’ev, I., 131, 139
aero-aquatic hyphomycetes, 295 Betancourt, D.A., 390
ascospores, 294 Bhan, U., 387
Canalisporium, 295 Bhargava, S., 531
dematiaceous hyphomycetes, 295 Bier, M.C.J., 535
freshwater ascomycetes, 294 Binder, M., 35
fungal adhesion and mucilage, 294 Bingley, G.D., 391
Helicoon species, 295 Biodiversity
hyphomycetes, 295 genetic lineages, 149
Ingoldian fungi, 295 molecular data analysis, 148
lignicolous hyphomycetes, 295 Pucciniomycotina, 148
propagules, 294, 295 research, 152–153
spores, 294 saprobic yeasts, 149
tetraradiate spores, 295 Septobasidium, 150
zoosporic fungi, 294 species-rich genera, 150
Arbuscular mycorrhizal fungi (AMF), 597 taxonomic distribution, 148
Arkansas Fungus (ARF), 594 Biofilms
Arthrobotrys oligospora, 584 infections, 502
Index 633
secondary metabolites, 503 Bursaphelenchus, 575

sinus mucous membranes, 502 Byndoor, M.G., 531
Biofuel production
biotechnology, 561–564
cellulases, 545–548 C
challenges, 564 Cai, L., 303
enzyme engineering, 559–560 Canopy plate technique, 133
fatty acid-derived bioproducts, 562 Carbon nanotubes (CNT), 563
genomics and metagenomics, 561 Carey, S.A., 388
lignocellulosic biodegradation, Carpenter ants, 400
545–547 Carter, M.V., 332
lignocellulosic biomass, 544 Carter, W.W., 579
system-level approaches, 560–561 Casciatori, F.P., 528, 531
Biofuels, 525 Castañeda-Ruiz, R.F., 197, 198, 204, 206–208,
Bioreactor 237–252, 254–257
cellulases, 532 CBS 518.82, 183
coal particles, 532 CBS 637.82, 183
Flammulina velutipes, 533 Cellulase system
fungal fermentations, 531 enzymatic cocktail, 549
Geotrichum candidum, 533 filamentous fungi, 548
hemicellulases, 532 Hypocrea jecorina (Trichoderma reesei),
Neurospora sitophila, 532 548, 549
packed-bed reactors, 532 lignocellulosic biomass, 548
rheological properties, 531 SSF, 549
Rhizopus arrhizus, 532 Certik, M., 555
Sclerotium glucanicum, 533 Chauvet, E., 302
SSF, 531 Chazalet, V., 395
Trametes hirsuta, 531 Chen, S., 554
Trichoderma reesei, 533 Cheng, A., 382
Bioremediation, 535, 536 Chiller, T.M., 397
Biotransformations Chimeras
Aspergillus niger, 534 artificial sequences, 52
chitosanases, 534 BLAST search, 53, 54
de novo pathway, 534 formations, 53
decolouration of dyes, 535 INSDC and UNITE exchange data, 55
Sporotrichum thermophile, 534 NGS datasets, 54
Swiss biotech company, 534 PCR/sequence assembly steps, 52, 53
terpenes, 535 perfect and poor alignment, 53
terpenoids, 534 screening small-to-midsize, 53
vanillin, 534 UNITE and the INDSC exchange data, 55
Blackwell, M., 272 Chlamydospore, 69, 70, 77, 80, 84–86, 93,
BLAST 96, 182–184, 186, 319, 377, 416,
chimera discovery, 53 591, 618
chimeric/authentic–nature of the Cho, E.J., 529, 531
sequence, 54 Cho, S.-H., 378
in INDSC, 58 Chunduri, J.R., 382
nonredundant databases, 51 Chung, Y.J., 387
reverse complementary insertions, 55 Chytridiomycetes, 269
reverse complementary sequences, 59 Chytridiomycota, 276, 277
UNITE/INSDC, 51 Classification, 65
Botrytis cinerea, 582 Kickxellomycotina (see Kickxellomycotina)
Bourne, J.M., 591 Mortierellomycotina (see
Brachyphoris oviparasitica, 594, 595 Mortierellomycotina)
Braun, U., 11, 14, 17, 206 Mucoromycotina (see Mucoromycotina)
Brewer, J.H., 388 Zoopagomycotina (see Zoopagomycotina)
634 Index
Claviceps purpurea physical or ecological stressors, 185

alkaloids ergocristine and ergocryptine, 622 Pleosporales, 186
pathology and life cycle, 622–623 secondary, 139
S. alterniflora, 620–622 self-interacting construction, 193
S. anglica, 621 self-reconstitution, 172
Spartina, 621 stability, 184
Coccidioides, 180 stripped-down Hyphomycetes, 178
Committee-dominated era substrate arthroconidia, 182
monophyletic clades, 11 superficial layers, 182
priority, 11 taxonomic characters, 171
Completoria complens, 132, 136 taxonomically revelatory analogues, 178
Completoriaceae, 132 Thallic conidia, 174
Conidial fungi, 170, 192, 326 vestigial characters, 175
Conidiobolus incongruus, 130 Consolidated bioprocessing (CBP), 563
Conidiogenesis Coprophilous fungi, 354–355
adaptations, 140 Coradin, J.H., 528
adapted processes, 175–177 Cornut, J., 334
aleurioconidi, 179 CREST, 50
Aspergillus, 171 Crous, P.W., 13, 15, 206
biological history, 170 Cryphonectria parasitica, 577
biosystematic history, 185 Cryptococcus species, 554–555
budding yeast structures, 180 Cryptomycota, 66, 277
class-level groups, 186 Crystallinity, 545
conscious decision processes, 174 Culture, 66
coordinating characters, 174, 175 Kickxellomycotina (see Kickxellomycotina)
coordination-optimizing Mortierellomycotina
changes, 192 (see Mortierellomycotina)
cut-and-paste individual figures, 186 Mucoromycotina (see Mucoromycotina)
dimorphic-systemic organisms, 180 Zoopagomycotina (see Zoopagomycotina)
and dispersal, 137–138 Cunninghamella echinulata, 555
disseminable cells, 173 Cutter, V.M. Jr., 141
DNA sequences, 171, 192
DNA-based biosystematics, 171
energy transfer, 172 D
Eurotiomycete lineages, 178 Dactylellina, 583
Eurotiomycetous dimorphic systemic Dactylellina ellipsospora, 585
pathogens, 181 Dactylospora, 271
fission cells, 181 Dai, Y., 26–36, 38–40
foot cells, 185 Damp buildings, mycotoxins
malleable Hyphomycetes, 184 adverse effects, 510
metaphor, 174 biocontaminants, 507, 508
microanatomy, 170 LC-MS/MS methodology, 509, 510
microsatellite repeat regions, 193 microbial communities, 508
microscopic conidial fungi, 170 satratoxins, 508
modifiable system, 172 secondary metabolites, 509, 510
monocots and dicots, 169 sterigmatocystin, DAS, DON and
morphogenesis, 171 verrucarol, 508
morphological complexity, 170 water, temperature and microbial
mutation, 193 interactions, 509
neutral alleles/mutations, 174 Dang, C., 334
opportunistic system, 172 Dannemiller, K.C., 388
phialides, 186 Dark septate endophytes (DSE), 299
phylogenetic arrangement, de Bary, A., 88
187–191 De Beer, Z.W., 16–18
physical factors, 185 de Hoog, G.S., 183
Index 635
De Kimpe, N., 535 vegetatively propagated crops, 320

