(Mycology Series 16) D.H. Howard-Pathogenic Fungi in Humans and Animals-Marcel Dekker (2003)

Pathogenic Fungi
in Humans
and Animals
Second Edition
edited by
Dexter H. Howard
UCLA School of Medicine
Los Angeles, California
Marcel Dekker, Inc. New York • Basel
Copyright © 2002 by Marcel Dekker, Inc. All Rights Reserved.

The first edition was published as Fungi Pathogenic for Humans and Animals (in three
parts), edited by Dexter H. Howard (Marcel Dekker, Inc., 1983).
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MYCOLOGY SERIES
Editor
J. W. Bennett
Professor
Department of Cell and Molecular Biology
Tulane University
New Orleans, Louisiana
Founding Editor
Paul A. Lemke
1. Viruses and Plasmids in Fungi, edited by Paul A. Lemke

2. The Fungal Community: Its Organization and Role in the Ecosystem, edited
by Donald T. Wicklow and George C. Carroll
3. Fungi Pathogenic for Humans and Animals (in three parts), edited by Dexter
H. Howard
4. Fungal Differentiation: A Contemporary Synthesis, edited by John E. Smith
5. Secondary Metabolism and Differentiation in Fungi, edited by Joan W.
Bennett and Alex Ciegler
6. Fungal Protoplasts, edited by John F. Peberdy and Lajos Ferenczy
7. Viruses of Fungi and Simple Eukaryotes, edited by Yigal Koltin and Michael J.
Leibowitz
8. Molecular Industrial Mycology: Systems and Applications for Filamentous
Fungi, edited by Sally A. Leong and Randy M. Berka
9. The Fungal Community: Its Organization and Role in the Ecosystem, Second
Edition, edited by George C. Carroll and Donald T. Wicklow
10. Stress Tolerance of Fungi, edited by D. H. Jennings
11. Metal Ions in Fungi, edited by Günther Winkelmann and Dennis R. Winge
12. Anaerobic Fungi: Biology, Ecology, and Function, edited by Douglas O.
Mountfort and Colin G. Orpin
13. Fungal Genetics: Principles and Practice, edited by Cees J. Bos
14. Fungal Pathogenesis: Principles and Clinical Applications, edited by
Richard A. Calderone and Ronald L. Cihlar
15. Molecular Biology of Fungal Development, edited by Heinz D. Osiewacz
16. Pathogenic Fungi in Humans and Animals: Second Edition, edited by
Dexter H. Howard
Additional Volumes in Preparation

Preface to the Second Edition
The major object of the first edition of Fungi Pathogenic for Humans and Animals
was a thorough review of the basic biology, host-parasite interactions, and current
method of detection and characterization of the zoopathogenic fungi. This is a
revision of the original book with a minor change in the title. Events since the
publication of the First Edition have made necessary the preparation of this sec-
ond edition.
Some chapters in the first edition remain adequate summaries of the topic
(e.g., ultrastructure, conidium formation, and subcellular particles) and will not
be repeated here. In other cases the original chapters have been expanded into
monographic treatments (e.g., dimorphism, antigens, and cell wall composition)
and a consideration of those topics would be inappropriate in a smaller context.
In many cases, knowledge in fields covered in the first edition has grown so much
that a single chapter is no longer adequate (e.g., nutrition, antifungal drugs, and
mycotoxins). Finally, the topics of immunology and pathogenesis have received
very recent coverage that makes inclusion of them in the second edition unnec-
essary. (The Mycota, VI, Human and Animal Relationship, D. H. Howard and
J. D. Miller, eds. New York: Springer Verlag, 1996; Fungal Pathogenesis, R. A.
Calderone and R. L. Cihlar, eds. New York: Marcel Dekker, Inc., 2002.)
The area of coverage from the first edition that has changed remarkably is
the topic of classification and nomenclature, for which there is not a current treat-
ment. In the years since the first edition many molecular techniques, upon which
taxonomic decisions are now based, have been introduced. Accordingly, Part
One of the second edition has been expanded to include peronosporomycetes, a
rearrangement of sections on the filamentous Ascomycetes, and a division of the
chapter on yeasts into two separate chapters—one on heterobasidiomycetes, and
a second covering both the endomycetes and the blastomyces.
iii
iv Preface to the Second Edition
The second edition has been further expanded to introduce a topic not con-
sidered in the first edition: fungal populations. Four topics are covered: Molecular
methods used in taxonomic decisions; Population genetics of Medically Impor-
tant fungi—phylogeny; population studies—DNA- and PCR-Fingerprinting of
Medically Important fungi; and population instabilities—the phenotyic variabil-
ity of Candida albicans.
Dexter H. Howard
Preface to the First Edition
PART A
In 1947, Walter J. Nickerson edited a volume on the Biology of Pathogenic Fungi,

which covered some aspects of the biology, physiology, and biochemistry of the
fungi pathogenic for man. Since then several fine textbooks have appeared, in
which are described the essential features of the clinical manifestation, epidemiol-
ogy, and pathology of fungous infections together with the identifying character-
istics of their etiologic agents. In addition, individual mycoses or single aspects
of all of the major mycoses have received abundant monographic treatment. How-
ever, a comprehensive consideration of the biology of the fungi pathogenic to
man and animals has not recently been offered. These volumes are an effort to
do so. It was not my object to present a series of “selected topics” or “recent
advances” but rather a thorough review of basic biology, host-parasite interac-
tions, and current methods of detection and characterization of the zoopathogenic
fungi.
Part A of this series deals with the basic biology of medically important
fungi. The first section comprises a discussion of the taxonomy and classification
of the zoopathogens. This section is followed by individual chapters on ultrastruc-
tural cytology, dimorphism, sporulation, blastoconidium germination, and the
transport of metabolites. Some additional topics which suggested themselves for
coverage could not be included in Part A because of size limitations. Other basic
biology topics will be included in subsequent parts of the series. (Members of
the bacterial order Actinomycetales, often included in various types of works on
medical mycology, will not be considered in this series.)
So much has been accomplished in each of these fields that it would be
v
vi Preface to the First Edition
impossible for any single author to give an in-depth presentation. Accordingly,

a compilation by many individuals was attempted despite the recognized difficul-
ties of variation and potential redundancy. The scope of these volumes is such
as to attract the attention of all engaged in research on fundamental aspects of the
fungi pathogenic to man and animals. However, the coverage will be of interest to
students who wish an up-to-date consideration of the frontiers of investigation
on these pathogens.
A special acknowledgment is required. All responsibility for necessary edit-
ing with regard to uniformity of format, proper references, and retyping of heavily
altered copy was assumed by my wife, Mrs. Lois F. Howard. She was, indeed,
the primary copyeditor of this work. She merits recognition for her unswerving
pursuit of excellence of presentation and the indefatigable insistence on perfec-
tion of textural detail which I had hoped from the outset would characterize these
volumes. Her efforts, in no small measure, and those of several of the authors,
have been supported by research grant AI 16252 from the National Institute of
Allergy and Infectious Diseases, National Institutes of Health, which is used to
fund the Collaborative California Universities–Mycology Research Unit (CCU-
MRU).
I am, of course, grateful to the splendid group of authors collaborating with
me to produce these volumes.
PART B
The original plan for a comprehensive coverage of the zoopathogenic fungi called
for a division of the work into three parts. The first part was to include chapters
on the basic biology of the fungi with special consideration classification, mor-
phology, and physiology. The second part was to cover aspects of pathogenicity
such as mechanisms of pathogenesis, host responses (cellular and humoral), tox-
ins, and antigens. The last part was to have dealt with practical matters of de-
tecting the fungi in nature and in clinical materials and with certain applications
such as vaccines and antifungal drugs.
In gathering the material, it became clear that the topical divisions would
have produced volumes of exaggerated disproportion. Far too much on the basic
biology was at hand and some of the topics in pathogenicity required preparations
that exceeded practical deadlines. Therefore, rearrangements were made. The
third volume of the series, labeled Part B:II, now contains two sections and an
Addendum. The first of these sections contains chapters on pathogenesis, includ-
ing aspects of the basic biology that have a direct relation to mechanisms of
tissue invasion, namely, cell wall composition, subcellular particles, and enzymes
involved in in vivo survival or tissue destruction. In addition, the rearrangement
has allowed for an updated coverage of cellular defense mechanisms involving
Preface to the First Edition vii
phagocytic cells and, in an Addendum, a reconsideration of the genus Exophiala,

a particularly troublesome member of the dematiaceous group.
The second section of Part B:II is more or less as originally planned and
covers such practical matters as epidemiology, detection of fungi in tissue, growth
of fungi in culture, and the development of fungal vaccines.
And thus the work is completed. I gratefully acknowledge the indispensable
assistance of Mrs. Lois F. Howard, who checked all bibliographical material,
read all of the manuscripts, and tried to maintain consistency throughout the three
parts of the work. I also thank Ms. Bette Y. Tang for the numerous typings and
retypings of edited manuscripts. Some support for this venture has been supplied
by research grant AI 16252 from the National Institute of Allergy and Infectious
Diseases, National Institutes of Health, which is used to fund the Collaborative
California Universities–Mycology Research Unit (CCU-MRU).
I am especially grateful to the group of splendid authors who collaborated
with me to produce this volume.
Dexter H. Howard
Contents
Preface to the Second Edition iii

Preface to the First Edition v
Contributors xi
Part One Classification and Nomenclature
1. An Introduction to the Taxonomy of Zoopathogenic Fungi 1

Dexter H. Howard
2. The Peronosporomycetes and Other Flagellate Fungi 17

Michael W. Dick
3. Zygomycetes: The Order Mucorales 67

M. A. A. Schipper and J. A Stalpers
4. Zygomycetes: The Order Entomophthorales 127

Shung-Chang Jong and Frank M. Dugan
5. Onygenales: Arthrodermataceae 141

Dexter H. Howard, Irene Weitzman, and Arvind A. Padhye
6. Ascomycetes: The Onygenaceae and Other Fungi from the

Order Onygenales 195
Lynne Sigler
ix
x Contents
7. Aspergillus, Fusarium, Sporothrix, Piedraia, and Their Relatives 237

Richard Summerbell
8. Yeasts: Blastomycetes and Endomycetes 499

Kevin C. Hazen and Susan A. Howell
9. Basidiomycetous Yeasts 535

Teun Boekhout and Eveline Guého
10. Dematiaceous Hyphomycetes 565

Wiley A. Schell
11. Miscellaneous Opportunistic Fungi: Microascaceae and Other

Ascomycetes, Hyphomycetes, Coelomycetes, and
Basidiomycetes 637
Lynne Sigler
Part Two Fungal Populations
12. Molecular Methods to Identify Pathogenic Fungi 677

Thomas G. Mitchell and Jianping Xu
13. Population Genetic Analyses of Medically Important Fungi 703

Jianping Xu and Thomas G. Mitchell
14. Genetic Instability of Candida albicans 723

Elena Rustchenko and Fred Sherman
Index 777
Contributors
Teun Boekhout, Ph.D. Centraalbureau voor Schimmelcultures, Institute of the

Royal Netherlands Academy of Arts and Sciences, Utrecht, The Netherlands
Michael W. Dick, D.Sc. Department of Botany at Earley Gate, School of Plant

Sciences, University of Reading, Reading, England
Frank M. Dugan, Ph.D. USDA-ARS Western Regional Plant Introduction

Station, USDA, Pullman, Washington, U.S.A.
Eveline Guého, Ph.D. Mauves sur Huisne, France
Kevin C. Hazen, Ph.D. Department of Pathology, University of Virginia

Health System, Charlottesville, Virginia, U.S.A.
Dexter H. Howard, Ph.D. Departments of Microbiology, Immunology, and

Molecular Genetics, Center for the Health Sciences, UCLA School of Medicine,
Los Angeles, California, U.S.A.
Susan A. Howell, B.Sc., Ph.D. Department of Medical Mycology, St. John’s

Institute of Dermatology, King’s College London, London, England
Shung-Chang Jong, Ph.D. Department of Microbiology, American Type Cul-

ture Collection, Manassas, Virginia, U.S.A.
xi
xii Contributors
Thomas G. Mitchell, Ph.D. Department of Genetics and Microbiology, Duke

University Medical Center, Durham, North Carolina, U.S.A.
Arvind A. Padhye, Ph.D. Mycotic Disease Branch, Division of Bacterial and

Mycotic Diseases, National Center for Infectious Disease, Centers for Disease
Control and Prevention, Atlanta, Georgia, U.S.A.
Elena Rustchenko, Ph.D. Department of Biochemistry and Biophysics, Uni-

versity of Rochester Medical School, Rochester, New York, U.S.A.
Wiley A. Schell, M.S. Department of Medicine, Duke University Medical Cen-

ter, Durham, North Carolina, U.S.A.
M. A. A. Schipper, Ph.D. Centraalbureau voor Schimmelcultures, Institute of

the Royal Netherlands Academy of Arts and Sciences, Utrecht, The Netherlands
Fred Sherman, Ph.D. Department of Biochemistry and Biophysics, University

of Rochester Medical School, Rochester, New York, U.S.A.
Lynne Sigler, M.S. Microfungus Collection and Herbarium, Devonian Botanic

Garden and Medical Microbiology and Immunology, University of Alberta, Ed-
monton, Alberta, Canada
J. A. Stalpers, Ph.D. Centraalbureau voor Schimmelcultures, Institute of the

Royal Netherlands Academy of Arts and Sciences, Utrecht, The Netherlands
Richard Summerbell, Ph.D. Centraalbureau voor Schimmelcultures, Institute

of the Royal Netherlands Academy of Arts and Sciences, Utrecht, The Nether-
lands
Irene Weitzman, Ph.D. Department of Pathology, Columbia University Col-

lege of Physicians and Surgeons, New York, New York, and Department of Mi-
crobiology, Arizona State University, Tempe, Arizona, U.S.A.
Jianping Xu, Ph.D. Department of Biology, McMaster University, Hamilton,

Ontario, Canada
1
An Introduction to the Taxonomy
of Zoopathogenic Fungi
Dexter H. Howard
UCLA School of Medicine, Los Angeles, California, U.S.A.
I. THE CLASSIFICATION OF FUNGI
The subject of this book is the classification of zoopathogenic fungi. The basic
units in classification are the species, and these are arranged into hierarchal
groups of genera, families, orders, classes, phyla, and kingdoms. The categories
may be subdivided (e.g., subphylum, subclass, suborder) to indicate degrees of
relationships. Populations within a given species that have some characteristics
in common may be set apart as tribes or varieties or some other subset designa-
tion. The delineation of the zoopathogen Ajellomyces capsulatus, anamorph: His-
toplasma capsulatum, goes as follows (1):
Kingdom: Fungi
Phylum: Ascomycota
Class: Ascomycetes
Order: Onygenales
Family: Onygenaceae
Genus: Ajellomyces
Species: Ajellomyces capsulatus
Variety: the varietal state applies to the anamorph (2):
Histoplasma capsulatum var. capsulatum, H.
capsulatum var. duboisii, and H. capsulatum
var. farciminosum
1
2 Howard
Table 1 The Kingdoms and Phyla

of Microorganisms Covered
in This Book
Kingdom: Fungi
Phylum: Chytridiomycota
Phylum: Zygomycota
Phylum: Ascomycota
Phylum: Basidiomycota
Mitosporic Fungia
Kingdom: Straminipila
Phylum: Heterokonta
Kingdom: Protoctista
Phylum: Protista
a
This group of anamorphic fungi—which
lack any known teleomorphic stage—was at
one time collected into a phylum Deuteromy-
cota, but this term, still used for convenience
in some arrangements (see, e.g., Chap. 8), has
been largely discontinued.
II. NOMENCLATURE OF HIERARCHAL GROUPS
The pathogenic organisms traditionally considered as fungi* are now distributed

among three biologic kingdoms: Fungi, Straminipila, and Protoctista (3). The
bulk of this book is devoted to the members of the kingdom Fungi. Also included,
however, is a consideration of the zoopathogenic members of the kingdoms
Straminipila and Protoctista. (See Chap. 2.)
The phyla to be considered in this book are listed in Table 1. Opinions on
the nomenclature of hierarchal groups vary somewhat among taxonomists. For
example, the terms Mycota and Fungi have both been used to designate the king-
dom to which most of the human pathogens belong (4). Likewise, at one time
mycologists used the term division instead of phylum (2) but more recently the
word phylum has been widely adopted in fungal taxonomy. Authors in this book
have used one term or the other as alternative words for the two highest hierarchal
categories. I have let stand whichever usage the author chooses for these catego-
ries. In this summary chapter, the various taxonomic groups are the ones that
* Fundamental descriptions of fungi may be found in the textbooks on medical mycology by K. J.