de Vries, G.A., 183 Ditylenchus, 576
Deacon, J., 336 Ditylenchus dipsaci, 582
Dearborn, D., 387 Diversity
Degawa, Y., 71, 76–79, 99 biotic and trophic relationships, 30
Delsuc, F., 33 DNA barcode, 32
Dematiae, 170 environmental samples, 31
Demyttenaere, J., 535 environmental species, 32
Denning, D.W., 394 fungal phylum, 32
Dennis, R.L., 241 GenBank, 31
Deoxynivalenol (DON) gene markers, 32
3-ADON and 15-ADON genotypes, genome, 32
471, 472 herbarium specimens, 31
F. graminearum, 471 inorganic and organic nutrition, 30
Fusarium head blight, 470, 471 mycorrhizal fungi, 30
genetic changes, 472 protein-coding genes, 33
mechanisms, 473 soil samples, 31
nivalenol, 473 DMSO. See Dimethylsulfoxide (DMSO)
oxygen tension, 483 DMSP. See Dimethylsulfoniopropionate
PMTDI, 473 (DMSP)
vomitoxin, Fusarium graminearum, 470 DNA barcoding
Deposition fungal community, 101
dense vegetation, 331 isolation and culture techniques, 100
gloiospores, 331 Kickxellomycotina, 100
gravity effect, 331 Mortierellales and Mucorales, 100
sedimentation, washout/impaction, 331 PCR primers, 101
spore clusters, 332 DNA shuffling approach, 559
waxy cuticle and trichomes, 332 Dörfelt, H., 242
Desirò, A., 85 Dornelo-Silva, D., 207
Desroches, T.C., 388 Dothideomycetes, 270, 271, 274–276, 278
De-Wei, Li, 1–5 Drechmeria balanoides, 588
Dianese, J.C., 207 Drechmeria coniospora, 587
Diaz, A.B., 531 Drechsler, C., 94–96
Diederich, P., 271 Dromph, K.M., 349
Dimethylsulfoniopropionate (DMSP), 620 DSE. See Dark septate endophytes (DSE)
Dimethylsulfoxide (DMSO), 620 Dual nomenclature, 7, 9
Dispersal, 424–425 Duddington, C.L., 95
Dispersal strategies, 320–327, 331–332 Duncan, R.A. Jr., 621
airborne transport, 327–330 Dunphy, G.B., 142
by animals, 340–355 Dust, 385, 386, 399
applications, 357 Dwyer, J., 132
deposition (see Deposition)
by indoor air, 330–331
liberation (see Liberation) E
plants and substrate particles, 339–340 Ecology, 422, 423
resurfacing, 332–333, (see also Spore Eilenberg, J., 138
dispersal pathway) Ekelöf, E., 338
in water, 333–339 Eleuterius, L.N., 621
Dispersal units, fungi Ellis, J.J., 87, 96, 97
cytoplasmic viscosity, 318 Elmer, W.H., 615–620, 622, 624–626
description, 318, 319 Endoparasites, 587
gloiospores, 318 Endophyte, 596
gradient steepness, 320 Endophytic and rhizospheric strains, 599
modes of, 318 Endophytic antagonists of nematodes, 598
morphology, 318 Endophytic fungi, 578
636 Index
Energy dissipation/circulation function F

(EDCF), 529 Farooq, A., 535
Entomopathogenicity, 133 Faulk, C.T., 395
Entomophthoromycetes: Ancylistaceae: Fermented food and condiments, 244–246
Conidiobolus, 131 Fisher, P.J., 295
Entomophthoromycetes: Entomophthoraceae: Fleischmann, A., 242
Zoophthoroid genera (subfamily Forcible spore discharge, 139
Erynioideae), 131, 132 Forest
Entomophthoromycota, 127–135, 140–142 and semiarid areas, 197
development, 136–137 streams, 198
host range, 133–134 tropical and subtropical, 204
infection Fourier Transform Infrared Spectroscopy
Basidiobolomycoses, 135 (FTIR), 553
germ tubes, 134, 135 French Distillers’ Association, 414
infective spores, 134 Freshwater fungi, 297–301
mycoses, 135 aero-aquatic fungi, 287
primary or secondary conidia, 135 aero-aquatic life strategy, 287
spore’s reaction, 134 aquatic ascomycetes, 296
wall-free protoplasts, 135 aquatic habitats, 285
nutritional habits, 133–134 aquatic hyphomycetes, 286
reproduction, 137 aquatic–terrestrial hyphomycetes, 290
conidiogenesis (see Conidiogenesis) asci and ascospores, 288
resting spores, 140–142 ascomycetes, 288, 296
sexuality, 140–142 Ascomycota and Basidiomycota, 296
Environmental Relative Moldiness Index biodiversity changes, 304
(ERMI), 400 Chromista, 290
Environmental sampling. See DNA barcoding Chytridiomycetes, 290
Enzyme cocktails, 559 chytrids, 290
Enzyme-Linked Immunosorbent Assays Cladosporium, Alternaria, and
(ELISA), 388 Penicillium, 290
Ergot alkaloids, 482 complex structures, 286
Esteya vermicola, 587, 589 conidial shapes and synanamorphs, 297
Ethanol Cryptomycota, 290
ethyl alcohol, 413 distribution pattern, 301–302
primary isolation, 418 Euascomycetes, 296
vapour, 423–424 Eumycota, 290
warehouses, 417 freshwater ascomycetes, 288
Ethnomycology freshwater comprises diverse habitats, 286
description, 243 freshwater habitats
edible mushrooms, 248–249 Aphanomyces and Saprolegnia, 301
fermented food and condiments, 244–246, aquatic ascomycetes, 297, 300
(see also Medicinal fungi) aquatic hyphomycetes, 299
microfungi as food, 249–250 aquatic–terrestrial fungi, 299
microfungi, human history, 243–244, (see aquatic–terrestrial–hyphomycetes,
also Yeasts and alcoholic beverages) 298, 299
EUBOLD, 50 basidiomycetes, 297, 298
Eurotiales, 182 cellulose and lignin, 298
Eurotiomycetes, 270, 271, 275 chemotaxis, 300
Evans, H.C., 91 chytrids, 300
Evolutionary landscape, 177 DSE and RAF, 299
Evolutionary divergences, 174 endomycorrhizal associations, 299
Evolutionary opportunity, 173 enzymatic activities, 298
Exophiala (Wangiella) dermatitidis, 396 freshwater ascomycetes, 298
Exopolysaccharide (EPS), 530 heterotrophs, 300
Index 637
host–parasite interactions, 301 and systematics

hydrolysis, 297 DNA sequence, 26
hyphae, 299 eukaryotes, 26
lignin, 298 fungal phyla, 27
Minutisphaera spp., 299 genomics, 27
mycoloop pathway, 301 global species, 26
mycorrhizal associations, 299 Life cycles, 26
parasitic zoosporic fungi, 300 mycelium, 26
plant materials, 297 phylum and class level, 27
primary ecological role, 297 single-celled and multicelled
Pythium phragmites, 301 organisms, 26
saprotrophic chytrids, 301 Fungal fragments
soft rots, 298 aerosolization, 378
soil-inhabiting fungi, 298 Alternaria and Botrytis, 378
vesicular–arbuscular mycorrhizas, 299 bioaerosols, 376
white- and brown-rot fungi, 298 gonomorphic and non-gonomorphic., 377
wood, 297 immunostaining, 377
zoosporic fungi, 301 nanotechnology, 379
zoosporogenesis, 300 respiratory deposition models, 377
fungal morphology, 285 size-selective studies, 378
helicosporous aero-aquatic fungi, 297 submicron, 377
human disturbance, 304 Fungal genomics
Ingoldian fungi, 286 evolution, 156
microhabitats, 286 Microbotryomycetes, 158
molecular studies, 297 Pucciniomycotina, 156
morphological and ecological groups, 286 Rhodotorula graminis, 158
Oomycetes, 290 rusted fungi, 156–158
phycomycetes, 290 sizes, 159
polyphyletic origin, aquatic Sporobolomyces roseus, 158
hyphomycetes, 296 Fungal infections, 401
properties, ecosystem functions, 304 Fungal metabolites, 525
ribosomal genes, 296–297 Fungal nomenclature, 8–10, 12–15
root-associated fungi, 297 anamorph-based names, 7
seasonal variation, 303–304 anamorph–teleomorph connections, 7
sporangiophores and sporangia, 285 classification, 18
substrate preference, 303 Dothideomycetes
transcriptomes, 305 Botryosphaeriales, 13
water-associated fungi, 286 Capnodiales, 13
zoosporic fungi, 288 Dothideales, 12
Fumonisin Pleosporales, 13
and aflatoxin, 476 Eurotiomycetes
corn consumption, 475 Chaetothyriomycetidae,
equine leukoencephalomalacia, 475 Chaetothyriales, 13
esophageal cancer, 475 Eurotiales, 14
Fusarium verticillioides, 474 legitimate names, 7
infants, 475 Leotiomycetes
insect damage, 474 Erysiphales, 14
maize genotypes, 474 Phacidiales, 15
NTDs, 475, 476 Melbourne, sessions
Fungal diversity discussions, 9
in Central and South American Neotropic, priority, 10
204–206 special Committee dealing, 9
hyphomycetes, 198 teleomorph-based names,
samples collection, 198–204 9, 10
638 Index
Fungal nomenclature (cont.) Fusarium palustre

monophyletic clades, 18 chlamydospores, 618
monophyletic genera, 17 distribution, 616–617
new species, 17 F. langsethiae, 618
Orbiliomycetes, 12 F. sporotrichioides, 618
Pezizomycetes, 12 macro- and meso-conidia, 618
sexual and asexual morphs, 17 pathology and life cycle, 618–620
Sordariomycetes Fusarium spp., 597
Xylariomycetidae, Xylariales, 15 Fusarium wilt, 579
suppressed names, 11, 17
teleomorph name, 7
unification G
anamorph and teleomorph Gabelle, J.C., 531
taxonomy, 8 Galper, S., 586
DNA data, 8 Gams, W., 7–19, 78, 79
GenBank, 8 Garcia-Carnelli, C., 535
unified nomenclature, 9, 17, 18 Gaspard, J.T., 585
Fungal pathogens Genome data
fungal identification, clinical setting, deep internodes, 35
452–453 gene sequencing and taxon sampling, 36
fungal nomenclature, 453–455 phylogenetic signal, 36
infections, 451 phylogenetic tree, 35
invasive fungal infections, 451 rRNA markers, 35
mucorales, 451 taxon-gene, 36
Fungal systematics Genomics
DNA sequences, 28 additional classes, 159
environmental samples, 30 characters of Pucciniomycotina, 157
environmental signals, 33 Geographic distribution, 150–152
fungal herbaria, 29 Geotrichum candidum, 530
genetic markers, 33 Gerdemann, J.H., 85
genome sequencing, 33 Gessner, R.V., 625
Geoglossum and Trichoglossum, 29 Gibbs, P.A., 531
lower-level phylogeny, 27 Global transcription machinery engineering
low-level classifications, 29 (gTME), 558–559
microfungi, 30 Globodera pallida, 580
multilocus phylogenies, 28, 29 Globodera rostochiensis, 579–580
multilocus phylogeny, 34 Glyoside hydrolase and carbohydrate esterase
nucleotide characters, 28 family, 550
phylogenetic informativeness, 29, 35 Goh, T., 285, 286, 288, 290, 294–305
phylogenetic markers, 35 Gold, W.L., 396
phylogenetics, 28 Gomori methenamine silver (GMS) stain, 179
polytomies, 34 Goodley, J., 395
taxa and genetic markers, 28 Goodman, P.J., 616
trial and error process, 34 Goos, R.D., 625
Fungal systematics and evolutionary Gräfenhan, T., 15, 98
functional gene families, 39 Gray, A.J., 622
inorganic and organic components, 39 Green, B.J., 377
organism groups, 38 Gregory, P.H., 320, 327
phylogenomics, 40 Grimsley, L., 384
toxic gene clusters, 38 Grove, W.B., 90
Fungi, evolution of Guarro, J., 208
dating fungal diversification, 238 Gueidan, C., 13
fungal lineages, 238 (see also Molecular Gulis, V., 297, 303
clock dating) Gupta, V.K., 543–545, 548–561, 563–565
molecular methods, 238 Gymnosporangium confusum, 158
Index 639
H Humber, R.A., 69, 127–142

Habitats, 150–152 Hyde, K.D., 13
Hallmann, J., 597 Hynes, H.B.N., 297
Halosphaeriaceae, 268, 272, 273, 276 Hyphal bodies, 136
Hansen, K., 12 Hyphomycetes
Hasna, M.K., 574 Central and South American forests, 197
Haverinen-Shaughnessy, U., 379 and coelomycetes, 207–209
Hawksworth, D.L., 11, 18 colonized materials, 198
Health effects ecological group, 201
Alternaria population, 389 fungal diversity, 198
asthma, 390 terrestrial samples, 198
chronic fatigue syndrome, 388 Hypogeous fungi
Cladosporium, 388 Collembola species, 349
CT genotype, 389 description, 346
DNA sequencing data, 388 Douglas squirrel, 346, 347
factors and indoor allergens, 389 mycophagous mammals, 347
guttation droplets, 387 small rodents diets, 347
heat-treated conidia, 387 subterranean rodent Ctenomys cf.
hypersensitivity pneumonitis, 387 knighti, 347
IgE levels, 389 wild boars, role of, 347
indoor fungi, 386
mycotoxigenic molds, 388
Penicillium, 390 I
pulmonary hemorrhage, 386 ICTF. See International Commission on the
slime molds, 389 Taxonomy of Fungi (ICTF)
Stachybotrys chartarum, 386 Ikonen, E.K., 574
stachybotrytoxicosis, 386 Incubation, 201–204
Wallemia sebi, 388 Indoor environments (microfungi)
Heath, M.C., 176 evolutionary directions, 399
Hemicellulases factors, 382
acetyl xylan esterase, 550 mattresses, 383
endo-1, 4-β-xylanase, 550 morphology-based methods, 399
psychrophilic fungi, 550 mycologists, 374
sugar units, 549 paint, 382
types, 549 plastic and glass, 381–382
xylan and glucomannan, 549 wallpaper and plasters, 381
Heo, J-.H., 530 wood, 381
Hesseltine, C.W., 87 wood-decaying fungi, 373
Heterodera daverti, 581 Induced systemic resistance (ISR), 598
Heterodera glycines, 580 Infections (indoor fungi)
Hevekerl, A., 531 A. fumigatus, 394
Hibbett, D.S., 28, 34, 127, 242 airborne spores, 395
High-throughput sequencing technology, 398 allergic diseases, 392
Hirose, D., 84 antifungal agents, 394
Hirst, J.M., 322, 326, 332 Aspergillus fumigatus, 394, 395
Hirsutella, 588 Aspergillus fumigatus meningitis, 397
Hirsutella rhossiliensis, 588 blastomycosis, 393
Hoffmann, K., 86, 87, 89, 92 corneal transplantation, 396
Holland, R.J., 594 endomycetous yeasts, 393
Howell, C.R., 596 fungal respiratory tract infections, 392
Hsu, N.Y., 389 Fusarium spores, 396
Huang, J., 557 healthcare facilities, 392
Huber, L., 326 human infections, 394
Hughes, S.J., 171 immunocompromised individuals, 394
Human activities, 4 Myxogastria, 392
640 Index
Infections (indoor fungi) (cont.) International Nucleotide Sequence Database