Kwon-Chung and J. E. Bennett (2) and on fundamental mycology by O. J. Alexopoulos, C. W.
Mims, and M. Blackwell (3). A glossary of terms employed in this introduction will be found at
the end of the chapter.
Introduction 3
contain animal pathogens (zoopathogens) and are covered in this book. The deci-
sions on coverage within these groups have been left up to the authors selected.
III. THE KINGDOM FUNGI

A. Chytridiomycota
This phylum comprises those members of the kingdom Fungi that produce motile
cells in their life cycles (3). There are five orders of such pathogens or commen-
sals within the single class of Chytridiomycetes (Table 2). The zoospores, pro-
duced by asexual reproduction of members of four of the five orders, have a
single, smooth, posteriorly directed whiplash flagellum. A single order, Neocalli-
masticales, contains forms that are polyflagellate. Only some examples of patho-
gens are included in this brief sketch of the phylum. For complete coverage of
the biology and host–parasite relations among the zoopathogenic chytrids, see
Chap. 2.
1. Neocallimasticales. The members of the order differ from other mem-

bers of the class by being composed of obligate anaerobes that have
multiple posterior flagella. These chytrids are commensals of the ru-
men and cecum of herbivores (3).
2. Blastocladiales. Important pathogens of mosquitoes and other insects
(Coelomomyces spp.) and of rotifers and nematodes (Caternaria spp.)
are contained in this order. The pathogenic species of the two genera
indicated are listed in Chap. 2.
3. Chytridiales. The genera Olpidium, Endochytrium, Rhizophydium, and
Phlyctochytrium variously contain important pathogens of nematodes,
rotifers, and amphibians.
Table 2 The Phylum

Chytridiomycota
Class: Chytridiomycetes
Order: Neocallimasticales
Order: Blastocladiales
Order: Chytridiales
Order: Spizellomycetales
Order: Monoblepharidales
Note: For a more complete treatment of this

phylum, see Table 1 of Chap. 2.
4 Howard
4. Spizellomycetales. The members of this order are diverse ecologically,

being found in soil and water, and include plant and fungal parasites.
5. Monoblepharidales. This order consists of but a few mostly sapro-
phytic species.
B. Zygomycota
The phylum Zygomycota (Table 3) contains those fungi that produce zygospores
as a result of sexual reproduction and consists of two classes: the class Trichomy-
cetes, which are obligate parasites of arthropods and will not be considered in
this book (3), and the class Zygomycetes, which contains several important patho-
gens. There are two orders in the class Zygomycetes—Mucorales and Ento-
mophthorales, both of which contain human and animal pathogens. The Mucor-
ales generally produce nonseptate hyphae, while the Entomophthorales are
usually septated.
1. Mucorales. (See Chap. 3.) The members of this order are grouped into
six families of agents of disease.
a. Mucoraceae. This family contains the genera Rhizopus, Absidia,
Apophysomyces, Mucor, and Rhizomucor, all of which are impor-
tant pathogens. These fungi reproduce asexually by means of spo-
rangia containing sporangiospores.
Table 3 The Phylum Zygomycota
Phylum: Zygomycota
Class: Trichomycetes
Class: Zygomycetes
Order: Mucorales
Family: Mucoraceae
Family: Syncephalastraceae
Family: Mortierellaceae
Family: Saksenaeaceae
Family: Thamnidiaceae
Family: Cunninghamellaceae
Order: Entomophthorales
Family: Basidiobolaceae
Family: Entomophthoraceae
Family: Completoriaceae
Family: Ancylistaceae
Family: Meristacraceae
Family: Neozygitaceae
Introduction 5
b. Syncephalastraceae. One species, Syncephalastrum racemosum,

has been reported in clinical materials. Asexual reproduction in
this family is different from that of other members of the Mucor-
ales. Sporangiospore-containing merosporangia are produced on
vesicles formed at the tips of sporangiophores. The sporangio-
spores are arranged in a row within the cylindrical merosporan-
gium (4).
c. Mortierellaceae. Mortierella spp. produce very small sporangio-
spores that are contained in a sporangium that lacks a columella.
Fungi thought to be species of Mortierella have been isolated from
human infections. M. wolfii has been found in bovine mycotic
abortion.
d. Saksenaeaceae. Members of this family produce sporangiospores
in flask-shaped sporangia that have long beaks. One species, Sak-
senaea vasiformis, is an important pathogen.
e. Thamnidiaceae. Single or multispored sporangiola formed at the
tips of branched sporangiophores characterize members of this
family. Cokeromyces recurvatus is an uncommon pathogenic spe-
cies within the family.
f. Cunninghamellaceae. In the genus Cunninghamella single-spore
sporangiola are formed on swollen, round vesicles that occur on
the tips of branched or unbranched sporangiophores. The family
contains at least one important pathogen, C. bertholletiae.
2. Entomophthorales. The zygospores of members of this order are simi-
lar to those of the Mucorales, but differ in morphological detail. The
asexual spores are called conidia, though with some species they ap-
pear to be monospored sporangia (2). The families within the order
Entomophthorales are shown in Table 3. Only the host–parasite rela-
tions of the families are given here. The additional biologic bases for
family separations are given in Chap. 4.
a. Basidiobolaceae. The family Basidiobolaceae comprises species
that are commensals of amphibians and reptiles, and parasites in
vertebrates. The human pathogen Basidiobolus ranorum is found
in this family.
b. Entomophthoraceae. The family Entomophthoraceae contains
pathogens of insects and other arthropods.
c. Completoriaceae. The family Completoriaceae has no animal para-
sites, but certain species are obligate intracellular parasites of fern
gametophytes.
d. Ancylistaceae. Conidiobolus spp. are contained in the family An-
cylistaceae. The genus contains at least two human pathogens, C.
coronatus and C. incongruus.
6 Howard
e. Meristacraceae. The family Meristacraceae contains obligate para-

sites of nematodes and tardigrades.
f. Neozygitaceae. Obligate pathogens of mites and other insects are
found in this family.
C. Ascomycota
This phylum is made up of fungi that reproduce sexually by means of ascospores
contained in an ascus. The morphology and arrangement of ascospores within
the ascus and the morphology of an ascus-bearing structure (ascoma), when pres-
ent, is one approach to the hierarchal arrangement of ascomycetes. The group is
divided into two classes: the Endomycetes and the Ascomycetes. Asexual repro-
duction in the class Endomycetes is by budding or fission of somatic cells and in
the Ascomycetes by formation of blastic or thallic conidia. Molecular phylogeny
studies have allowed associations to be realized even when a known teleomorph
for a given anamorph has not been revealed. Some of these associations are sug-
gested throughout the coverage in this section.
1. Endomycetes
a. Saccharomycetales. Ascomata are not formed. Ascospores are of
various shapes. Asci are formed singly or in chains.
(1) Saccharomycetaceae. This family contains those yeasts that
reproduce by budding (blastoconidia). The colonies are ac-
cordingly mainly unicellular, though some species produce
pseudohyphae.
(2) Dipodascaceae. This family includes the genus Dipodascus,
which produces a Geotrichum anamorph with arthroconidia.
Other pathogenic yeasts that reproduce by fission (arthroconi-
dia) are included in this family. (See Chap. 8.).
2. Ascomycetes.
a. Onygenales. The order consists of four families. The members of
these families produce ascomata called cleistothecia, the peridium
of which is composed of a loose network of hyphae (2). The term
gymnothecium is sometimes applied to this type of cleistothecium.
(1) Arthrodermataceae. (See Chap. 5.) This family comprises
parasites known as the dermatophytes (ringworm fungi) and
saprophytes that are morphologically similar. The family is
represented by the single genus Arthroderma, whose asexual
states are in the genera Microsporum and Trichophyton. An
additional member of the group known only in its conidial
state is Epidermophyton.
(2) Onygenaceae. (See Chap. 6.) Two important systemic patho-
Introduction 7
gens of humans are found in this family: Ajellomyces capsu-

latus (anamorph, Histoplasma capsulatum) and A. dermatit-
idis (anamorph, Blastomyces dermatitidis). In addition, one
rare pathogen, Ajellomyces crescens (anamorph, Emmonsia
crescens), belongs in this family. A relationship to the Ony-
genaceae is inferred for other important human pathogens,
such as Coccidioides immitis and Paracoccidioides bra-
siliensis. Emmonsia parva, a pathogen of small mammals, is
also thought to belong in the family. Several other pathogens
and saprophytes (contaminants of clinical materials) are
found in the family. The anamorphic forms of the latter are
placed in the genera Chrysosporium and Malbranchia. (See
Chap. 6.)
(3) Gymnoascaceae and Myxotrichaceae. A number of uncom-
mon pathogens and saprophytes encountered as contaminants
in clinical materials are found in these two families. (See
Chap. 6.).
b. Eurotiales. This order contains a number of cleistothecial fungi
whose anamorphs are usually phialidically formed conidia. The
most important family is the Trichocomaceae (see Chap. 7), which
includes the teleomorphs of Aspergillus and Penicillium. There are
a number of species in these asexual genera that are not known
to produce a sexual state. Nevertheless, their affinity to the Euroti-
ales is clear. There are other important opportunistic pathogens of
Eurotialean affinity known only in their anamorphic forms. (See
Chap. 7.) The other families in the order Eurotiales (Table 4) con-
tain only rare causes of disease.
c. Ophiostomatales. A perithecium is the ascoma of the sexual state
of members of this order. One important pathogen in this group
is thought to have close affinity with Ophiostoma (Family Ophio-
stomataceae), but is currently known only in its anamorphic form,
Sporothrix schenckii.
d. Dothideales. The members of this order produce cleistothecial as-
comata. The order comprises six families.
(1) Didymosphaeriaceae—Neostestudina rosatii, an agent of
mycetoma.
(2) Piedraiaceae—Piedraia hortae, the cause of black piedra.
(3) Herpotrichiellaceae—no teleomorphs that are known as zoo-
pathogens but a number of anamorphs whose cells are mela-
nized are thought to belong in this ascomycete family (e.g.,
Phialophora, Cladophialophora, Exophiala, and Fonse-
caea). (See Chap. 10 on the Dematiaceous Hyphomycetes.).
8 Howard
Table 4 The Phylum Ascomycota
Phylum: Ascomycota
Class: Endomycetes
Order: Saccharomycetales
Family: Saccharomycetaceae
Family: Dipodascaceae
Class: Ascomycetes
Order: Dothideales
Family: Didymosphaeriaceae
Family: Herpotrichiellaceae
Family: Piedraiaceae
Family: Dothideaceae
Family: Botryosphaeriaceae
Family: Mycosphaerellaceae
Order: Eurotiales
Family: Trichocomaceae
Family: Pseudoeurotiaceae
Family: Eremomycetaceae
Family: Thermoascaceae
Order: Microascales
Family: Microascaseae
Order: Onygenales
Family: Arthrodermataceae
Family: Onygenaceae
Family: Gymnoascaceae
Family: Myxotrichaceae
Order: Ophiostomatales
Family: Ophiostomataceae
Order: Hypocreales
Family: Hypocreaceae
Order: Pleosporales
Family: Leptosphaeraceae
Family: Pleosporaceae
Order: Sordariales
e. Microascales. This order, which produces ascomata that are cleis-

tothecia or perithecia, contains a single family of pathogens, Mi-
croascaceae, which houses the important agent of mycetoma,
Pseudallescheria boydii (anamorph, Scedosporium apiospermum),
and common clinical isolates in the anamorphic genus Scopulari-
opsis. (See Chap. 11.)
f. Hypocreales. The ascoma of this order is a perithecium. This order
Introduction 9
contains many important opportunistic pathogens of humans and

common clinical contaminants. One example is Fusarium spp. (tel-
eomorphs Gibberella spp. and Nectria spp., family Hypocreaceae).
(See Chap. 7.).
g. Pleosporales. The ascomata of this order are cleistothecia. The or-
der comprises two families: Leptosphaeraceae, which contains
Leptosphaeria senegalensis and L. thompkinsii, agent of myce-
toma, and Pleosporaceae, which contains Cochliobolus spp. (ana-
morphs: Curvularia and Bipolaris) and other dematiaceous patho-
gens best known by their anamorphic names. (See Chap. 10.)
h. Sordariales. Ascomata are perithecia or cleistothecia. Perhaps the
best-known member of this order is Neurospora. Rare human
pathogens include Chaetomium spp. (2).
D. Basidiomycota
The distinctive feature of the members of the phylum Basidiomycota is the pro-
duction of basidiospores (sexual spores) on the outside of a club-shaped to elon-
gate structure called the basidium. The members of the basidiomycota considered
in this book will be the zoopathogenic yeasts found in the phylum and some rare
causes of infectious diseases found among the ‘‘mushrooms’’ (Table 5). Yeast
forms are found in all three main phylogenetic lines of basidiomycetes, namely
the Hymenomycetes (Cystofilobasidiales, Trichosporonales, Tremellales, and Fi-
lobasidiales), Urediniomycetes (Sporidiales), and the Ustilaginomycetes (Malas-
seziales). Medically important basidiomycetous yeasts belong to the genera
Cryptococcus, Trichosporon (Hymenomycetes), and Malassezia (Ustilaginomy-
cetes). Other basidiomycetous yeasts reported in clinical material occur in the
genera Rhodotorula and Sporobolomyces (Urediniomycetes). This topic is cov-
ered in Chap. 9.
The class Hymenomycetes also contains forms known colloquially as
mushrooms (e.g., the orders Agaricales and Aphyllophorales contain such fungi).
The former contains Coprinus cinereus, and the latter houses Schizophyllum com-
mune, both of which have been reported to cause rare infection in humans. It is
ordinarily the toxic or hallucinogenic aspects of mushrooms that involve human
disease. These topics were covered in the first edition of this work (5), but will
not be considered in the second edition. Two excellent references on the topics
are Arora (6) and Lincoff and Mitchell (7).
E. Mitosporic Fungi: The Fungi Imperfecti