pathogenic species, 397 Collaboration (INSDC)
preservative-free MPA injections, 397 curators, 49
respiratory infections, 395 developments, 60
Sporothrix schenckii, 393 fungal endophyte, 50
water-damaged books, 395 interactive and users, 49
Ingham, R.E., 574 invaluable function, 49
Ingold, C.T., 320, 324, 336 multiple sequence alignments, 62
Inhalation exposure, mycotoxins, 499, 502, and NGS, 60
506, 507, 510 scientific community, 48
acute renal failure, 507 substandard entries, 56
aerodynamic behavior, 499 taxonomic re-annotation,
airborne particles, 499 51, 52
alveolar macrophages, 500 and UNITE, 51, 55, 61
bronchioles, 499 Internet, 65, 95
damp indoor spaces, complexity Invasive aspergillosis
(see Damp buildings, mycotoxins) A. fumigatus, 457, 458
DNA adducts, 507 allogeneic stem cell transplant
exotoxins, 497 recipients, 455
lung antifungal therapy, 456
bleeding, 506 Aspergillus species, 457
cells of, 507 beta-tubulin region, 458
gliotoxin, 506 chromogenic assay, 456
satratoxins, 506 chronic pulmonary aspergillosis, 456
spores and mycelia, 506 Fusarium and Trichosporon species, 457
T-2 toxin, 507 galactomannan, 456, 457
worsening and asthma, 507 hematopoietic stem cell transplantation, 455
mucociliary clearance mechanism, 500 hemodialysis, 457
nose (see Nasal exposure) immunosuppressive chemotherapies, 455
particle deposition (see Particle radiographic studies, 456
deposition) solid organ transplant recipients, 455
primary function, respiratory system, 496 treatment, 456
respiration rate and breathing, 501 Ippen, R., 132
respiratory system, 495 ISHAM, 50
risk assessment (see Risk assessment) Islam, Z., 387
spore measurement, 497 Isolation, 66
spores and mycelia fragments, Kickxellomycotina (see Kickxellomycotina)
497, 498 Mortierellomycotina
sterigmatocystin, 498 (see Mortierellomycotina)
T-2 toxin, 496 Mucoromycotina (see Mucoromycotina)
toxic symptoms, 495 Zoopagomycotina (see Zoopagomycotina)
Internal transcribed spacer (ITS), 453 ITS. See Internal transcribed spacer (ITS)
chimera-free reference file, 54 ITSX, 55
chimeric unions, 52
genetic marker, 48
mycological community, 48 J
non-ITS sequences, 58–59 Jabaji-Hare, S., 85
nuclear ribosomal, 48 Jaffee, B.A., 589
public sequences, 48 Jaklitsch, W.M., 12
Sanger sequence, 48 James, T.Y., 26–36, 38–40, 66
UNITE, 49, 60 Jansson, H.B., 586
International Code for Botanical Javanovic, 557
Nomenclature, 453 Jeffries, P., 98
International Commission on the Taxonomy of Jessup, D.A., 132
Fungi (ICTF), 11 Johnson, H.E., 331
Index 641
Johnson, T.W., 268 sexual reproduction, 73

Johnston, P., 12, 14 zygospores, 75
Jonczyk, P., 531 Kickxellales
Jones, E.B.G., 267–269, 271–274, 276–278 Coemansia, 76
Julella avicenniae, 275 culture of, 77
Jun, O.K., 577 fatty acids, 76
isolation, 77
Linderina, 76
K Myconymphaea yatsukahoi, 76
Kang, X., 533 Ramicandelaber, 76
Kaplan, W., 132 sexual reproduction, 76
Karim, N.J., 575 spores, 76
Karpov, S.A., 66 orders, 69
Kasuhiro, O., 76 sexual reproduction, 69
Kaur, R., 625 Kim, Y.H., 577
Kaushik, N.K., 297 Kimura, M., 174
Keller, S., 131 Kirk, P.M., 65–112
Kelly, J.J., 299 Klink, J.W., 577
Kendr, W.B., 207 Kluth, S., 341
Kendrick, B., 219, 220, 224, 230, 232, 234, 297 Kluyveromyces marxianus, 525, 526
Kenneth, R.G., 131, 139 Koehn, R., 335
Kepler, R., 16 Koehsler, M., 95
Khan, A., 593 Kohlmeyer, E., 267, 268
Kick sampling technique, 70 Kohlmeyer, J., 267, 268
Kickxellomycotina, 69–77 Kohout, P., 297
Asellariales Kõljalg, U., 47–62
Baltomyces, 71 Konova, I.V., 76
chlamydospores, 70 Krug, J.C., 77, 93, 100
digestive tract, 70, 71 Kuehn, K., 335
forceps, isopods, 70 Kuhlman, E.G., 78
holdfast morphology, 70 Kumar, S., 553
infiltration, 71 Kuo, A., 101
isopods, 70 Kurihara, Y., 77
mycelium, 69 Kurtzman, C.P., 245
springtails, 71 Kwaśna, H., 76
classification, 67–77
culture collections, 66
Dimargaritales L
culture of, 72 Laboulbenia marina, 271
Dimargaris and Tieghemiomyces, 72 Laboulbeniomycetes, 270, 271
Dimargaris cristalligena, 72, 73 Lackner, M., 16
isolation, 72 Laegaard, S., 299
sexual reproduction, 72 Lagerlof, J., 577
Spinalia radians, 73 LaMondia, J., 237–252, 254–257, 618
Tieghemiomyces, 72, 73 Lectics, 169–193
Harpellales Leidy, J., 95
Harpellaceae, 73 Lesher, J.L. Jr., 395
in vitro conditions, 75 Levinson, H.Z., 352
Legeriomycetaceae, 74 Li, D-W., v–vi, 1–6, 197–217, 237–252,
life cycle, 75 254–257, 397
lipid content, Smittium culisetae, 75 Liberation
molecular techniques, 75 active and passive mechanisms, 320
monitoring, fungal growth, 74 airborne concentration, ascospores, 326
ovarian cysts, 75 airflows, 320
room temperature, 74 Alternaria spp., 322
642 Index
Liberation (cont.) classification, 268