This group comprises the fungi that have no known teleomorphic stage and repro-
duce only by means of asexual conidia. Although molecular methods have sug-
10 Howard
Table 5 The Phylum Basidiomycota
Phylum: Basidiomycota
Class: Hymenomycetes
Order: Cystofilobasidiales
Order: Trichosporonales
Order: Tremellales
Order: Filobasidiales
Order: Aphyllophorales
Order: Agaricales
Class: Urediniomycetes
Order: Sporidiales
Class: Ustilaginomycetes
Order: Malasseziales
Note: Taxonomic ordering of the higher taxa

of basidiomycetous fungi has been subject to
much change over the years (3). The scheme
given here is based on morphology and mo-
lecular approaches (3). The major focus in
this book is on the groups containing yeasts
(see Chap. 9), but two orders of mushrooms,
Aphyllophorales and Agaricales, which con-
tain rare human pathogens, are also included.
(See Chap. 11.)
gested teleomorphic association for these anamorphs, it is often convenient to

discuss them as a group of anamorphs. (See, e.g., Chap. 10.) At one time these
anamorphs were collected into a phylum designated the Deuteromycota and ar-
ranged into appropriate hierarchal subdivisions. Such arrangements were subse-
quently recognized as inappropriate, and the terms ‘‘form phylum,’’ ‘‘form
class,’’ etc. were adopted (2). These artificial names were subsequently aban-
doned, and the term Mitosporic Fungi has been used instead (4,8). There are a
number of subdivisions that are nevertheless useful in arranging such imperfect
fungi (Table 6).
The Blastomycetes are those unicellular fungi that reproduce by budding
or fission but for which no teleomorphic stage has been identified. These forms
obviously have ascomycete or basidiomycete affinities but are separated into this
group of mitosporic fungi in most treatments (see Chaps. 8 and 9), although some
taxonomists indicate apparent teleomorphic associations in their treatment of the
subject (9). The Hyphomycetes are hyphal fungi for which no teleomorph stage
has yet been revealed. Again, teleomorph associations for such imperfect fungi
have been suggested by some investigators (9), but usually they are referred to
as mitosporic fungi and are not arranged into a hierarchal organization in the
Introduction 11
Table 6 Mitosporic Fungi
A. Blastomycetes—anamorphic yeasts with no known teleomorphic state

B. Coelomycetes—fungi producing a pycnidium or acervulus
C. Hyphomycetes—fungi with no pycnidium or acervulus
1. Moniliaceous—hyphae and conidia hyaline
2. Dematiaceous—hyphae and conidia dark
D. Agonomycetes (Mycelia Sterilia)—fungi producing hyphae only. No reproductive
propagules
Note: The fungi imperfecti, which produced only conidia were gathered in the past, into the phylum
Deuteromycota (3). This practice has been abandoned in recent taxonomic treatments of zoopatho-
genic fungi.
literature on medical mycology (8). It is customary to divide the Hyphomycetes

into Dematiaceous (i.e., dark-spored; see Chap. 10) and Moniliaceous (i.e.,
hyaline-spored; see Chap. 11). The pathogenic Dematiaceous Hyphomycetes are
involved in the phaeohyphomycoses. Two other categories are considered among
the mitosporic fungi: the Coelomycetes (i.e., those fungi that form their conidia
on acervuli or within pycnidia) and Agonomycetes (Mycelia Sterilia; i.e., these
fungi that produce no reproductive propagules of any kind). (N.B.: they may
form chlamydospores.) One member of this latter group, Rhizoctonia sp., was
reported in a case of human keratitis. (See Ref. 9.)
Some interesting new work on three pathogens, Rhinosporidium seeberi
(rhinosporidiosis), Pneumocystis carinii (pneumocystosis), and Lacazia (Loboa)
loboi, has revealed previously unknown taxonomic associations. Rhinospori-
diosis presents as tumorlike masses, commonly in the nasal mucosa or conjunc-
tiva of humans and animals (2). The taxonomic position of R. seeberi has always
been uncertain. Recently studies on 18S r RNA genes from tissue infected with
the fungus has shown a phylogenetic relationship to a novel group of protists
that infect fish and amphibians. The fungus is ‘‘the first known human pathogen
from the DRIPs clade, a novel clade of aquatic protistan parasites (Icthyasporea)’’
(10).
Pneumocystis carinii was originally considered a protozoan, and most of
the information about this important opportunistic pathogen is found in the litera-
ture on animal parasites. Studies on the ribosomal RNA sequences, however,
established that P. carinii was a member of the kingdom Fungi (11). The system-
atic position of P. carinii within the kingdom Fungi is uncertain, but the best
evidence indicates that it is an ascomycete (12).
Lobomycosis is a chronic cutaneous and subcutaneous infection in humans
and dolphins whose etiologic agent Lacazia (Loboa) loboi has remained in an
uncertain taxonomic position since its original description (2). Recently a phylo-
12 Howard
genic analysis by molecular means has confirmed earlier studies that indicated
the fungus belongs in the family Onygenaceae of the order Onygenales (Ascomy-
cota) (13).
IV. THE KINGDOM STRAMINIPILA

A. Phylum Heterokonta
Only one phylum, Heterokonta, in the kingdom Straminipila, will be considered
in this book. The phylum is diverse, and only a brief summary indicating the
various hosts of these parasites is given here. (See Chap. 2 for a consideration
of their biology.) The phylum comprises fungi whose zoospores have two fla-
gella, one directed anteriorly that is adorned with two rows of tripartite hairs,
and one directed posteriorly that is smooth. There are two classes (Labyrinthista
and Peronosporomycetes) in the phylum that contain animal parasites. Only one
genus, Labyrinthriloides, in the class Labyrinthista, that contains pathogens of
certain shell-less mollusks, will be considered in this book. The class Peronospor-
omycetes contains three subclasses: the Peronosporomycetidae, which is mainly
restricted to phytopathogens but also contains a mammalian pathogen, Pythium
insidiosum; the Rhipidiomycetidae, whose members are saprophytes; and the Sa-
prolegniomycetidae, which includes parasites of animals. There are four orders
of pathogens within the last subclass (Table 7).
1. Subclass Peronosporomycetidae.
a. Pythiales. This order contains a pathogen of mammals including
humans, Pythium insidiosum.
2. Subclass Saprolegniomycetidae.
a. Saprolegniales. A number of important fish, crustacean, and insect
pathogens are contained in this order.
b. Salilagenidiales. This order comprises parasites of crustacea and
mollusks.
c. Myzocytiopsidiales. Pathogens of nematodes, rotifers, and mos-
quitoes and other insects are contained in this order.
d. Leptomitales. This group comprises pathogens of nematodes, roti-
fers, and insects.
V. KINGDOM PROTOCTISTA
Only the phylum Protista of the kingdom Protoctista will be considered. The
members of the class Plasmodiophoromycetes are characterized by zoospores
with two unornamented flagella that are of unequal length. A single genus in the
Introduction 13
Table 7 The Kingdoms Straminipila and

Protoctista
Kingdom: Straminipila
Phylum: Heterokonta a
Class: Labyrinthista
Order: Thraustochytriales
Class: Peronosporomycetes
Subclass: Peronosporomycetidae
Order: Pythiales
Subclass: Saprolegniomycetidae
Order: Saprolegniales
Order: Leptomitales
Order: Salilagenidiales
Order: Myzocytiopsidales
Phylum: Protista b
Class: Plasmodiophoromycetes
Order: Haptoglossales
a
The only phylum in the kingdom Straminipila to be
considered here. The hierarchal considerations have
been simplified. See Tables 1 and 2 of Chap. 2 for a
complete treatment of this phylum.
b
The only phylum of the Protoctista to be considered.
order Haptoglossales, Haptoglossa, a pathogen of rotifers will be considered in

this book.
VI. POPULATIONS
Groups of individuals will be considered in Part 2 of this book. Populations of

individual species that are separated from other populations by physical or biolog-
ical barriers will diverge from one another (14). Examples of such divergence
among fungi are accumulating. In one recent example, RFLP analysis of 32 iso-
lates of Paracoccidioides brasiliensis indicated at least five geographically dis-
tinct groups of strains. The groups of strains corresponded to country borders
(15). Such divergence is one possible basis for the emergence of new species
(evolution). Molecular techniques applicable to such descriptions are covered in
Chap. 12, and the emergence of a new field—molecular epidemiology, which is
based on molecular data—is considered in Chap. 13.
Divergence in populations may also have a genetic basis (14). One example
is Candida albicans. Until recently, this fungus was considered an amictic ana-
14 Howard
morph. It has been shown that the fungus spontaneously produces a high fre-
quency of chromosomal aberrations (16). Such alterations have been suggested
to be a means of achieving genetic variability in an organism that lacks a teleo-
morphic form (16). Such variation could be a basis for speciation in a group of
fungi like the unicellular yeasts in which taxonomic characters are limited and
depend to a large extent on utilization of various metabolites (17). Very recently
it has been shown that C. albicans can be induced to mate (18,19), but the extent
to which mating occurs in populations of C. albicans remains to be evaluated.
The genetic variability of C. albicans and the recent studies on mating will be
covered in Chap. 14.
GLOSSARY
Acervulus (pl. acervuli). A fruiting body without a covering tissue, usually a

discoid or flat mass of conidiophores producing conidia in a moist mass.
Anamorph. The forms representing asexual reproduction.
Arthroconidium (pl. arthroconidia). A conidium produced by septation of a
hypha and fragmentation at the septa.
Ascocarp. See ascoma.
Ascoma (pl. ascomata). A fungus structure containing asci, which in turn bear
ascospores. Also called an ascocarp.
Ascospores. A sexual spore borne within an ascus. The definitive structural
element of the phylum Ascomycota.
Ascus (pl. asci). An ascospore-bearing saclike structure.
Asexual reproduction. Reproduction that does not involve fusion of nuclei
and meiosis.
Basidiospores. Sexual spores borne on a basidium which define the phylum
Basidiomycota.
Basidium (pl. basidia). The fungus structure upon which basidiospores are
borne.
Blastoconidium (pl. blastoconidia). A conidium produced by budding.
Clade. Literally a branch. A monophyletic group. The word is used in studies
on phylogeny (evolution) of a species.
Cleistothecium. A completely closed ascoma.
Colony (pl. colonies). In the unicellular yeasts, a collection of yeast cells (blas-
toconidia, q.v. or arthroconidia, q.v.). In the molds, a collection of hyphae,
also called a mycelium (pl. mycelia) or a thallus (pl. thalli).
Columella. The sterile inflated end of a sporangiophore that extends into the
sporangium.
Conidiophores. A specialized hypha that bears conidia.
Conidium (pl. conidia). An asexual reproductive propagule.
Introduction 15
Dermatophyte. Literally, ‘‘skin plant.’’ A group of pathogenic fungi that cause

the clinical conditions known as Tinea (ringworm).
Dimorphic. Having two forms. In medical mycology the term has been used
to describe a duality of form associated with the parasitic and saprophytic
phases of the life cycle of a zoopathogenic fungus.
Flagellum (pl. flagella). A hairlike structure that propels a motile cell.
Gymnothecium (pl. gymnothecia). An ascoma whose wall is composed of
loosely woven hyphal elements.
Hypha (pl. hyphae). A tubular filament—usually branched—that makes up
the vegetative portion of a mold. Two forms of hyphae occur: nonseptate, in
which the cytoplasm of the hypha is uninterrupted by cross walls (septa), and
septate, in which the cytoplasm of the hyphae is regularly interrupted by
septa.
Merosporangium (pl. merosporangia). A cylindrical sporangium in which
the sporangiospores are arranged in a row.
Mitospore. A nucleated spore formed by mitosis.
Ostiole. A hole in an ascoma or a pycnidium.
Peridium (pl. peridia). The outer wall of a fruiting body.
Perithecium. A closed ascoma with a hole (ostiole) at the top and a wall of
its own.
Pycnidium (pl. pycnidia). An asexually produced flasklike body with an osti-
ole and containing conidia.
Sexual reproduction. Reproduction that includes fusion of nuclei and meiosis.
Sporangiolum (pl. sporangiola). A small sporangium containing a few spor-
angiospores.
Sporangiophores. The modified hyphal element that bears the sporangium.
Sporangiospores. Asexual spores produced within a sporangium.
Sporangium (pl. sporangia). A saclike structure within which asexual spores
(sporangiospores) are formed by progressive cleavage.
Teleomorph. The forms representing sexual reproduction.
Zoospore. A motile asexual spore.
Zygospore. A resting spore that develops from a zygote formed by the union
of two similar gametes. The spore that defines the phylum zygomycota.
REFERENCES
1. DH Howard, HM Kokkinos. Classification of fungi. In: RD Fegin, JD Cherry, eds.

Textbook of Pediatric Infectious Diseases. vol 2. 4th ed. Philadelphia: Saunders,
1998, pp. 2287–2288.
2. KJ Kwon-Chung, JE Bennett. Medical Mycology. Philadelphia: Lea & Febiger,
1992.
16 Howard
3. CJ Alexopoulos, CW Mims, M Blackwell. Introductory Mycology. 4th ed. New