Ascomycota, discharge of, 324 Dactylospora species, 271
ballistospore ejection, 322–324 deliquescing asci, 268
drying-induced hygroscopic movements, 325 Dothideomycetes, 271, 274
“puff” and “tap” mechanisms, 326 Eurotiomycetes, 271
vegetative symbiotic propagules, 321 Halosphaeriaceae, 272
wind, in spore release, 321 Julella avicenniae, 275
Lichinaceae, Lichinomycetes, 271 Kohlmeyeriella, Lindra, and Lulworthia, 272
Lichtwardt, R.W., 73 Laboulbeniomycetes, 271
Lignin peroxidases (LiPs), 551 Lichinaceae, Lichinomycetes, 271
Lignin-modifying enzymes (LMEs), 551 lineages, 267
Lignocellulosic biomass, 550 Morosphaeriaceae, 275
Lin, S.-J., 85 morphological characters, 269
Linde, T., 531 oil globules, 268
Linder, D.H., 278 organic materials, 267
Lindmark-Henriksson, M., 535 physical conditions, 267
Linford, M.B., 577 Pleosporales, 274
Liu, C.Q., 528 Pleosporomycetidae, 274
Lombard, L., 183 salinity and temperature, 267
Lonicer, A., 253 Savoryella, 272, 273
Luo, H., 584, 585 Sordariomycetes, 271, 273
Luo, J., 15, 16 Swampomyces, 273
Lutzoni, F., 28 taxonomy, 268
Lyons, J.I., 625 TBM clade, 272, 273
Verrucariales, 271
yeasts, 269
M zygomycetes, 269
Macleod, D.M., 136 Marra, R.E., 616–620
Madden, L.V., 326 Maser, C.A., 348
Madelin, T.M., 331 Mating system, 421–422
Madyastha, K.M., 535 MBL. See Mannose-binding lectin (MBL)
Magyar, D., 315–371, 350 McCabe, D.E., 141
Manganese peroxidases (MnPs), 551–552 McCook, K.P., 535
Mankau, R., 585 McGee, P.A., 340
Man-made spore dispersal, 355–356 McNeill, J., 9
Mannose-binding lectin (MBL), 440 Media
Manzanilla-Lopez, R.H., 593 Backusella, 86 (see also Mucoromycotina)
Mariano, G-.R., 528 nutrient-poor culture, 86, 91
Marine fungi nutrient-rich, 78
adaptive features, 272 pH, 74
Agaricomycetes, 269 Syzygites megalocarpus, 91
Aigialus spp., 275 Medicinal fungi
Amylocarpus encephaloides, 271 alkaloid ergotamine, 256
ascomycetes, 269 The Beggars (painting), 252, 253
Ascomycota, 268, 270, 271, 273 in Chinese medicine, 251
ascospores, 268 “convulsive ergotism”, 254
asexual forms, 268 DNA sequencing, 250
asexual fungi, 269 ergotism, 252
asexual marine fungi, 275, 276 Hypocrella bambusae, 251
basal lineages, 276–277 in modern medicine, 250
basidiomycetes, 269 polypores, 250
Basidiomycota, 269 smut diseases, 251
chytridiomycetes, 269 technological biosynthesis, 256–257
chytrids, 276 Theatri Botanici, 253, 254
Index 643
Medium-density fiberboard (MDF), 381 energy consumption, 543

Meloidogyne incognita, 590 fungal metabolism, 525
Meloidogyne spp., 593 genetic engineering, 564
Mendoza de Gives, P., 585 host plants, 3
Meredith, D.S., 322 lignocellulosic biomass, 544
Mesofungi lipid accumulation and degradation, 564
Claviceps purpurea, 224, 226 lipid-based bioproducts, 565
Coccomyces dentatus, 221, 223, 224 living plants, 206, 207
Colpoma crispum, 224, 228 microbial systems, 564
description, 220 oleaginous fungi, 544
Diatrype stigma, 224, 230 oleaginous microorganism, 565
Helminthosphaeria, 221 saccharification, 526
Heyderia abietis, 232, 234 secondary metabolites, 526
Hypomyces luteovirens, 224, 227 sexual and asexual states, 1
Ilex aquifolium, 221, 222 soil, plant debris and submerged plant
Leptonia cephalotrichum, materials, 207–213
232, 233 TAGs, 544
meiofauna/mesofauna, 220 taxonomic groups, 2
Mucronella pulchra, 230, 232 wastewater treatment, 526
mycotoxins, sources of, 219 Microfungi and plant-parasitic nematodes
myxomycetes and mycetozoa, 220 Aphelenchoides, 574
natural habitats, 231 biocontrol fungi, 578
Nectriopsis violacea, 224, 227 Ceratocystis fungus, 575
Onygena equina, 224, 229 Ditylenchus, 576
Phyllactinia (Erysiphales), 230, 231 Ditylenchus dipsaci, 582
Pilobolus, 232, 233 endoparasitic nematodes,
Rhytisma punctatum, colonies of, 221 579, 581
Saccobolus, 232, 234 endophytic fungi, 578
substrates, 224 ESTs, 575
Taphrina alni, 224, 225 fungal antagonism, 582
Taphrina populina, 221, 225 fungi producing traps, 583–586
Trochila ilicina, 221–223 fungivorous nematodes, 573
Uncinula (Erysiphales), 230, 231 hypovirulent strains, 577
Metabolic engineering Meloidogyne incognita, 580
biofuels, 557 motile nematode stages, 583
ethanol fermentation, 558 nematode-induced root wounding, 579
genetic techniques, 558 nematophagous fungi, 590
microbes, 557 organic matter, 586
molecular biology techniques, 558 pinewood nematode, 575
SCALEs, 558 plant-parasitic nematodes, 578
Metadata Rhizoctonia solani, 577
ecological and geographical, 60 rhizosphere, 585
molecular (DNA sequence) data, 59–60 root wounding, 580
Metagenomic, 561, 562 sedentary nematode stages, 590
Methylprednisolone acetate (MPA) in soil, 575
injections, 396 soybean cyst nematode, 580
Meyers, S.P., 621 trapping fungi, 586
Microbial interactions, 509, 512, 515 Microsporidia, 277
Microbial lipid production, 554 Mitchell, D.A., 528
Microbiome, 442, 443 Mites, 380, 399
Microfungi Mitochondrial genome, 421
bioethanol, 525 Mitotic/nuclear characters, 129–130
biofuels, 543 Miura, A., 302
Cryptomycota, 2 Mold, 379
644 Index
Molecular clock dating and Mucoromycotina, 80

ascomycete lineages, 240 sexual reproduction, 77
conidiophores, 242 sporangiophores, 77
ecological functions, 242–243 Moss, S.T., 75, 76
fossil records, 239 Mucedineae, 170
fossilized basidiomata, 242 Mucormycosis and order Mucorales
fungal “sporocarps”, 239 aggressive treatment, 460
fungal fossils, 239 diabetic patients, 460
lichen fossils, 240 and entomophthoromycosis, 461
Mesozoic and Cenozoic ambers, 241 infections, 460
mycorrhizal fungi, 240 organisms, 460
Molecular techniques in mycological studies, phylum Zygomycota, 461
48, 55–57 Rhizopus and Mucor species, 461
chimeras, 52–55 zygomycosis, 460
community, 61 Mucoromycotina, 86–92, 106–112
data sharing, 60–61 Calcarisporiella thermophila, 83
fungi and fungal diversity, 48 classification, 80–93
INSDC, 49 Endogonales, 85
ITS (see Internal transcribed spacer (ITS)) Mucorales
long-term stability and immediate Backusellaceae, 86
flexibility, 49 Choanephoraceae, 86
metadata, 59–60 culture media, 86, 106–110
morphological characters, 47 Cunninghamellaceae, 87
morphological structures, 47 isolation and transfer, 110
names and taxonomic re-annotation, 51–52 Lentamycetaceae, 87
non-ITS sequences, 58–59 Lichtheimiaceae, 87, 88
nutrient cyclers, 61 microscope slides, 111–112
outcome, 60–61 Mucoraceae, 88, 89
research, 48 Mycocladiaceae, 89
researchers recover, 61 Mycotyphaceae, 89
reverse complementary sequence, 58–59 Phycomycetaceae, 90
soil and wood, 47 Pilobolaceae, 90
species traits, 59–60 Radiomycetaceae, 90, 91
technical quality Rhizopodaceae, 91
BLAST search, 55–57 Saksenaeaceae, 91, 92
DNA ambiguity codes, 55 sexual reproduction, 86
homopolymer regions, 55 Syncephalastraceae, 92
Sanger sequences, 55 Umbelopsidaceae, 92
UNITE, 49–51 sexual reproduction, 80
Morosphaeriaceae, 274, 275 Sphaerocreas pubescens, 84
Mortierella isabellina, 555 Multiscale analysis of library enrichment
Mortierellomycotina, 78–80 (SCALEs), 558
classification, 77–80 Münchberg, U., 80
description, 65 Murthy, N., 535
Dissophora decumbens, 79 Mutualism in fungi
ergosterol, 80 description, 349
Mortierella jet and infrabuccal pocket, ant, 350
crude glycerol, 80 mold spores dispersal, dust mites, 352, 353
Echinochlamydosporium variable, 79 southern pine beetle, 351, 352
isolation and culture, 78 Xyleborus spp., 351
Lobosporangium transversale, 79 “zombie ant” dispersing pathogenic
Mortierella capitata spores, 78 Ophiocordyceps spores, 349
Mortierella wolfii, 80 MVOCs, 390, 391
zygospore formation, temperature, 79 Myceliophthora thermophila I-1D3b, 528
Index 645
Mycoparasite Neural tube defects (NTDs), 475, 476