York: Wiley, 1996.
4. DL Hawksworth, PM Kirk, BC Sutton, DN Pegler. Ainsworth and Bisby’s Diction-
ary of the Fungi. 8th ed. Cambridge, UK: International Mycological Institute, CAB
International., University Press, 1996.
5. PP Vergeer. Poisonous fungi: Mushrooms. In: DH Howard, ed. Fungi Pathogenic
for Humans and Animals. part B. New York: Marcel Dekker, 1983, pp. 373–412.
6. D Arora. Mushrooms Demystified. 2nd ed. Berkeley, CA: Ten Speed Press, 1986.
7. G Lincoff, DH Mitchell. Toxic and Hallucinogenic Mushroom Poisoning. New
York: Van Nostrand Reinhold, 1977.
8. DA Sutton, AW Fothergill, MG Rinaldi. Guide to Clinically Significant Fungi. Balti-
more: Williams and Wilkins, 1998.
9. GS de Hoog, J Guarro, J Gené, MJ Figueras. Atlas of Clinical Fungi. Utrecht, the
Netherlands: Centraalbureaus voor Schimmelcultures, 2000.
10. DN Fredricks, JA Jolley, PW Lepp, JC Kosek, DA Relman. Rhinosporidium seeberi:
A human pathogen from a novel group of aquatic protistan parasites. Emerging Infec
Dis 6:273–282, 2000.
11. JC Edman, JA Kovacs, H Mansur, DV Santi, HJ Elwood, ML Sogin. Ribosomal
RNA sequence shows Pneumocystis carinii to be a member of the Fungi. Nature
334:519–522, 1988.
12. JW Taylor, E Swann, ML Berbee. Molecular evoluation of ascomycete fungi. In:
DL Hawksworth, ed. Ascomycetes Systematics: Problems and Perspectives in the
Nineties. New York: Plenum, 1994.
13. RJ Tarcha, RA Herr, JW Taylor, L Ajello, L Mendoza. Phylogenetic analysis of the
ITS 1, 5.85 and ITS 2 rDNA sequences of Lacazia loboi confirms placement of this
unique fungal pathogen within the Onygenales. Abstr. 101st Meeting of the Ameri-
can Society for Microbiology, F 131:381, 2001.
14. E Mayr. Systematics and the origin of species from the viewpoint of a zoologist:
A reprint with a new introduction by the author. Cambridge, MA: Howard University
Press, 1999.
15. GA Niño-Vega, AM Calcagno, G San-Blas, F San-Blas, GW Gooday, NAR Gow.
RFLP analysis reveals marked geographical isolation between strains of Paracocci-
dioides brasiliensis. Med Mycol 38:437–441, 2000.
16. EP Rustchenko-Bulgac, F Sherman, JB Hicks. Chromosomal rearrangements associ-
ated with morphological mutants provide a means for genetic variation in Candida
albicans. J Bacteriol 172:1276–1283, 1990.
17. NJW Kreger-van Rij, ed. The Yeasts. Amsterdam: Elsevier, 1984.
18. CM Hull, RM Rainer, AD Johnson. Evidence for mating of the ‘‘asexual’’ yeast
Candida albicans in a mammalian host. Science 289:307–310, 2000.
19. BB Magee, PT Magee. Induction of mating in Candida albicans by construction of
MTLa and MTLα strains. Science 289:310–313, 2000.
2
The Peronosporomycetes
and Other Flagellate Fungi
Michael W. Dick
School of Plant Sciences, University of Reading, Reading, England
I. BASIC BIOLOGY
There are two major phylogenetic lines of flagellate fungi: those with zoospores
having a single, posteriorly directed, smooth whiplash flagellum and those with
zoospores having two (heterokont and anisokont) flagella, one directed anteriorly
and clothed with two rows of tubular tripartite hairs (straminipilous ornamenta-
tion) and one directed posteriorly, smooth and with an acronema. The first line
(the Chytridiomycetes) constitutes a near-basal clade on the animal/mycote phy-
logeny; the second line (the Peronosporomycetes) is a major clade with the chro-
mophyte algae (Table 1). The Plasmodiophoromycetes (Haptoglossa) is charac-
terized by zoospores with two homokont but anisokont unornamented flagella.
The Peronosporomycetes are probably the largest and are certainly the most
diverse monophyletic class of flagellate fungi. Originally separated from other
flagellate fungi by their oogamous sexual reproduction, the Peronosporomycetes
are now primarily distinguished from other fungi by the distinctive biflagellation
of the zoospore. Peronosporomycetes are fungi on both physiological and mor-
phological criteria; that is, they are eukaryotic with uninucleate or coenocytic
protoplasts bounded by cell walls in their assimilatory states and are thus obli-
gately osmotrophic heterotrophs (1,7,8).
The peronosporomycete/chromophyte algae/straminipilous-heterotroph
monophyletic line includes gut commensals such as Blastocystis and Proteromo-
nas (Slopalinida), which are not fungi (2), and animal parasites such as Laby-
rinthuloides (Labyrinthista), which are occasionally treated as fungi but which
I do not regard as constituting a fungal class (3,8). A few organisms, such as
17
18 Dick
Table 1 Classification of Flagellate Fungal Parasites and Commensals of Animals
Kingdom: Mycota
Class: Chytridiomycetes
Order: Chytridiales
Order: Spizellomycetales
Order: Neocallimasticales: Neocallimaticaceae; Neocallimastix IB Heath, Caeco-
myces JJ Gold, Piromyces JJ Gold et al., Anaeromyces Breton et al., Orpino-
myces Barr et al.
Order: Blastocladiales: Coelomomycetaceae; Catenariaceae; Oedogoniomycetaceae
Coelomomyces Couch, Catenaria Sorokin, Oedogoniomyces Kobayasi & M.
Ôkubo
Order: Monoblepharidales
Kingdom: Straminipila (subkingdom Chromophyta)
Phylum: Heterokonta (other phyla omitted from consideration in this chapter)
Class: Labyrinthista
Order: Labyrinthulales (one doubtful species—L. thais)
Order: Thraustochytriales
Thraustochytriaceae: Labyrinthuloides F. O. Perkins
Subphylum: Peronosporomycotina
Order: Lagenismatales
Class: Hyphochytriomycetes
Class: Peronosporomycetes
Phylum: Protista (other phyla omitted from consideration)
Class: Plasmodiophoromycetes
Order: Plasmodiophorales
Order: Haptoglossales
Haptoglossaceae: Haptoglossa Drechsler
Coelomomyces (Chytridiomycetes), do not fit the above definition of a fungus,

lacking a cell wall in the assimilatory phase (4). The Neocallimastigales (Chytrid-
iomycetes) are divergent within their class, being obligate anaerobes with zoo-
spores possessing multiple posterior flagella (5).
Within the Peronosporomycetes three subclasses can be recognized (Table
2). The Peronosporomycetidae are species-rich (60% of the total number of spe-
cies in the class) but mainly restricted to phytopathogens. The Rhipidiomycetidae
are saprobic on vegetable substrata, but the taxa of the Saprolegniomycetidae
have diverse habitats and substrata and include the most important of the flagel-
late fungal parasites in animals. In addition, the peronosporomycete order incer-
Peronosporomycetes and Other Flagellate Fungi 19
Table 2 Classification of the Class Peronosporomycetes (orders and families

containing parasites of animals in boldface)
Peronosporomycetes
Subclass: Peronosporomycetidae
Order: Peronosporales
Order: Pythiales
Pythiaceae: Pythium Pringsh., Lagenidium Zopf
Subclass: Rhipidiomycetidae
Order: Rhipidiales
Subclass: Saprolegniomycetidae
Order: Saprolegniales
Saprolegniaceae: Saprolegnia Nees, Achlya Nees, Sommerstorffia Arnautov,
Hydatinophagus Valkanov, Couchia W. W. Martin
Leptolegniaceae: Leptolegnia de Bary, Aphanomyces de Bary
Order: Sclerosporales
Order: Leptomitales
Apodachlyellaceae: Eurychasmopsis Canter & M. W. Dick
Leptolegniellaceae: Aphanomycopsis Scherff., Nematophthora Kerry & D. H.
Crump
Order: Salilagenidiales
Salilagenidiaceae: Salilagenidium M. W. Dick (36)
Haliphthoraceae: Haliphthoros Vishniac, Atkinsiella Vishniac, Halodaphnea M.
W. Dick
Order: Olpidiopsidales
Order: Myzocytiopsidales
Myzocytiopsidaceae: Myzocytiopsis M. W. Dick, Gonimochaete Drechsler, Chla-
mydomyzium M. W. Dick
Crypticolaceae: Crypticola Humber et al.
Genera Incertae Sedis: Blastulidium Pérez, Ciliomyces I. Foissner & W. Foissner,
Endosphaerium D’Eliscu
tae sedis, the Myzocytiopsidales, contains important parasites of nematodes. Al-

though most species of Peronosporomycetes are freshwater or terrestrial, a few
are oligohaline or marine, but it is yet to be established whether these taxa repre-
sent primarily or secondarily marine evolutionary developments.
The evolution of the Peronosporomycetes differs from that of eumycote
fungi and the uniflagellate class Chytridiomycetes in that it is based on vegetative
diploidy, not haploidy. The nuclear cycle is haplomitotic B (6), in which mitosis
is confined to the diploid phase. Haploid mitosis does not occur. Both haplomi-
totic A and diplomitotic ploidy cycles are absent from the Peronosporomycetes,
20 Dick
although they occur elsewhere in the phylum and kingdom (e.g., Fucophyceae).
Diplomitotic ploidy cycles occur in the Blastocladiales (Chytridiomycetes).
II. HOST–PARASITE RELATIONSHIPS

AND INTERACTIONS
Parasitism of aquatic animals and the saprobic existence of these fungi on dead
animals and sloughed animal remains has been recognized for 250 years (7,8).
However, the range of animals parasitized is both wide yet restricted, including
vertebrates, crustaceans, insects, and aschelminths (nematodes and rotifers). Para-
sites of crustaceans bridge the freshwater and marine environments, albeit with
different and possibly phylogenetically distantly related species.
Fungus/host-animal relationships are sometimes fairly tightly circum-
scribed, as with the Salilagenidiales parasitic in marine crustaceans and the Myzo-
cytiopsidales parasitic in aschelminths. However, in both groups there are well-
documented occurrences of parasitism outside this normal range. Myzocytiopsis
can infect tardigrades (9). Another species of the same genus has been reported
on a gasterotrich protoctist (see Table 3). One species of Halodaphnea has been
described from a marine rotifer rather than a crustacean (see Key in Section
IV,B,6), and other within-habitat/cross-host boundary parasitisms can be found
in the literature. On the other hand, animal groups are often parasitized by a range
of unrelated fungi: for example, nematodes by Ascomycetes, Peronosporomy-
cetes, and Plasmodiophoromycetes; insects (Diptera) by Peronosporomycetes
(Pythiales and Saprolegniales) and Chytridiomycetes (Blastocladiales).
By far the most noteworthy crustacean parasites are Aphanomyces (Sapro-
legniales) on freshwater crayfish (10,11,220) and Salilagenidium and Halodaph-
nea (Salilagenidiales) on marine crabs and prawns (12–14). Entire populations
of European crayfish have been eliminated from many river systems in Europe
following the introduction and spread of Aphanomyces astaci (Krebspest dis-
ease), and recovery is improbable (11,15,220). Mariculture of crabs, prawns, and
shrimps in Asian coastal waters is subject to epidemics caused by various species
of the Salilagenidales (12,13).
Although there are several examples of insect parasitism, Lagenidium
(Pythiales), which is endoparasitic in mosquito larvae, has received a consider-
able amount of research funding for biological control of mosquito populations
(see, e.g., 16–19), but Crypticola (Myzocytiopsidales), on mosquito and blackfly
larvae (see Ref. 14), and Leptolegnia chapmanii (Saprolegniales), also in mos-
quito larvae, have not been studied as widely (20,21). Coelomomyces (Blastoclad-
iales) was the subject of a number of papers in the early quest for biological
control of mosquitoes (22–24).
The disease of salmonid fish commonly referred to as UDN (ulcerative dermal
Table 3 Peronosporomycetes, Chytridiomycetes, and Plasmodiophoromycetes associated with Aschelminthes
Nematode hosts Rotifer hosts
Catenaria allomycis (175) In Allomyces anomalus R. Emers. (Blas-

tocladiales) from soil
Catenaria anguillulae (176) In sheep liver fluke eggs (and nematodes—
later reports)
Catenaria auxiliaris (J. G. Kühn) (177) In Heterodera schachtii Schmidt
Catenaria vermicola (178) In Xiphinema chambersi Thorne (from St.
Augustine grass)
Chlamydomyzium anomalum (G. L. In an unidentified nematode (from barnyard
Barron) (171) soil)
Chlamydomyzium aplanosporum (171) In Distylae rotifers (from rotting straw)
Chlamydomyzium internum (171) In Distylae rotifers (from organic debris
with pine needles)
Chlamydomyzium oviparasiticum (171) In eggs of Adineta spp. [from farmyard soil]
Chlamydomyzium septatum (Karling) In rotifer eggs (and sporangia of Catenaria
(171) anguillulae Sorokin on snake skin, from
soil sample)
Peronosporomycetes and Other Flagellate Fungi
Chlamydomyzium sphaericum (55) Adult Rhabditis nematodes and rarely, tardi-

grades
Endochytrium oophilum (De Wild) In various algae, protists, and rotifer and
(179) nematode eggs (according to later reports)
Endochytrium operculatum (De Wild) In various algae, protists, and rotifer and
(179) nematode eggs (according to later reports)
Gonimochaete horridula (180) In Acrobeloides sp. [affin. A. buetschlii (De
Man) Thorne on decaying leaves of Acer
rubrum L.]
Gonimochaete latutubus (181) In Rhabditis marina Bastian
Gonimochaete lignicola (182) In nematodes (from riverbank soil)
21
Table 3 Continued 22
Gonimochaete pyriformis (183) In Diploscapter spp. (from soil in a topical

greenhouse)
Halodaphnea parasitica (K. Nakam. In Brachionus plicatus Müller (marine)
& Hatai) (14)
Haptoglossa elegans (184) In bdelloid rotifers (from debris under a tree
fern)
Haptoglossa heterospora (185) In many species of nematodes found in
agar–water–soil litter cultures
Haptoglossa humicola (186) In bdelloid rotifers (from soil in a tropical
greenhouse)
Haptoglossa intermedia (187) In bdelloid rotifers and nematodes (from
woodland soil)
Haptoglossa mirabilis (188) In Adineta spp. (from soil in a tropical
greenhouse)
Haptoglossa zoospora (189) In nematodes (from farmyard soil)
Hydatinophagus americanus (190) In Monostyla sp.
Aphanomyces americanus (115,190)
Hydatinophagus apsteinii (115,191) In Epiphanes senta (Müller as its synonym,
Hydatina senta Ehrenb.)
Aphanomyces hydatinae Valkanov,
Archiv für Protistenkunde 74: 17
(1931)
Myzocytiopsis bolata (55) In adult Rhabditis nematodes; some eggs
parasitized
Myzocytiopsis distylae (Karling) (171) In eggs of Distyla sp. (from a water sample)
Dick
Myzocytiopsis elegans (Perronc) In Philodina roseola Ehrenb.
(171)
Myzocytiopsis fijiensis (Karling) In eggs of Distyla sp. (soil sample)
(171)
Myzocytiopsis glutinospora In Rhabditis terricola Dujardin (from barn-
(G. L. Barron) (171) yard soil)
Myzocytiopsis humicola From soil in a cattle pen
(G. L. Barron & Percy) (171)
Myzocytiopsis indica (U. P. Singh) In rotifers
(171)
Myzocytiopsis intermedia (G. L. Barron) In Rhabditis sp. (soil sample from a birch–
(171) maple–poplar wood)
Myzocytiopsis lenticularis In nematodes (from soil in a cattle pen)
(G. L. Barron) (171)
Myzocytiopsis microspora (Karling) In bodies of Distyla sp. (from a soil sample)
(171)
Myzocytiopsis oophila (Sparrow) (171) In eggs and embryos of rotifers (from
aquatic debris)
Myzocytiopsis osiris (36) Infecting a large proportion of the popula-
tion of adult Rhabditis nematodes and
some juveniles
Myzocytiopsis papillata (G. L. Barron) In Rhabditis terricola Dujardin (from barn-
(171) yard soil)
Myzocytiopsis parthenospora (Karling) In eggs and bodies of Distyla sp. and Phi-
(171) lodina sp.
Bodies of Heterodera sp. and eggs of Chae-
tonotus larus O. Müller (Gasterotricha
from a soil sample)
Myzocytiopsis subuliformis (E. Maupas) In Rhabditis teres Schn. and Rhabditis giar-
(171) dii E. Maupas
23
Myzocytiopsis vermicola (Zopf ) (171) In Anguillula sp.