biocontrol agents, 73 Neutral theory of evolution, 174
Chaetomium, 72 Next-generation sequencing (NGS) methods, 48
Dicranophora, Chaetocladium and NFAT. See Nuclear factor of activated
Parasitella, 88 T cells (NFAT)
Piptocephalidaceae, 97 Niche, 180
Mycoses, 451, 455 Nilsson, R.H., 26–36, 38–40, 47–62
Mycotoxins, 470, 495 Nivalenol, 471, 473
aflatoxin, 476–479 Nolan, R.A., 142
climate change, 482 Nomenclature Committee for Fungi (NCF), 9, 11
distribution, 470 Non-ITS sequences, 58–59
DON (see Deoxynivalenol (DON)) Norvell, L.L., 9
ergot alkaloids, 482 NTDs. See Neural tube defects (NTDs)
ergotism, 469 Nuclear cytology, 129, 130
fumonisin, 474–476 Nuclear factor of activated T cells (NFAT), 440
inhalation exposure (see Inhalation Nuclear genome, 420
exposure, mycotoxins) Nuclear-associated organelles (NAO), 129, 130
OTA, 479–481 Nutritional habits, Entomophthoromycota,
patulin, 481 133, 134
Penicillium, 470 host range
secondary metabolites, 469 acarine hosts, 134
T-2/HT-2 toxins, 481 Basidiobolus ranarum, 133
canopy plate technique, 133
fatal disease, 133
N Massospora, 134
Naár, Z., 349 nematodes, 133
N-acetylhexosaminidase (NAHA), 378 Neozygites, 134
Nag Raj, T.R., 318 Pandora neoaphidis, 134
Nasal exposure Parasites, 133
bacteria, 502 Pathogens infect, 133
biofilms, infections, 502 phylogenetic data, 133
breathing, 505 Saprobes, 133
chronic rhinosinusitis, 502, 503 saprobic species, 133
CNS disorders, 503 soil saprobe, 134
β-D-glucan, 505 Strongwellsea, 134
intra-nasal instillation, 505 Zoophthora radicans, 134
olfactory nerve, 503
oxidative stress and inflammation, 506
radio-labeled particles, 504 O
roridin A, 504 O’Malley, M.A., 330
rostral migratory stream, 504 Oboirien, B.O., 532
satratoxin G, 504 Occupational asthma (OA), 379
Stachybotrys spores, 505 Ochratoxin (OTA)
National Center for Biotechnology agricultural products, 479
Information, 453 carcinogenicity, 480
Natural disasters and indoor molds, 383–385 citrinin, penicillic acid and
NCF. See Nomenclature Committee for Fungi naphthoquinone, 480
(NCF) coffee and cocoa, 479
Nematode pathogens, 132–134, 139 evaluation, 480
Nematodes, 400 human disease, 480
Nematode-trapping fungi, 584 nephrotoxin, 480
Neocosmospora cyanescens, 183 Penicillium verrucosum, 479
Neotyphodium spp., 596 pork and pig-blood-based products, 480
Neozygitomycetes, 128, 130, 131 wheat and barley, 479
646 Index
Olfactory nerve, 503, 504 Phakopsora pachyrhizi, 158

Onygenales, 182 Phenol oxidases (laccases), 552
Operational taxonomic units (OTUs), 3, Phillips, A.J.L., 13
385, 398 Phospholipo-mannans (PLMs), 441
informative name, 52 Phycomycetes, 278
ITS sequences, 49 Phylogenetic diversity, 267
SHs, 49 marine fungi (see Marine fungi)
Ostrander, G.K., 177 Phylogenetic reclassifications,
OTA. See Ochratoxin (OTA) Entomophthoromycota
AFTOL project, 127
Basidiobolaceae, 132
P Basidiobolomycetes, 128, 130
Padhyay, A.A., 92 Basidiobolus, 127, 128
Page, R.M., 139 biological and gene-based information, 132
Pang, K.-L., 267–278 Blastocladiomycota, 127
Pante, E., 28 Chytridiomycota, 127
Paparu, P., 599 Completoriaceae, Completoria
Paranasal sinuses, 502 complens, 132
Parasite entomophthoraceous nuclei, 130
Ascomycota, 97 Entomophthorales, 127
Chaetomium, Mortierella and Mucorales, 72 entomophthoroids, 130
Cochlonemataceae, 95 Entomophthoromycetes, 128, 131, 132
development, 72 fungi in phylum, 127, 128
Mucor hiemalis, 73 Meristacraceae, 132, 133
mushroom, 89, 91 mitotic/nuclear characters, 129–130
Penicillium and Trichoderma, 97 Neocallimastigomycota, 127
Piptocephalis, 98 Neozygitomycetes, 128, 131
Zoopagaceae, 95 polyploidization, 130
zoopagalean, 94 taxonomic value, 129
Parasitize nematodes, 591 Zygomycota, 127
Particle deposition Phylogenetics
aerodynamic behavior, 499 higher-level relationships, 153–154
alveoli, 500 ITS region, 48
dose determination, 501 molecular data, 48
epithelium, 499 MycoBank, 155
gravity, 500 Rhodotorula, 155
mucociliary escalator, 500 sequence data, 155
respiration rate and breathing, 501 Sporobolomyces, 155
Pathogen taxonomic improvements, 154–155
immunocompromised individuals, 394 Phylogeny, Aigialus spp., 275
indoor fungi, 392 Piattoni, F., 347
Patulin, 481 Picioreanu, C., 338
Pažoutová, S., 621 Piriformospora indica, 598
PCR. See Polymerase chain reaction (PCR) Plant debris
Pedrotti, L., 599 microfungi, 207–213
Penicillium chrysogenum, 533 paper towel, 201
Penicillium spp., 555–556 Plant parasites, 573
Perkins, A.J., 340 Pleosporales, 274–276
Perlin, D.S., 394 Pleosporomycetidae, 274
Persoon, C.H., 219 PLMs. See Phospholipo-mannans (PLMs)
Persson, C., 586 PMTDI. See Provisional maximum tolerable
Petkovits, T., 79, 84 daily intake (PMTDI)
Phaeosphaeria spartinicola Pneumocystis, 180, 181
distribution and description, 625–626 Pneumocystis carinii pneumonia (PCP), 393
Index 647
Pochonia chlamydosporia, 592 Rhoades, H.L., 577

Poinar, G.O. Jr., 242 Rhodosporidium toruloides, 553
Polymerase chain reaction (PCR), 3 Rhodotorula glutinis, 556
Polyploidization, 130 Risk assessment
Pratylenchus penetrans, 581 dampness and mold, 514
Pratylenchus-Verticillium system, 581 guidance, 511
Prochazka, K., 334 public health protection, 510
Protoplastic development, 136 reference concentration (RfC)
Provisional maximum tolerable daily intake development, 511, 512
(PMTDI), 473, 475 safe levels, 513
Pseudallescheria/Scedosporium, 458–460 satratoxins G and H, 513
Pseudaphelidium drebesii, 276 screening tools, 512
Puccinia chrysanthemi, 158 sterigmatocystin and aflatoxin, 512
Puccinia sparganioidis T-2 toxin, 512, 513
description, 624–625 Rosling, A., 3
distribution, 623–624 Rossman, A.Y., 9, 15
pathology and life cycle, 624–625 Rotem, J., 322
Pucciniales Rotylenchulus reniformis, 586
biodiversity research, 148 rRNA, 271, 273, 277
disc-like spindle pole bodies, 147 Rueda, M.G.M., 535
microbotryales, 147 Ruiz, H.A., 532
pathological and systematic research, 148 Rybarczyk-Mydłowska, K., 574
phylogenetic relationships, 148 Ryberg, M., 59
Pucciniomycotina species, 147
Purpureocillium lilacinum, 592, 594
S
Saccardo, P.A., 170
Q Saccharomyces cerevisiae, 526
QIIME, 50 Sadowski, E.M., 242
Quandt, A., 16 Saikawa, M., 95, 96
Quantitative real-time polymerase chain Sakayaroj, J., 272
reaction (QPCR), 400 Salt marshes
carbon sequestration, 615
fungal pathogens, natural ecosystems, 615
R Fusarium spp., 616
Radopholus similis, 586 microfungi, 616
Raghuwanshi, R., 543–545, 548–561, 563–565 S. alterniflora, 616
Rahardjo, Y.S.P., 528 S. anglica, 616
Raja, H.A., 302 Spartina and Juncus, 615
Raman Microspectroscopy-Based Sample drying, 201
Identification, 398 Sample washing, 198–201
Ranzoni, F.V., 91 Samson, R.A., 91
Rational design approach, 559 Samuels, G.J., 12
Ray, J., 169 Satratoxin-G (SG), 387
Raybould, A.F., 622 Savoryella, 273
Réblová, M., 13 Savoryellales, 268, 272, 273
Redhead, S.A., 8, 9 Saxena, A., 543–545, 548–561, 563–565
Relative humidity (RH), 379 SCATA, 50
Resting spores (RS), 129, 140–142 Scedosporiosis, 458–460
Reverse complementary sequence, 58–59 Schadt, C.W., 3
Rhexolytix conidiogenesis, 377 Schizolysis, 181, 182
Rhizoctonia fragariae, 581 Schizolytic conidiogenesis, 181, 182
Rhizoctonia solani, 577, 580 Schmidt, A.R., 242
Rhizosphere colonization, 593 Schoch, C.L., 8, 180, 185, 271, 273
648 Index
Scott, J.A., 413–421, 423, 425, 426 microbiological processes, 526

Secondary conidiogenesis, 139 micro-scale effects, 528
Secondary metabolites xylanase production, 529
biofilms, 503 Sondergaard, M., 299
biosynthesis, 503 Sordariomycetes, 15–17, 270–273, 275, 276, 278
damp indoor spaces, 509 Hypocreomycetidae
ecologic niche, 508 Glomerellales, 16
toxin concentrations, 512 Hypocreales, 15–16
Seely, J.C., 132 Microascales, 16
Seifert, K.A., 7–9, 17, 186 Sordariomycetidae
Selosse, M.A., 335 Diaporthales, 16
Senthilkumar, S.R., 528, 529 Ophiostomatales, 17
Septobasidiales, 147 Xylariomycetidae, Xylariales, 15
Sequence Sparrow, F.K., 268
BLAST searches, 53 Spatafora, J.W., 272
chimeras, 52 Species hypotheses (SHs)
DNA, 48, 51, 61 data sharing, 60
INSDC, 51, 62 definition, 49
ITS, 48, 49, 59, 60 genus-level cluster, 50
names and classification of Index names, 52
Fungorum, 51 reference data/taxonomic progress, 51
non-ITS sequences, 58–59 UNITE, 61
protein sequences, 49 Spirit aging, 423
public DNA sequences, 48 Spore dispersal pathway
reference, 47, 51 aerobiology pathway, 316, 317
technical quality, 55–58 K-/r-strategy, 316
UNITE, 49, 50 in microfungi biology, 315
Sexuality primary and secondary inocula, 316
ancient abandonment, 141 space and time scales, 316
ascomycete and basidiomycete fungi, 140 Stabilizing selection
definitions, 141 coordination-optimizing changes, 192
morphological and developmental developmental momentum, 184
hallmarks, 141 Stajich, J.E., 106
parasexuality, 142 Stirling, G.R., 595
patterns, 141 Stress responses, 420
Shearer, C.A., 302, 335 Submerged fermentation (SF)
Siemensma, F., 95 airlift bioreactors, 530
Sigler, L., 183 mathematical model, 529
Sikora, R.A., 579, 597 morphology, 529
Silva, M., 206 strain and bioreactor, 529
Simon-Nobbe, B., 435 xylanase level, 530
Singh, S., 543–545, 548–561, 563–565 Sudova, R., 299
Slime molds, 400, 401 Suetrong, S., 274, 275
Slippers, B., 13 Summerbell, R., 169–178, 180–186,
Smith, M.E., 65–111 192, 193
Solid-state fermentation (SSF), 549 Sung, G.H., 250, 251
aerial hyphae, 528 Sutherland, J.R., 575
batch processes, 527 Sutton, D.A., 451–453, 455–462
bioprocess engineering, 527 Suyama, Y., 99
bioreactors, 527 Swampomyces, 273
enzyme production, 529 Sympodial proliferation, 171, 186, 190, 192
fungal morphology, 528 Synnemata, 171, 187, 291, 295, 341
high-added-value products, 527 Systematics
“low-technology” processes, 527 Acrocalymma, 376
mathematical model, 528, 529 Balaniopsis triangularis, 375
Index 649
Cladosporium cladosporioides s.l., 375 U