Table 3 Continued

24
Myzocytiopsis zoophthora (Sparrow) in rotifers and rotifer eggs

(171)
Nematophthora gynophila (192) In females of Heterodera avenae Wollen-
weber
Olpidium entophytum (A. Braun) Ra- In rotifer eggs
bench., Flora Europaea Algarum 3:
283 (1868) var. intermedium J. C.
Constantineanu, Revue Générale de
Botanique 13: 371 (1901) (37:137)
Olpidium granulatum (37:145) In rotifer eggs
Olpidium gregarium (Nowak.) J. Schröt, In rotifer eggs
Kryptogamen-Flora von Schlesien
3(1): 182 (1885) (37: 144)
Olpidium incognitum (193) ‘‘Parasitic in the egg of a rotifer’’
Olpidium longum (193) ‘‘Parasitic in the eggs of certain [aquatic] an-
imalcules’’
Olpidium macrosporum (Nowak.) J. In rotifer eggs
Schröt, Kryptogamen-Flora von
Schlesien 3(1): 182 (1885) (37: 155)
Olpidium nematodeae Skvortzov, Archiv In nematodes
fur Protistenkunde 57:204 (1927)
(37:147)
Olpidium paradoxum (194) From pond water
Olpidium poreferum (195) [see also Parasitic in rotifers
Olpidium allomycetos Karling, Ameri-
can Journal of Botany 35: 508, 1948
(37: 143)]
Rarely (experimentally) in rotifer eggs
Dick
Olpidium rotiferum Karling, Lloydia 9: In adult rotifers and rotifer eggs

6 (1946) (37:146)
Olpidium sparrowii (196) In eggs of Lecane spp. (from cultivated
soil)
Olpidium vermicola (197) Endoparasitic in nematode eggs (from rot-
ting wood)
Olpidium zootoctum (A. Braun) (37: In Anguillula sp.
155)
Phlyctochytrium nematodae Karling, Parasitic in adults, eggs, and cysts of nema-
Lloydia 9: 7 (1946) (37: 337) todes
Pseudosporopsis rotatoriorum (198) An anisokont biflagellate monad parasitic in
nematodes [probably a Haptoglossa]
Pythium caudatum (G. L. Barron) (36, In nematodes (from soil in a cattle pen)
55)
Legenidium caudasum G. L. Barron,
Antonie van Leeuwenhock 42: 134
(1976)
Sommerstorffa spinosa (199) Rotifers [thallus epiphytic on Cladophora
(Chlorophyceae)]
Synchaetophagus balticus (200) In Synchaeta monopus Plate
Rhizophydium gibbosum (Zopf) A. Parasitic on rotifer eggs (and in various nem-
Fisch., Rabenhorst’s Kryptogamen- atodes and algae according to other au-
Flora, 2 Aufl., Bd 1, Abt. 4: 102 thors)

(1892) (37: 237)
Rhizophydium vermicola (37: 277) Nematodes infected by Harposporium, Apha-
nomyces, or Lagenidium (⫽ Myzocyti-
opsis)
Rhizophydium zoophthorum (P. A. In cultures of rotifers (adults and eggs)
Dang.) A. Fisch., Rabenhorst’s Kryp-
togamen-Flora, 2 Aufl., Bd 1, Abt. 4:
94 (1892) (37: 285)
Note: listed in alphabetic order with nomenclatural citation (and reference number, page number appended for Sparrow, 1960) with the type host and habitats
25
where noted. Many of the species of Myzocytiopsidales and Haptoglossales can be transferred between nematodes and rotifers in laboratory culture.
26
Key to the Species of Salilagenidiaceae and Haliphthoraceae
1 Thallus more or less mycelial, branched, mean hyphal diam. ⬍24 µm, usually ⬍15 µm, septate or sparingly septate; non-
rhizoidal, culturable; eucarpic or holocarpic with time; sexual reproduction present or absent. 7
1′ Thallus more or less inflated, lobed, branches with mean diam. ⬎25 µm; septa rare or absent; rarely rhizoidal (in culture),
culturable; holocarpic with time (doubtfully eucarpic); sexual reproduction absent. 2
2(1′) Thallus irregularly and broadly tubular, rhizoids absent; sporangiogenesis not reported to have a centripetal contraction
phase; zoospores with lateral flagellar insertion; mean diameter of zoospore cysts ⬍6 µm (volume equivalent ⬍120
µm 3), or if greater then parasitic in mollusks [these species may be closer to Haliphthoros, but resemble A. dubia in
their habit and the long tapering exit tubes]. (Halodaphnea) 3
2′(1′) Thallus inflated, septa normally absent; sporangiogenesis intrasporangial, often with a late, marked centripetal contraction,
initials amoeboid at first on fine cytoplasmic strands; mean diameter of zoospore cysts ⬎7 µm (volume equivalent ⬎180
µm 3) [parasitic in eggs of crabs Pinnotheres pisum (L.) and other crustaceans (Crangon, Gonoplax, Leander, Macropo-
dia, Paguristes, Portunus, and Typton; sometimes regarded as saprobic); rhizoids cut off by septa occasionally present in
culture but not observed in natural substrata; thallus lobes stout, 27–50 µm, tips swollen, up to 100 µm diam., zoosporan-
gia 50–400 ⫻ 10–30 µm, with one or more exit tubes, distal part (up to 50 µm) hyaline; occasionally proliferous; zoo-
spores pyriform or slipper-shaped (with lateral flagellation?), diplanetic (but not dimorphic?), 10–12 µm long (4.0–6.0 ⫻
6.0–8.2 µm; (4.0)4.8(6.0) ⫻ 8.7(10.0) µm), first-formed cysts 7.0–8.0(9.0) µm (7.4 µm) diam., second-formed cysts
6.0–7.0 µm (6.8 µm) diam., germ tube 1.7 µm diam., gemmae present] [this genus may not belong in the Haliphthora-
ceae]. Atkinsiella dubia (D. Atkins) Vishniac (monotypic genus in this text)
3(2) Zoospore cysts approximately 5.0 µm diam. (volume equivalent ⬍75 µm 3); colonies more or less saccate with 1–3 zoospo-
rangial exit tube(s); optimum temperature for growth ⬎24°C [parasitic in various Crustacea]. 4
3′(2) Zoospore cysts 8.0 µm diam. (volume equivalent ⬎250 µm 3); colonies filamentous with 1 (rarely 2) exit tube(s) produced
from each zoosporangium; optimum temperature for growth 20°C (5–25°C) [parasitic in Haliotis sieboldii Reeve (aba-
lone); hyphae stout, irregular 16–41(140) µm diam., branched, nonseptate, becoming septate to delimit zoosporangia; zoo-
spores pyriform 4.0–8.0 ⫻ 7.0–12.0 µm, diplanetic, isokont; zoospore cysts germinating by means of a fine filament
62–295 µm long before broadening to form the thallus]. Halodaphnea awabi (N. Kitancharoen et al.) M. W. Dick
4(3) Zoosporangial exit tubes 1–3, always unbranched. 6
4′(3) Zoosporangial exit tubes normally single, sometimes branched. 5
Dick
5(4′) Colony pigmentated, gray to light brown; optimum temperature for growth 30–32°C; [parasitic in eggs of Scylla serrata
(Forsskål); hyphae (12)26(40) µm diam., zoosporangia 42–1150 ⫻ 5–15 µm; zoospores pyriform or slipper-shaped,
diplanetic but not dimorphic, (3.8)4.5(5.0) ⫻ (5.0)6.3(10.0) µm, cysts (4.5)5.0(7.5) µm diam.].
Halodaphnea hamanaensis (Bian & Egusa) M. W. Dick (type species)
5′(4′) Colony hyaline; optimum temperature for growth 25°C (15–30°C) [parasitic in Panulirus japonicus (von Siebold); holocar-
pic, hyphae stout, branched and septate, 10–22(64) µm diam., subthalli transformed into zoosporangia or gemmae; zoo-
spores pyriform or reniform 4.0–5.0 ⫻ 7.0–10.0 µm, diplanetic; zoospore cysts 5.0–7.0 µm diam., germinating by
means of a fine filament 14–253 µm long before broadening to form the thallus].
Halodaphnea panulirata (N. Kitancharoen & Hatai) M. W. Dick
6(4) Zoosporangia with 1-several broad discharge tubes, (6)8–9(10) ⫻ (40)200–300(510) µm containing several ranks of zoo-
spores [parasitic in Portunus pelagicus L.; holocarpic, hyphae stout, becoming septate with age, 10–38 µm diam., sub-
thalli transformed into zoosporangia or thick-walled gemmae, 22–190 µm diam.; zoosporogenesis intrasporangial; zoo-
spores pyriform or subglobose (4.0)4.7(6.5) ⫻ (5.0)6.3(8.0) µm, diplanetic; zoospore cysts (4.0)5.2(7.0) µm diam.,
germinating by means of a fine filament 5–190 µm long before broadening to form the thallus; optimum temperature for
growth 25°C (20–30°C)]. Halodaphnea okinawaensis (K. Nakam. & Hatai) M. W. Dick
6′(4) Zoosporangia with 1–2 infrequently-branched discharge tubes [discharge tubes straight, wavy or coiled, usually with a cone-
like base, 6–14 ⫻ 20–780 µm, parasitic in Brachionus plicatilis Müller (Rotifera); usually holocarpic (eucarpic with age
or in suboptimal growth temperatures), hyphae stout, saccate, becoming septate with age, 15–50 µm diam., subthalli
transformed into sporangia or developing into thick-walled gemmae, 40–200 µm diam.; zoosporogenesis intrasporangial;
zoospores pyriform (4.0)4.6(5.6) ⫻ (4.8)6.0(7.4) µm, monoplanetic, isokont; zoospore cysts (4.8)5.5(6.0) µm diam., ger-
minating by means of a fine filament 8–250 µm long before broadening to form the thallus; optimum temperature for
growth 25°C (20–30°C)]. Halodaphnea parasitica (K. Nakam. & Hatai) M. W. Dick
7(1) Thallus septate but not usually with endothallial contraction and new wall formation; zoospore size large (volume equiva-
lent ⬎275 µm3); sexual reproduction present or absent (Salilagenidium) 10
7′(1) Thallus elements rounding up to form new endothallial walls; zoospore size variable (volume equivalent ⬍250 µm3); sexual
reproduction absent. (Haliphthoros) 8
8(7′) Parasitic in crustaceans or mollusks; zoospore size medium-large (volume equivalent ⬎100 µm3); resting bodies not known.
9
27
Key to the Species of Salilagenidiaceae and Haliphthoraceae 28
8′(7′) Parasitic in larvae of mollusks (Venus); zoospore size small (volume equivalent ⬍75 µm 3); ‘‘resting bodies’’ 40 ⫻ 45 µm
up to 80 ⫻ 90 µm formed as lateral diverticula (oogonia?) [thallus initially filamentous, 10–15 ⫻ 82 µm, becoming both
inflated and with tapered extremities, resembling rhizoids; intrathallial walled bodies (31–33 ⫻ 46–56 µm); zoosporan-
gia intramatrical, 5 ⫻ 15–142 µm; zoospores 2.0 ⫻ 5.0 µm, anisokont, with a shorter straminipilous flagellum].
Haliphthoros zoophthorum (Vishniac) M. W. Dick
9(8) Parasitic in eggs of mollusks (Urosalpinx (U. cinerea (Say)), Haliotis), and crustaceans (Penaeus); sporangia with short
exit tubes (7.0)8.0(14.0) µm long; zoospores monoplanetic [holocarpic; hyphae (7.0)14.0–18.8(40.0) µm; zoospores pyri-
form, subspherical or elongate, (5.0)6.8–7.2(10.6) ⫻ (6.7)8.5(12.2) µm, monoplanetic, cysts (6.8)7.8–8.5(8.6–20.0) µm,
very slender germ tube 0.5(1.8–2.2) µm diam.]. Haliphthoros milfordensis Vishniac (type species)
9′(8) Parasitic in larvae of prawns (Penaeus monodon Fabricius); sporangia with long exit tubes 620 ⫻ (7.50)–(12.5) µm; zoo-
spores polyplanetic, dimorphic(?) [hyphae stout, branched, irregular, nonseptate (10.0)21.0(37.5) µm, intrathallial bodies
(190 ⫻ 100 µm) not disarticulating, remaining connected like beads; first-formed zoospores pyriform, subspherical or
elongate, with lateral flagellar insertion, 5.0–7.5 ⫻ 7.5–12.5 µm, second-formed zoospores slipper-shaped and slightly
shorter (10.0 µm), cysts 5.0–7.5(12.5) µm, very slender germ tube] Haliphthoros philippinensis. Hatai et al.
10(7) Sexual reproduction not known. 11
10′(7) Sexual reproduction known. 13
11(10) Zoospores of medium size (volume equivalent ⬍400 µm3). 12
11′(10) Zoospores of large size (volume equivalent ⬎500 µm3) [parasitic in eggs and larvae of crabs (Scylla serrata)].
Salilagenidium scyllae (Bian et al.) M. W. Dick
Salilagenidium thermophilum (K. Nakam. et al.) M. W. Dick
These two species are scarcely separable from the published descriptions:
Salilagenidium scyllae: hyphae thick, irregular, branched, 7.5–17.0(40.0) µm diam., sparingly septate, nonsegmented, thalloid ele-
ments becoming sporangia; zoosporangial discharge tubes short or long, 37–500 ⫻ 4–10 µm, apex dilating to form a deliquescent
vesicle; cytoplasm not filling vesicle prior to cleavage, vesicle not persistent, zoospores reniform, pyriform, ovoid or oblong,
(7.0)10.0(15.0) ⫻ (8.0)12.5(17.5) µm, released by deliquescence of vesicle or singly through a pore on the vesicle, monoplanetic;
cysts (7.5)10.0(15.0) µm diam., cyst wall 1.5 µm thick.
Salilagenidium thermophilum: hyphae thick, irregular, branched, 8.0–24.0(40.0) µm diam., nonseptate, becoming sporangia; zoospo-
rangial discharge tubes 34–440 ⫻ 6–14 µm, vesicular membrane not apparent; cytoplasic cleavage completed after discharge,
Dick
‘‘vesicle’’ 36–80 µm diam.; zoospores pyriform to subglobose, laterally biflagellate, 8.0–14.0 (mean 10.3) ⫻ 10.0–16.0 (mean
13.3) µm; monoplanetic; cysts 6.0–16.04 µm diam.; thermotolerant (15)30–40(45)°C.
12(11) Parasitic in muscles and swimmerets of crustaceans (shrimps) (Pandalus borealis Krøyer), holocarpic, culturable; hyphae
wide, irregular, branched, 7.0–10.0 µm diam., partial cleavage within the sporangium, discharge tube 86–240 ⫻ 7–10
µm, vesicle formed, zoosporogenic protoplasm not filling vesicle, zoospores released by rupture of vesicle, vesicle persis-
tent, 9.6 ⫻ 12.9 µm globose, reniform, pyriform or elongate, cysts 5.5–12.0 µm.
Salilagenidium myophilum (Hatai & Lawhav.) M. W. Dick
12′(11) Parasitic in stomach of crayfish (Penilia); mycelium nonseptate, of uniform diameter, 4.2–5.2(7.0) µm, with homogeneous
protoplasm; zoosporangia spherical or ellipsoidal, on short side branches, smooth walled; zoosporogenesis intrasporan-
gial, released through a pore over a period of 2 min, swimming away 30 min later; zoospores 30–50 in a zoosporan-
gium, of irregular shape, with an anteriorly inserted flagellum [known only from original locality].
‘Hyphochytrium peniliae N. J. Artemczuk & L. M. Zėlėzinskaja’ nom. illeg.
13(10′) Zoosporogenesis partly extrasporangial in a vesicle; antheridia absent; culturable. 14
13′(10′) Zoosporogenesis intrasporangial; zoospore initials in a single row distally; antheridia present, hypogynous [parasitic in eggs
of crabs (Mytilus edulis L.) and possibly lamellibranchs Barnea and Cardium; hyphae 7.5–20.0(40.0) µm diam.; zoospo-
rangia undifferentiated from hyphae, occasionally proliferous; zoospores pyriform, 8.0–14.0 µm long, cysts 6.0–11.0 µm
diam., diplanetic; oogonia rare, oospores single, nearly plerotic, 17.5–30.0(37.0) µm diam., oospore wall two layered, 7.5
µm thick]. Salilagenidium marinum (D. Atkins) M. W. Dick
14(13) Parasitic in eggs and larvae of crabs [Callinectes (C. sapidus Rathbun), Limulus], and barnacles (Chelonibia); hyphae spar-
ingly septate, extramatrical hyphae (5)8–14(50) µm diam., 1030 µm long, aseptate; zoosporangium with vesicle persis-
tent after discharge; tip of exit tube gelatinizing and contents flowing into the thick gelatinous envelope, never filling it,
vesicle persistent, zoospores 9.3 ⫻ 12.5 µm; cysts 8.0–10.0(11.3) µm diam., monoplanetic, germ tube 2.5 µm diam., oo-
gonia intercalary, oospores (18)25(36) µm diam., [wall 3 µm thick, subeccentric oil reserve].
Salilagenidium callinectes (Couch) M. W. Dick (type species)
14′(13) Parasitic in barnacles [Chthamalus (C. fragilis Darwin)]; hyphae stout, irregular, branched, highly vacuolate 10–18(39) µm
diam., becoming segmented, segments behaving as zoosporangia, zoosporogenesis within a vesicle formed from the swell-
ing of the exit tube apex, cytoplasm entering the vesicle only after the latter is completely developed, cytoplasm not fill-
ing the vesicle, vesicle disappearing immediately after discharge, zoospores reniform, 6.8–8.5 ⫻ 8.5–10.2 µm, oogonia
intercalary or terminal, 19–47 µm diam., oospores 1(2), aplerotic (16)21–25(27) µm diam.
Salilagenidium chthamalophilum (T. W. Johnson) M. W. Dick
Note: Supplementary information in brackets does not form part of the key dichotomy. For taxonomy see Refs. 14, 36, 37.
29
30 Dick
necrosis) or EUS (epizootic ulcerative syndrome), caused by Saprolegnia parasitica,

occasionally reaches epidemic levels, and the disease continues to be under investiga-
tion because it has commercial importance in relation to fish farming (25,26). Another
disease of vertebrates is Pythiosis insidiosi, caused by Pythium insidiosum (27).
Other parasites of invertebrates include the endoparasites of the Aschel-
minthes (Table 3), such as Myzocytiopsis, Gonimochaete, Chlamydomyzium (My-
zocytiopsidales), Nematophthora (Leptomitales: Leptolegniellaceae), Catenaria
(Chytridiomycetes: Blastocladiales) Olpidium, Endochytrium, Rhizophydium and
Phlyctochytrium (Chytridiomycetes: Chytridiales), and Haptoglossa (Plasmodio-
phoromycetes), but with the exception of Nematophthora (28), their use in bio-
logical control has yet to be assessed. Although a few species have an extensive
bibliography, the information for most of the species is restricted to the type
descriptions, which are therefore cited in Table 3.
The fungal parasites of noncellular (protozoan) Protoctista are even less
well known, with reports again limited to (often early) original descriptions.
Parasite/host relationships are listed in Table 4.
The gut commensals placed in the Neocallimastigales have been reviewed
by Li and Heath (29), Trinci et al. (30), and Ho and Barr (5).
III. METHODS OF DETECTION

A. Gross Features
Parasites on fish, crustacea, and insects are immediately obvious and ultimately
destructive. Extramatrical hyphae are usually abundant on the surface of diseased
fish and insects and are visible with the unaided eye. Willoughby (31) has pro-
vided a summation of the different peronosporomycete infections of fish, but
caution is still necessary in determining the distinction between aggressive obli-
gate parasitism and casual wound parasitism of moribund fish. The fungal thalli
of the Salilagenidiales may be largely intramatrical, but these can readily be seen
in whole mount preparations of the host limbs and gills. Microscopically obvious
intramatrical disease is also apparent in aschelminths. In both Aschelminthes and
marine Crustacea the diseased animal shows little sign of distress initially, even
though the thallus development may be extensive. This is particularly true of
rotifers, which continue to move and feed even though the major part of the body
cavity may be occupied by the fungus. The gross features of Aphanomycosis of
crayfish (Krebspest disease) are somewhat less obvious, but the disease causes
rapid population extermination (11,15).
B. Disease Development
In fish, the disease most commonly encountered is an initially superficial ulcer-
ation, which rapidly becomes more deep-seated, causing histologically recogniz-
Table 4 Peronosporomycetes and Chytridiomycetes associated with Protozoan Protoctista
Aphanomyces acinetophagus (190, 37: 843) In Acineta flava Claparède & Lachmann (Suctoria)
Aphanomycopsis cryptica (Canter, in Ref. 201) In Ceratium hirundinella (O. F. Müll). Bergh. (Dinomastigota,
Gonyaulacales)
Aphanomycopsis peridiniella (Boltovskoy and Aramb, in Ref. In cysts of Peridinium willei Huitf.-Kass (Dinomastigota)
202)
Ciliomyces spectabilis (203) In cysts of Kahiella simplex (Horvath, Ciliat) from air-dried
meadow soil, after remoistening)
From fallen inflorescences of Cecropia sp. (Moraceae)
Endemosarca anomala (204) In Colpoda spp. (Ciliata)
From old fruits of Annona muricata L. (Annonaceae)
Endemosorca hypsalysis (205) In Colpoda spp. (Ciliata)
From fallen flowers of Althaea sp. (Malvaceae)
Endemosarca ubatubensis (205) In Colpoda spp. (Ciliata)
Eurychasmopsis multisecunda (Canter, in Ref. 42) In Podophrya sp. (Suctoria) parasitic in
Myzocytiopsis parthenospora (Karling, in Ref. 171) In eggs of Chaetonotus iarus O Müller (Gasterotricha; see also
Table 3)
Nucleophaga amoebae (206) In the hypertrophied nucleus of Thecamoeba verrucosa (Gläser;
as its synonym, Amoeba verrucosa Gläser)

Nucleophaga hypertrophica (207) Type material not verified
Nucleophaga peranemae (208) In Peranema trichophorum (Ehrenb.) Stein (Sarcomastigophora,
Euglenida)
Olpidiomorpha pseudosporae (209, 37: 123) Within the zoocyst of Pseudospora leptoderma Scherff.
Olpidium arcellae (210) Saprobic (1) on Arcella sp.
Olpidium difflugiae (211, 37: 154) In Diffugia sp.
Olpidium leptophrydis (211, 37: 154) In zoocysts of Leptophrys vorax (Cienk.)
Olpidium pseudosporeanum (211, 37: 146) In zoocysts of Pseudosporopsis bacillariacearum Scherff. and
Pseudospora parasitica Cienk.
31
32
Table 4 Continued
Olpidium vampyrellae (211, 37:146) In zoocysts of Vampyrella spp.

Pseudosphaerita drylii (212) In Phacus acuminatus Stokes (Sarcomastigophora, Euglenida)
Pseudosphaerita euglenae (206, 37: 963) In Euglena sp. (Sarcomastigophora, Euglenida)
Pseudosphaerita radiata (P. A. Dang., in Ref. 213, 37: 963) In Cryptomonas ovata Ehrenb. (Cryptophyceae, Cryptomona)
Rhizoblepharis amoebae (214) In Amoeba binucleata Grüb. (Rhizopoda)
Sphaerita amoebae (215) In Thecamoeba sphaeromucleolus [Greeff; as ‘‘Amoeba
sphaeronucleolus’’ (Rhizopoda)]
Sphaerita dangeardü (37: 126) Not determinable
Sphaerita endogena ( pro parte) (37: 126) In Euglena sanguinea Ehrenb. (Sarcomastigophora, Euglenida)
Sphaerita entamoebae (216) In Entamoeba citelli E. R. Becker (Rhizopoda; from the ground
squirrel Citellus)
Sphaerita minor (217) In Trichomonas sp. (Parabasilia)
Sphaerita normeti (218) In Entamoeba histolytica Schaedinn [as ‘‘Entamoeba coli’’
(Rhizopoda)]
Sphaerita nucleophaga (215) In Thecamoeba sphaeronucleolus [Greeff; as ‘‘Amoeba
sphaeronucleolus.’’ also in ‘‘Amoeba terricola’’ (Rhizopoda)]
Sphaerita phaci (219) In Phacus pleuronectes (O. S. Müller) Dujardin
(Sarcomastigophora, Euglenida)
Sphaerita plasmophaga (215) In Thecamoeba sphaeronucleolus [Greeff; as ‘‘Amoeba
sphaeronucleolus’’ (Rhizopoda)]
Sphaerita trachelomonadis Skvortzov (37: 127) In Trachelomonasteres Maskell var. glabra Skvortzov (1927) and
T. swirenkoi Skvortzov (Sarcomastigophora, Euglenida)
Spirospora paradoxa (209) In cysts of Vampyrella sp. (possibly Vampyrella spirogyrae
Cienk.)
Note: listed in alphabetic order with nomenclatural citation (expanded in References) with the type host and habitats where noted. (Page numbers in brackets
refer to Sparrow, 1960.) Note that the spelling of authorities for taxa may not correspond with that in the References list.
Dick
able granulomas composed of host macrophage cells and hyphae (31). It is

thought that initial entry is through weakened or less-protected dermis on vulnera-
ble extremities, such as fins. The disease is progressive through muscle tissue,
resulting in massive loss or necrosis, eventually causing death after several days
and severe mutilation. A less common disease syndrome is caused by initial infec-
tion in the gill/pharynx/gut tissues. Asphyxiation and starvation result in more
rapid death with reduced superficial symptoms.
Melanization of cuticle tissue in the region of hyphal penetration is a char-
acteristic defense symptom to Aphanomycosis of crayfish (Krebspest disease)
(32–34). Melanization will effectively immobilize but not kill the infective hy-
phae; the extent to which this melanization is effective in constraining the fungus
will determine the success or failure of the infection. Söderhäll and Cerenius (35)
have related the immune reaction of crayfish to the elicitation of defense pro-
cesses by β-1, 3-glucans, which are, of course, characteristic wall carbohydrates
of peronosporomycetes.
IV. CHARACTERIZATION
A. Methods for Taxonomic Decision Making Including MB
The morphology and the morphogenesis of the Peronosporomycetes are of taxo-
nomic and phylogenetic importance. The most obvious characters relate to thallus
form, but for identification to class and lower hierarchical levels zoospore mor-
phology and morphogenesis are normally essential. (Many of the pathogens do
not—or do not readily—reproduce sexually.) Transmission electron microscopy
(TEM) is diagnostic at class level, particularly with respect to mitochondrial pro-
files and vesicular inclusions. Several 18S rDNA sequences are now available
(21,36) for representatives of the Saprolegniales and Pythiales, but identification
probes for animal parasites have yet to be published. Diagnostic features are
summarized below under the subheads thallus morphology and protoplasmic fea-
tures, zoosporangia and zoosporogenesis, zoospore and zoospore cyst morphol-
ogy, sexual reproduction, biochemistry, and molecular biology.
1. Thallus Morphology and Protoplasmic Features

The thalli of the Peronosporomycetes may be filamentous, composed of hyphae
forming a mycelium; or coralloid (eucarpic or holocarpic), allantoid, or ellipsoid
(holocarpic) (8,36). Chytridiales (37) are monocentric with or without an assimi-
lative system composed of branched rhizoids. Blastocladiales may be pseudo-
filamentous or composed of catenulate chains of cells. All hyphae show tip
growth. Only with holocarpic thalli (both septate and nonseptate) may there be
superficial resemblance between the Peronosporomycetes and the Chytridiomy-
34 Dick
cetes. Obligate parasites may be entirely confined within a single host protoplast
(endobiotic parasites of protozoa) or intercellular within tissues or in the haemo-
coel of arthropods and aschelminths.
In the Peronosporomycetes the vegetative thallus is bounded by a wall
membrane at maturity, but may be naked initially in some endobiotic parasites.
Septa are normally only present to delimit reproductive structures or act as retrac-
tion septa (as in old mycelia of Pythium). In the Saprolegniaceae there is fre-
quently excessive synthesis of wall material at the septum, resulting in the devel-
opment of an irregular peg or callus on one or both sides of the septum. Hyphae
may be of relatively narrow diameter (10–20 µm in Aphanomyces) or broad diam-
eter (20–40 µm in Saprolegnia). Generalized intussusception of wall material
occurs in older hyphae of Saprolegnia, and these hyphae may have diameters up
to 120 µm.
The appearance of the protoplasm, using light microscopy, can often pro-
vide distinctive diagnostic features to an experienced worker, but it is difficult—
and may be misleading—to describe these differences for a novice. Note should
be made of the presence and kind of cytoplasmic streaming and the granulation
of the cytoplasm, particularly the coarseness of the granulation and the ‘‘glassy’’
character of the groundplasm. (Contrast the gravelly appearance of the proto-
plasm in hyphae of Saprolegnia with the sparce large inclusions suspended in a
translucent matrix in Halodaphnea.)
The thallus is initiated from a uninucleate zoospore cyst or an aplanospore,
from a multinucleate asexual spore or propagule, or from a uninucleate sexually
produced oospore. In most of the endoparasitic fungi that have been studied by
TEM, an extremely fine penetration tube of approximately 0.1–0.2 µm in diame-
ter enters the host, and subsequent tip expansion enables the formation of the
first unit of the thallus.
At the ultrastructural level mitochondria are conspicuous in TEMs. Mito-
chondrial cristae of Peronosporomycetes appear either as longitudinal cylindrical
profiles with unconstricted connections to the inner mitochondrial membrane or
as transverse circular sections, while in the Chytridiomycetes (with the exception
of the obligately anaerobic Neocallimastigales) the cristae are platelike with vari-
ous oblique—but never circular—profiles. Dictyosomes (homologues of the
Golgi bodies of animals) are also well developed, but with relatively few cisternae
in each stack. Of the remaining vesicular systems, the two most abundant are
those of the lipid vesicles, and in the Peronosporomycetes, the Dense Body Vesi-
cles (DBVs), which constitute a ‘‘family’’ of vesicles of different appearence or
characteristic size. Dense body vesicles are implicated in diverse functions in the
life history of the Peronosporomycetes, and while they are often prominent in
reproductive morphogenesis, they can be found at all developmental stages.
Dense body vesicles are characterized by the possession of an electron-opaque
core or inclusion surrounded by a more electron-lucent zone, the boundaries be-
tween which may be more or less blurred by myelin-like configurations (1).