microfungi-related genera, 374 UCHIME
name systems, 376 and ITSX, 55
Tritirachium spp., 375 NGS datasets, 54
Wallemia sebi, 375 UNITE
BLAST, 50, 51
curation and quality control
T processes, 50
Tahara, S., 535 data export files, 60
Tanabe, Y., 95 download page, 54
Taxonomic composition, 425–426 genus-level alignment, basidiomycete
Taxonomy, 2, 8, 9, 11, 15, 17, 27–30, 38, 40, genus Meira, 50
51, 52, 129, 148, 154, 156, 170, hypothesis alignments, 61
171, 185, 248, 273, 286, 290, 399, and INSDC, 55, 56
401, 433, 441, 451–462, 583, 595 ITS sequences, 49, 60
re-annotation and names limited-taxon approach, 49
BLAST search, 51 molecular identification, 60
DNA sequences, 51 NGS, 50
Index Fungorum, 51 OTUs, 49
INSDC entry FR731412, 51, 52 public sequences, 49
non-conspecific sequences, 51 SHs, 49, 52
organisms and organism groups, 51 taxonomic re-annotation, 51
OTUs, 52 taxonomic scope, 49
precise reference, 51 web-based sequence management
SH, 52 environment, 49
uncultured fungus, 51 Untereiner, W.A., 13
UNITE, 51 Upadhyay, R.S., 543–545, 548–561,
Taylor, J.W., 8 563–565
TBM clade, 272, 273 Urabe, J., 302
Tedersoo, L., 2, 3, 12, 59 UTAX, 50
Termites, 400
Thambugala, K.M., 13
Thaxter, R., 79 V
Thermoascus aurantiacus, 532 Van Dyke, C.G., 625
Thomas, G.M., 242 Varga, J., 349
Thomas, K., 303 Verrucariales, 271
T-2/HT-2 toxins, 470, 481 Versatile peroxidases (VPs), 552
Timper, P., 573–599 Vertebrate mycoses, 133
Toll-like receptor (TLR), 440 Verticillium, 581
Toome-Heller, M., 147, 148, 150–156, Větrovský, T., 3
158, 159 Vijaykrishna, D., 273
Torula conglutinata, 414, 415 Voglmayr, H., 12
Torula herbarum, 299 Voigt, K., 87
Townsend, J.P., 26–36, 38–40 Vu, V.H., 528
Tremella fuciformis, 530
Tretter, E.D., 65–111
Triacylglycerols (TAGs), 544 W
Trichoderma, 595 Wagner, L., 79, 84
Trichoderma reesei, 528, 557 Wang, Y., 535
Trichothecium indicum, 178 Wang, Z., 26–36, 38–40
Trytek, M., 535 Warehouse staining
Tsui, C.K.M., 285, 286, 288, 290, 294–305 disfigurement, 413
Turkey X disease, 495 French Botanical Society, 415
Turland, N.J., 9 verification, 416
Tyrrell, D., 136 Webster, J., 302, 334, 335
650 Index
Weete, J.D., 80 carbonized grape berries, 247

Wellcome, H.S., 256 fungal species, 246
Whiskey fungus Yike, I., 387
industrial revolution, 413 Young, T.W.K., 76
microbial biofilm, 413 Yu, K-.P., 398
nineteenth Century, 414–415
physiology, 419, 420
twentieth century, 415–417 Z
twenty-first century, 417–419 Zambettakis, C., 375
White, M.M., 65–112 Zasoski, R.A., 589
White-rot fungi, 535 Zearalenone, 470, 473, 474, 482
Wiederhold, N.P., 451–453, 455–462 Zhang, N., 9
Wijayawardene, D.N.N., 7 Zhou, Z.Y., 244, 245, 247, 249
Wijayawardene, N.N., 13 Zhuang W.-Y., 15
Will, K.W, 28 Zoopagomycotina, 94, 95, 97, 98
The Wisdom of God Manifested in the Works classification, 93–99
of the Creation, 169 Cochlonemataceae
Wong, Y., 531 Acaulopage, 95
Wood-Eggenschwiler, S., 302, 330 Amoebophilus simplex, 95
Work-exacerbated asthma (WEA), 379 culture, 94
World Energy Council (WEC), 543 description, 95
Wu, G.-G., 85 growth, 94
Würtz, H., 385 Pythium, 95
Wurzbacher, C.M., 336 spore chains, 94
description, 94
Helicocephalidaceae, 96
X Piptocephalidaceae
Xiang, M., 589 Ascomycota, 97
cultures, Cokeromyces recurvatus, 98
isolation, 97, 98
Y Kuzuhaea, 98
Yang, C., 373–401 sporophores, 97
Yang, E., 249 sexual reproduction, 93
Yang, X., 553 Sigmoideomycetaceae, 98, 99
Yang, Y.T., 584 Zoopagaceae, 95, 96
Yao, Y.-J., 85 Zoutman, D.E., 183
Yarrowia lipolytica, 553–554 Zygomycetes, 269
Yeasts and alcoholic beverages Zygomycota, genome sequencing,
alcoholic fermentation, 246 101–106
in ancient Chinese culture, 247 Zygosporogenesis, 141

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Fungal Biology

Maria G. Tuohy, PhD

More information about this series at http://www.springer.com/series/11224

Co-Innovation Center for Sustainable

ISSN 2198-7777 ISSN 2198-7785 (electronic)

Library of Congress Control Number: 2016933679

Springer Cham Heidelberg New York Dordrecht London

Printed on acid-free paper

Springer International Publishing AG Switzerland is part of Springer Science+Business Media

1 Introduction: Advances and Predicament ............................................ 1

9 Fungal Diversity of Central and South America.................................. 197

23 Interactions of Microfungi and Plant-Parasitic Nematodes ............... 573

Index ................................................................................................................. 631

Mohd. Aamir, Ph.D. Department of Botany, Banaras Hindu University, Varanasi,

Wade H. Elmer, Ph.D. Department of Plant Pathology and Ecology, The

Donát Magyar, Ph.D. Department of Air Hygiene and Aerobiology, National

Eric D. Tretter, M.S. Department of Biological Sciences, Boise State University,

What Are Microfungi?

D.-W. Li, Ph.D. (*)

© Springer International Publishing Switzerland 2016 1

Ainsworth GC (1976) Introduction to the history of mycology. Cambridge University Press,

The Previous Situation

As documented in the International Code of Botanical Nomenclature (the ICBN,

W. Gams, Ph.D. (*)

© Springer International Publishing Switzerland 2016 7

Preparations for a Uniﬁcation

In Studies in Mycology 45 (Seifert et al. 2000), several authors explored how

The Crucial Sessions at Melbourne

participants, the ﬁrst vote ended immediately in an overwhelming yes result.

This is a remnant of the previous time-honored rule of precedence for

These paragraphs imply that the previous, entirely rule-dominated nomenclature

The Committee-dominated Era

Discussion papers (published or unpublished) with lists of preferred generic names

Thambugala et al. (2014) delimit the Dothideaceae (including the Dothioraceae)

Réblová and Untereiner (2013) introduce the new anamorph-based family

families (Adelococcaceae, Celotheliaceae, Chaetothyriaceae, Cyphellophoraceae,

Braun (2013) proposes conservation of the teleomorph-based name Blumeria over

name Volutellonectria illegitimate. For the difﬁcult complex of Acremonium–

The older genus name Colletotrichum is to be protected against the teleomorph

Acknowledgments For critical reading of the manuscript, I am indebted to Keith A. Seifert,

Johnston P, Seifert K, Stone J, Rossman A, Marvanová L (2014) Recommendations for generic

Zheng Wang, R. Henrik Nilsson, Timothy Y. James, Yucheng Dai,

Fungal Diversity and Systematics

Z. Wang, Ph.D. (*)

© Springer International Publishing Switzerland 2016 25

Integrative Taxonomy and Current Fungal Systematics

Traditionally, morphological and sometimes ecological traits have been used to

widely applicable, facilitating convenient naming of species or strains, especially

Systematics and Classiﬁcation for Invisible Diversity

situation where next-generation sequencing efforts of environmental substrates

species-poor group Taphrinomycotina of the Ascomycota. The recognition of the

approach toward recovering an effective diversity of protein-coding genes that will

Fungal Genomes, Phylogenomics, and Phylotranscriptomics

Approaches for selecting robust sets of phylogenetic markers based on sequenced

Experimental Design and Analysis for Systematics Using

Phylogenetic inference can be improved either by use of better models or by obtain-

Averaged support (PLRS)

optimized by taxonomically sparse genome-scale data (Lopez-Giraldez et al. 2013;

Fungal Systematics in the Future: Integration of Fungal

Systematics is fundamental to organismal biology and is the discipline that synthe-

cially interesting in efﬁciently addressing the genetic basis of various phenotypes

Further extension of the broad impacts of fungal systematic research requires

Bruto M, Prigent-Combaret C, Luis P, Moenne-Loccoz Y, Muller D (2014) Frequent, independent