Dense body vesicles (DBVs) can vary in the proportions of core to matrix; at
different phases of the life history and organ development they may show differ-
ent and developmentally intergrading ultrastructure. The prominence and pre-
sumed importance of DBVs may be related to the phosphate/polyphosphate stor-
age differences between Eumycota (including the Chytridiomycetes) and the
Peronosporomycetes (38).
2. Zoosporangia and Zoosporogenesis

Asexual reproductive units in eucarpic fungi may develop in terminal, lateral,
or intercalary positions. The adaptive diversity of asexual reproduction in the
Peronosporomycetes is considerable with respect to the shape and dimensions of
the zoosporangium, the determinate or indeterminate zoosporangial renewal, the
presence of a discharge vesicle, the site and mechanism of protoplasmic cleavage,
and zoospore discharge. Molecular biology does not support a phylogenetic, as
opposed to a taxonomic, significance for zoosporangial morphogenesis. (See
below.)
For most species, a more or less differentiated zoosporangium is delimited
from the vegetative system by a septum, and the vegetative system is eucarpic.
Alternatively, the entire thallus may assume the role of a zoosporangium or series
of zoosporangia (holocarpic development, irrespective of whether the sporangia
mature simultaneously or sequentially), frequently with little morphological mod-
ification other than the formation of an exit tube and dehiscence papilla. Cleavage
of zoospore initials occurs either within the zoosporangium (intrasporangial zoo-
sporogenesis) or after discharge of the sporangial protoplasm (extrasporangial
zoosporogenesis). Intrasporangial zoospores may be released by enzymatic decay
of a papilla or a circumcissile ring, leaving an operculum in both phylogenetic
lines.
In the Saprolegniaceae (Saprolegnia) zoosporogenesis is intrasporangial,
and cleavage furrows developed from dictyosome-derived cisternae become con-
fluent first with the prominent central tonoplast vacuole and eventually breach
the plasmamembrane. There is a consequent loss of volume (approximately 10%)
of the zoosporangium as turgor is lost. Zoospore discharge is achieved by imbibi-
tion of water through the zoosporangial cell wall in response to the release of
osmotically active β-1,3-glucans within the confines of the zoosporangium (39).
In the Leptolegniaceae (Leptolegnia, Aphanomyces) the zoosporangial contents
are released before the zoospores are fully formed and discrete (40,41). In the
Pythiales (Pythium) the extrusion of uncleaved multinucleate protoplasm takes
place into an extra-plasma-membranic, membranous, glucan-polymer vesicle (the
homohylic vesicle) that is formed simultaneously with discharge and that is con-
fluent with the sporangium wall. In the Myzocytiopsidales a single tonoplast is
36 Dick
not present, but a few tonoplastlike vacuoles become apparent early in the prespo-
rangial stage and persist while the cleavage cisternae reach an advanced stage of
orientation (9). In Blastulidium and Eurychasmopsis planonts become parietally
rearranged prior to intrasporangial encystment (42). In the Salilagenidiales most
of the cytoplasm is peripheral at the midcleavage stage prior to discharge, and
large vacuoles probably merge with the cleavage cisternae so that the develop-
ment shares similarities with but is not identical to those of the Saprolegniaceae,
Leptolegniaceae, or Myzocytiopsidaceae. Coincident with this zoosporogenesis
is the presence or absence of a gelatinous matrix surrounding the planonts as
they are discharged (10,43). A ‘‘vesicle’’ develops from the exit tube apex as a
gelatinous matrix prior to the extrusion of the protoplasm. [See also some Myzo-
cytiopsidales (44).] In the Salilagenidiales, in contrast to Pythium, the protoplasm
does not fill this clearly defined vesicle. At maturity the gelatinous matrix (the
vesicle) becomes partially inverted and collapses down the outside of the exit
tube during zoospore maturation, often remaining as a sleeve after discharge. The
boundary of a gelatinous matrix can often be distinct, and in light microscopy it
may be difficult to distinguish between such a boundary and the presence of a
membranous vesicle. A ‘‘membrane’’ (a precipitative vesicle) may thus be
formed as a precipitation reaction between such a colloidal matrix and the envi-
ronment.
If hyphal regrowth and zoosporangial renewal takes place, it may be
through the zoosporangial septum (internal renewal ), with the successive
zoosporangial septa formed above or below the primary zoosporangial septum.
Zoosporangia may also be produced in sequence on the same determinate axis
(basipetal development) or by a lateral branch (cymose renewal ). Conversely,
sporangial development may be arrested to produce resting bodies (hyphal bodies
in Pythium or gemmae in Saprolegnia), which may either germinate to produce
zoospores after the manner of the species or germinate directly, producing a
hypha.
3. Zoospore and Zoospore Cyst Morphology

The fungal zoospore is a normally uninucleate, motile naked cell or planont.
Substantial differences in zoospore volume and concomitant microtubular cyto-
skeletal array and complexity may influence zoospore shape and ultrastructure.
In the great majority of the Peronosporomycetes there is only one zoospore form:
This is the principal zoospore form (8,45), which is reniform or bean-shaped
with flagella laterally inserted in a groove. The flagella are inserted on a protuber-
ance, the kinetosome boss, which may bridge the flagellar groove. The angle
of divergence between the two flagella (and their subtending kinetosomes) is
approximately 130–150° (46).
The flagella are of different lengths (anisokont) and of different ultrastruc-
tural morphology (heterokont). The anteriorly directed flagellum (the straminipi-

lous flagellum) is ornamented with stiff tubular tripartite [flagellar] hairs
(TTHs), which are usually in two rows (see Ref. 14). The posteriorly directed
flagellum is unornamented or with a fibrillar surface coat. In the Peronosporomy-
cetes the flagella may either be subequal or markedly anisokont, and the strami-
nipilous flagellum are shorter. The two rows of stiff TTHs reverse the thrust of
quasi-sinusoidal beat so that the straminipilous flagellum pulls the zoospore
through the water. (For further references see Ref. 7.)
Sometimes a sequence of two or more zoosporic phases may be inter-
spersed by encysted phases. This phenomenon is known as polyplanetism. The
principal-form zoospore exhibits polyplanetism in both the Saprolegniomycetidae
(Saprolegnia) and Peronosporomycetidae (Pythium). In Saprolegnia the more or
less ovoid zoospore formed within the zoosporangium may develop flagella that
are subapically inserted (the auxiliary zoospore) (45), but the flagellar root system
for the straminipilous flagellum of the auxiliary zoospore is deficient. The auxil-
iary zoospore is not known to be polyplanetic. When the polyplanetism involves
the production of an auxiliary-form zoospore followed by one or more planetic
phases of the principal-form zoospore, the zoospore production is termed dimor-
phic. The cysts formed from each kind of zoospore are also often morphologically
distinct. Zoospore cysts may have a variety of ornamentation on their surfaces.
The principal-form zoospores of some species of Saprolegnia have distinctive
split-ended hairs (the boat-hook hairs), which are derived from preformed struc-
tures in cytoplasmic vesicles of the zoospore (47).
The zoospore cysts of auxiliary-form zoospores are formed after retraction
of the flagella so that tufts of TTHs are left on the outside of the cyst at the point
of flagellar insertion and retraction. In contrast, the flagella of principal-form
zoospores are shed and leave no plasma-membranic ornamentation. However,
both kinds of cysts for different species of peronosporomycetes show considerable
diversity with respect to protoplasmically originating cyst wall ornaments (7,36).
The most remarkable of these ornaments are the boat-hook hairs, which have
split-ended tips. The length and arrangement of the boat-hook hairs is thought
to be diagnostic (very long and in tufts for Saprolegnia parasitica) (7,48,49).
The flagellar base is composed of the kinetosome and the attached roots
of microtubules. Between the kinetosome and the axoneme there is a transitional
zone (1,50,51). The root system is composed of six units; each of the kinetosome
bases has two roots. Independent variation in the ultrastructure of each of these
four roots occurs between taxa, and sometimes within a species. Variation in the
detailed ultrastructure of the transitional zone also occurs, but in each case there
is a transitional plate, attached to the kinetosome at the end distal to the nucleus.
The transitional plate transects the axoneme core in a plane above that of the
cell plasma membrane and extends to the flagellar plasma membrane (50,51).
Within the axoneme core (i.e., inside the cylinder of the nine doublets) and distal
38 Dick
to the transitional plate there is an electron-opaque structure appearing in longitu-

dinal sections like a concertina or double helix of repeating units.
The flagellar root systems of the Chytridiomycetes are quite distinct
(51,52), often with prominent striated fiber roots and a proximal mitochondrion.
The transitional plate is on a plane corresponding to the cell plasma membrane.
Intergeneric diversity of the flagellar root systems also occurs in this class.
Many protists possess extrusomes (peripheral vesicles) that extrude or evert
their contents to the exterior but that are not involved with wall synthesis. The
Peronosporomycetes have a highly developed complex of extrusomes associated
with encystment and germination (53,54). Prominent peripheral vesicles have
been figured for zoospores of Eurychasmopsis (42) and Myzocytiopsis (9,55).
The shedding or retraction of flagella at encystment is an important and
possibly fundamental diagnostic criterion, but is rarely observed or recorded (56).
There may be total detachment and loss (principal-form zoospores) or a range
of retraction mechanisms. The method of contact with the host is a diagnostic
characteristic. Contact may be established by the flagellar tip, but in most peron-
osporomycetes the zoospore comes to rest with the ventral groove adjacent to
the substratum or host (42), followed by encystment.
4. Sexual Reproduction
Peronosporomycetes have oogamous sexual reproduction in which the production
of an egg (oosphere or female gamete) in an oogonium (receptor gametangium)
is generally—but not necessarily—accompanied by an antheridium (donor gam-
etangium). Neither flagellate gametes nor flagellate zygotes are formed. Gametan-
gia may be developed terminally, subterminally, in an intercalary position on
main or branch hyphae, as terminal or lateral appendages to a nonmycelial thallus,
or from the entire thallus. When an antheridium is present and fertilization occurs,
there is the injection of a small part of the antheridial protoplasm into the oogo-
nium through a fertilization tube. In the Saprolegniaceae the fertilization tube
may be branched, although there is no evidence that the number of branches ever
equates with the number of apparently mature oospores; it is usually much less.
In the holocarpic, nonseptate and therefore heterothallic Eurychasmopsis a fertil-
ization hypha develops when each cell of a chain of endogenous cells within the
donor gametangium ‘‘germinates’’ to cross the space between noncontiguous
gametangia. However, the majority of species of most of the genera are homothal-
lic and heterothallism may be secondarily derived. In myceliar species having
antheridial production, the subtending antheridial branch grows toward the oogo-
nium under hormonal attraction (57–60) and the antheridium is differentiated
after contact with the oogonium. The oogonial initial develops as a swelling with-
out septation or by a transformation of a gametangial segment. The first trigger
for this morphogenetic change must be endogenous, either from a nutritional or

biochemical stimulus.
Gametangia of the Peronosporomycetes are meio-gametangia in which
meiosis occurs. Synchronous meioses occur in the coenocytic gametangia. In
paired gametangia the meioses are also either simultaneous, or nearly so, between
the two protoplasts. This unique feature makes it possible for karyogamy to take
place between two haploid nuclei from adjacent meioses in the same gametan-
gium (automictic sexual reproduction). Sexual reproduction can thus occur with-
out a separate male gametangium or antheridium (3, Fig. 2). Because of this
phenomenon, the absence of a male gametangium does not necessarily indicate
parthenogenetic (i.e., no meiosis, no karyogamy) development so that automictic
sexual reproduction and parthenogenesis cannot be distinguished without cyto-
logical evidence (61). In Nematophthora the nonseptate ellipsoid thallus appar-
ently functions as an automictic gametangium, eventually containing several oo-
spores. In the Myzocytiopsidales and a few Pythiales the thallus becomes septate,
and adjacent segments assume the function of gametangia. In such cases the
thallus segment is the site of meiosis (thalloid meiosis). Gametangial copulation
(Myzocytiopsidales) occurs when the two gametic protoplasts condense to a com-
mon pore in the contiguous walls of the gametangia; the condensation is depen-
dent on vesicle expansion distal to the point of union in both gametangia (3).
The fertilization tube, which forces the oosphere away from the point of union,
and the conjugation pore, toward which both gametangial protoplasts converge,
appear to be quite different morphogenetic processes.
After meiosis the contents of the receptor gametangium (oogonium) be-
come separated as one or several uninucleate and initially unwalled gametes;
however, there is no differentiation of the donor gametangial contents into dis-
crete uninucleate gametes (but note the ‘‘endocellular’’ antheridia of Eurychas-
mopsis). The sexual process (meiosis and karyogamy) and the morphogenesis of
the two kinds of gametangia should therefore be considered as separate criteria.
Many species have oogonia with a smooth, more or less spherical outline,
but many others are ornamented. The pattern of development of wall ornamenta-
tion depends on three criteria: initial expansion, secondary primordial initiation,
and wall deposition. The diversity of oogonial form depends on the sequential
or simultaneous expression of these criteria. The deposition of an internal layer
or layers results in the thick oogonial walls, particularly characteristic of the
Saprolegniales, but the deposition of this inner layer is often excluded from cer-
tain regions to form the simple pits (Saprolegnia parasitica).
When the antheridium is of regular occurrence, the shape, origin, and posi-
tion of the antheridium can be useful taxonomic criteria. The mode of application
of the antheridium to the oogonium is distinctive for species and sometimes
groups of species, but the continuous interspecific cline of variation between fully
40 Dick
apical and completely lateral attachments makes reference to accurate illustra-

tions essential. Antheridial origin can either be characteristic for a species, or
highly variable within a species. Antheridia may arise from just below the oogo-
nium (closely monoclinous), from the hypha subtending the oogonial branch (mo-
noclinous), or from a different hyphal system (diclinous).
The morphogenesis of the oosphere is of major phylogenetic significance
in the Peronosporomycetes in two ways. First, the protoplasmic organization sub-
sequent to meiosis follows one of two alternatives: centripetal oosporogenesis
(vacuolar or tonoplast involvement) or centrifugal oosporogenesis (protoplasmic
rearrangement frequently with residual periplasm). Cleavage in the polyoospor-
ous taxa of the Saprolegniales is essentially centrifugal and similar to cleavage
in zoosporangia, with a tonoplast vacuole fusing with cleavage cisternae to form
peripheral mounds of presumptive oosphere protoplasts. When the oogonial
plasma membrane is finally breached, the oospheres tumble to the center of the
oogonium. The extent to which the oogonial cavity is filled by the oosphere is
variable and can be taxonomically diagnostic. In the Pythiales the oogonioplasm
does not contain a tonoplast vacuole, and the peripheral oogonioplasm gradually
loses its organelles to define a centripetal oosphere. Subsequently, the oospore
(zygote) wall forms at this boundary, so that the spore is more or less aplerotic
within the oogonium. In Pythium the concept of an aplerotic index has been
proposed to aid taxonomic assessment of species differences (62,63).
Second, deposition of the oospore wall and distinctive cytoplasmic reorga-
nization occurs during the development of the oospore. The oospore wall itself
is complex; possibly up to seven or more layers can be distinguished, but not all
organisms have all layers. Functionally, the most important layer is the deposit
of resorbable glucan polymers outside the plasma membrane forming the endo-
spore, analagous to the albumin of a bird’s egg. The endospore usually shows
concentric layers (64). Within the oospore, the protoplasmic contents become
reorganized. Two complementary processes of vesicular coalescence proceed si-
multaneously. The DBVs gradually coalesce to form a large single membrane-
bound structure, the ooplast, which can be seen clearly in the oospores of almost
all Peronosporomycetes. At the same time there may be a variable degree of
coalescence of lipid globules. Both contribute to the characteristic bubbly appear-
ance of the partially mature oospore. One or more transparent ellipsoid zones in
the mature oospore mark the position(s) of nuclei (nuclear spots). At the most
extreme state of coalescence, seen in some species of Achlya, the mature oospore
contains a single ooplast and a single lipid globule. The patterns of lipid coales-
cence (centric, subcentric, subeccentric, and eccentric states) have taxonomic
value and possibly significance in both ecology and phylogeny.
Under both light and electron microscopy the ooplast has different charac-
teristics within the class. In most of the Saprolegniales the ooplast is fluid, with
Brownian movement of granules, but in the Pythiales the ooplast appears to be
homogeneous under light microscopy. Using TEM, the ooplast of the Pythiales
is electron-opaque with a dispersed electron-lucent phase. The phases are re-
versed in the Saprolegniales. In the Salilagenidiales the ooplast descriptions sug-
gest closer similarities to the Saprolegniales.
Parity between estimates of chromosome number as obtained from light
microscopical methods and pulsed gel electrophoresis might be difficult to estab-
lish because of the possibilities of autopolyploidy, polysomy, and chromosome
polymorphy (65).
Many Chytridiomycetes have no known sexual system (37). In the Blasto-
cladiales, however, the resistant sporangium functions as a meiosporangium, pro-
ducing haploid planonts (gametes). The haploid and diploid phases of the nuclear
cycle may occur in different hosts. (See Coelomomyces, below.)
5. Biochemistry
Cantino (66) was the first to incorporate a range of biochemical criteria into the
systematics of the flagellate fungi, emphasizing that the genetic loss of a biochem-
ical pathway or attribute was unlikely to be restored. Such pathway loss is still
considered to be of phylogenetic importance (7,8). Three kinds of organic synthe-
sis have diagnostic value.
The amount of fibrillar wall material is much less in the fungi, and in the
Peronosporomycetes cellulose (β-1,4-glucan) tends to be masked by the much
larger amounts of β-1,3- and β-1,6-glucans. The fibrillar component of the walls
of Chytridiomycetes is chitinous, but glucosamine also occurs in walls of the
Peronosporomycetes, and the presence of polymerized chitin has been confirmed
for Saprolegniaceae (67).
There are two fundamental lysine synthesis pathways (68). The Perono-
sporomycetes are characterized by possession of the DAP (α,⑀-diaminopimelic
acid) pathway, while the Chytridiomycetes possess the AAA (α-aminoadipic
acid) pathway. The evolution of the DAP pathway is thought to antedate that of
the AAA pathway (the occurrence of which correlates with chitinous cell walls)
because the DAP pathway interferes with that for chitin biosynthesis (69). Al-
though the AAA lysine synthesis is correlated with the presence of mitochondria
with flat, platelike cristae (and chitin synthesis), DAP lysine synthesis may be
associated with a range of mitochondrial ultrastructure.
Detailed requirements for exogenous sterols differ between genera, as does
the ability to make and utilize sterols with certain substituents. Similarities be-
tween Lagenidium and Phytophthora can be contrasted with the similarities be-
tween Salilagenidium, Achlya, and Plerogone (70). Sterol metabolism is consid-
ered to be an important factor in the survival of Lagenidium giganteum. Polyene
antibiotics act on Eumycota but not the Peronosporomycetes; since these antibiot-
ics are thought to function by acting on membrane-bound sterols, the selectivity
42 Dick
of the antibiotics suggests that there is a difference between these two groups of
heterotrophs with respect to their membrane-bound sterols (71–73).
6. Molecular Biology
Molecular biological investigations of animal parasites have yet to provide a
sufficient body of comparative data. However, comparisons of the data for the
saprobic and plant pathogenic taxa indicate that there will be considerable scope
for developing clear protocols for identification.
The total genomic contents vary widely: the length of the nuclear genome
has been shown to vary between approximately 60 and 250 Mb in Phytophthora
(74); the length of the mitochondrial genome has been shown to vary between
36.4 and 73.0 kb with the presence of an inverted repeat, ranging in length from
approximately 10 to 30 kb (75,76). Data for 18S rDNA (21) and mitochondrial
phylogeny (77) have confirmed the dichotomy of the subclasses Peronosporomy-
cetidae and Saprolegniomycetidae, but also indicate polyphyly at the genus level.
Existing genera have been defined solely by zoosporangial morphology and mor-
phogenesis. Restriction mapping has revealed variability in the intergenic regions
of the ribosomal DNA; both the position (within the NTS of the rDNA repeat
unit) and the occurrence of inverted copies of 5S rDNA genes show variation
within the subclass Peronosporomycetidae and within the subclass Saprolegnio-
mycetidae.
Molecular biological analyses are accumulating so rapidly that this sum-
mary is inevitably ‘‘dated.’’ Nevertheless, it is noteworthy that sequences for the
ribosomal gene alone may not be sufficient to clarify phylogenetic relationships
(21), even though these data will aid identifications.
B. Orders, Families, and Genera Covered

The following more detailed accounts do not follow a common pattern because
the amount of information available is most variable. Some species (including
Saprolegnia parasitica, Aphanomyces astaci, Pythium insidiosum, and Lageni-
dium giganteum) are treated in more detail, but the remaining pathogens are dealt
with in collective groups by means of tabulation or key. Tables 1 and 2 provide
the systematic framework, but for most of the pathogens a host/habitat subhead
is more convenient.
1. The Freshwater Fish Pathogens

Saprolegnia parasitica Coker (78). Illustrations: (47, 49, 78, oogonia).
Thallus mycelial, extensive, hyphae 30–118 µm diam. Zoosporangia cylin-
drical with internal renewal, (75)150–200(1050) µm ⫻ (20)30–45(80)
µm. Zoosporogenesis intrasporangial. Zoospores dimorphic and polypla-

netic. Zoospore cysts 9–11 µm diam. with tufts of long, often recurved
boat hook hairs. Oogonia seldom formed in disease situations and not
always capable of induction in culture; when formed, terminal, or inter-
calary, spherical or pyriform-ellipsoidal, 54–146 ⫻ 18–72 µm, wall with
or without relatively small simple pits.
Antheridia diclinous. Oospores (2)14–23(40) per oogonium, (16)18–
24(28) µm diam. The small mean size of the oospore is considered diag-
nostic.
The disease of salmonid fish was first reported by fly fishermen in the nine-
teenth century in Irish and Scottish river systems, where it caused periodic but
not always annual damage to game-fish stocks (salmon and trout). Accounts of
the disease have accumulated over a long period, and the disease has a large
bibliography. (See Refs. 31, 79, 80.) Saprolegnia parasitica is now a noteworthy
disease in fish farms and hatcheries in Europe, North America, and southeast
Asia (31). Subsequent to Coker’s description in 1923 of the species causing the
disease, several other related fish parasites (81–84) and fish hosts have been
reported (49,81,82,85–87). Of the additional genera reported, the more common
are Achlya and Aphanomyces. The species of Achlya are usually from the lacus-
trine, eccentric-oospored group (45). Aphanomyces is discussed separately below.
Other species of Saprolegnia (S. shikotsuensis Hatai et al.) (88,89) are also associ-
ated with the disease. Although the most common disease syndrome is of massive
superficial lesions as described in the introductory paragraphs, visceral mycoses
have also been reported (90,91). The geographic range is now worldwide (31),
with additional reports from India (93) and Australia (94).
One of the most difficult problems in the pathology of this disease is the
determination of those organisms that are causal and the dismissal of those organ-
isms that are opportunistic wound-site secondary invaders. Many of the associ-
ated saprolegniaceous fungi fall into the latter category or have not been unequiv-
ocally shown to be primary pathogens. The greatest confusion has arisen between
the disease-causing Saprolegnia parasitica and the opportunist saprobe S. diclina.
The distinctions between these two species have been discussed at the physiologi-
cal (48,95–98), ultrastructural (7,47,49), and molecular biological (99) levels.
The cyst ornamentation of the principal-form zoospore, consisting of tufts of
long, flexuous ‘‘boat hook’’ hairs, is distinctive and diagnostic (47,49), although
other Saprolegnia saprobes may have intermediate ornamentation (7) and not all
species of saprobic Saprolegnia species have yet been examined. Similarly, the
problem with molecular methodology will be the ability to screen the causal agent
from opportunists when so few species of the genus have been analysed.
Study of the environmental constraints on disease establishment has only
just been touched upon (100). Much work has still to be carried out on the mycotic
44 Dick
aspects of the disease when it is epidemic in fish-farm culture (101). For many
years the standard treatment for this disease has been the application of the toxic
Malachite Green (102), but a more sophisticated approach to fungicide applica-
tion is now being put forward (103).
Aphanomyces pisci [nom. illeg. (no Latin)] (104). Illustrations: (104).
Thallus mycelial with delicate, profusely branched hyphae, 7.2–9.0 µm
diam. Zoosporangia formed from undifferentiated lengths of mycelium,
filamentous, unbranched. Zoosporogenesis with up to 40 spores per zoo-
sporangium. Zoospores of the principal form. Zoospore cysts 7.0–7.5
µm diam. Gemmae abundant, elongate and lobed (reminiscent of lobulate
Pythium sporangia). Oogonia absent. Antheridia absent. Oospores absent.
Aphanomyces piscicida (105) [no illustrations].
Aphanomyces invadans Willoughby et al. (as ‘‘invaderis’’) (106) Illustra-
tions: (106)
Thallus mycelial, hyphae moderately branched, 11.7–16.7 µm diam.,
young hyphae narrower (8.3 µm diam.). Zoosporangia formed from un-
differentiated lengths of mycelium, filamentous, often complex, branched
systems, approximately 330–930 µm in length. Zoosporogenesis-
producing spores encysting at lateral orifices, cysts 6.7–10 µm diam.
Zoospores of the principal form. Oogonia unknown. Antheridia un-
known. Oospores unknown.
Aphanomyces frigidophilus (107). Illustrations: (107).
Thallus mycelial, hyphae moderately branched, 7–10 µm diam. Zoosporan-
gia formed from undifferentiated lengths of mycelium, filamentous, un-
branched. Zoosporogenesis incomplete at time of discharge, encysting
at mouth of zoosporangium. Zoospores of the principal form. Oogonia
abundant, lateral on short hyphae, pyriform or subspherical, 16–25 µm
diameter, with crenulate wall. Antheridia absent. Oospores single, 14–
22 µm diameter.
The first record of another fish disease caused by a straminipilous fungus
was in 1944 (108). The disease is apparently due to penetration from the gut
(compare Refs. 90, 91), presumably caused by ingestion of the pathogen spores.
The infected fish develops an abnormal dorsal hump and the abdominal cavity
becomes grossly swollen. This is followed by obvious mycelial development in
the dorsal musculature; eruption of hyphae to the outside has not been observed,
so the source of the infective unit has not been established. Fish-to-fish transfer
via fungal propagules apparently does not occur (106). It is possible that zoospo-
rogenesis only occurs after death and initial decay of the host, but such sporula-
tion, which has been sought, has not been found either; the source of infection
remains an enigma. More recent studies (109–111) of the disease syndrome have
revealed systemic granulomas.
Epizootic ulcerative disease syndrome (EUS) due to Aphanomyces may be

caused by the same organism (92), and has also been reported (93) in estuarine
conditions (compare diseases caused by Saprolegnia, reviewed above). There
may therefore be some confusion between the identities of the pathogens and the
disease symptoms. Enhanced salinity tolerance may be a factor in the disease
caused by Aphanomyces (112).
The species taxonomy of the causal organism or organisms assigned to the
genus Aphanomyces is not at all clear. Oospores, which would be diagnostic,
have not been described for all binomials, but the fungus is known to produce
Aphanomyces-like zoosporangia according to some cultural studies (108,110).
The growth rates and temperature optima of pathogenic Aphanomyces spp. (in-
cluding A. astaci; see below) are different and the southeast Asian fish-pathogenic
isolates are thermophilic, growing well at 30°C. Neither isozyme nor molecular
biological surveys of numerous isolates have been carried out. Such studies might
resolve whether the causal agent is in fact a distinct species or more than one
species. It has also been suggested (31) that the causal agent might be a Leptoleg-
nia, a genus that is now placed in a separate family together with Aphanomyces
(21).
2. The Freshwater Crustacean Pathogen

Aphanomyces astaci (113).
Thallus mycelial with hyphae of limited extent, of uniformly narrow width,
7.5–9.5 µm diam. Zoosporangia formed from undifferentiated lengths of
mycelium, filamentous, unbranched. Zoosporogenesis almost completed
intramatrically, planonts formed in single file, remaining connected by
protoplasmic strands during discharge, up to 40 per zoosporangium. Zoo-
spores of the principal form. Zoospore cysts 8.0–9.5 µm diam. Oogonia
not normally formed. [NB: the description by Rennerfelt (114) of mi-
nutely spiny-walled, uniovulate oogonia 41–48 µm diam. Antheridia
monoclinous, 1–2 per oogonium. Oospores 22.4–28.83 µm diam. should
be treated with caution.]
The disease of crayfish was first reported in 1903 (see Refs. 113, 115, 220)
and was apparently the result of the introduction of the American crayfish into
European river systems before 1860. At some stage some of the Californian stock
is presumed to have been contaminated with its benign parasite, Aphanomyces
astaci. The European crayfish lacks immunity to this necrotic parasite and the
European and Asia Minor commercial production of crayfish has fallen dramati-
cally and probably irreparably (11,15,220). The fungus has an extensive bibliog-
raphy (see Refs. 115, 116), and considerable work has been published on its
physiology (117,118), biochemistry (35) (including an extensive bibliography),
and pathobiology (119).
46 Dick
The disease of the European crayfish is noteworthy because of the sudden

and total destruction of the population in any given river and lake system, the
streambeds being strewn with dead crayfish. There are a few isolated river sys-
tems in which the European crayfish is as yet uninfected, but quarantine protocols
are liable to be circumvented by the irresponsible introduction of nonnative cray-
fish. Once stock of moderately resistant crayfish are present, the fungal inoculum
will remain, so that any reintroduction of the European crayfish stock will imme-
diately become annihilated. The spread of the disease throughout European river
systems has been traced from Italy and central Europe into northern Europe and
Russia, Scandinavia, Iberia, and Asia Minor.
In moderately resistant North American crayfish, the disease starts as super-
ficial lesions on the exoskeleton, particularly in thinner areas such as the limb
and abdomen joints. However, the hyphal infections which result from encysted
zoospores, are arrested, but not killed, by melanization (33,119,120). In the sus-
ceptible European crayfish this melanin reaction is weak and develops slowly,
so that the fungus is able to penetrate the haemocoel, rapidly causing death. The
haemocytes of the haemocoel are thought to be responsible for the defense reac-
tion whereby the activation of the crayfish prophenoloxidase enzyme by the fun-
gal infection (35,121) results in the melanin deposit around, and stasis of, the
fungal hyphae.
The range of hosts and their relative susceptibilities have been discussed
on a number of occasions (11,15,32,34). In Asia Minor the causal agent of the
disease, in at least some cases, appears to be Saprolegnia parasitica (49); how-
ever, the pathobiology appears to be identical.
3. The Warm Temperate/Tropical Mammalian Pathogen

Pythium insidiosum (27,121,122). Illustrations: (27).
Thallus mycelial, hyphae 4–6 (10) µm diam., branches almost at 90°, some-
times septate, more so in vivo than in vitro; sometimes with club-shaped
appressoria. Zoosporangia produced only in water cultures, filamentous
and undifferentiated from assimilative hyphae, 45–700 µm ⫻ 3–4 (dis-
tally 5–8) µm. Zoosporogenesis extrasporangial in a vesicle 20–60 µm
diam. Zoospores of the principal form, 12–14 ⫻ 6–8 µm. Zoospore cysts
8–12 µm diam., ultrastructural morphology undescribed. Oogonia sel-
dom formed, subglobose, intercalary with septa at a distance from the
swelling, (19)23–30(36) µm diam. Antheridia 1(2–3), diclinous. Oo-
spores aplerotic, single, (17)20–25(27) µm diam. with a distinct ooplast.
Cardinal temperatures min 10°C, optimum 34–36°C, max 40–45°C.
The disease was originally reported from India under the name burusattee
as a disease of horses grazing in or near stagnant water, but the fungal nature of
the causal organism was not noted until 1901 (123). The disease has subsequently
been described as ‘‘phycomycosis of horses’’ (124), ‘‘swamp cancer’’ (125),

‘‘granular dermatitis’’ (126), ‘‘equine pythiosis’’ (122), finally being named as
‘‘pythiosis insidiosi’’ (127). The causal organism was initially regarded as non-
sporulating mycelium, but zoospores were reported in culture in 1974 (125) when
the organism was regarded as a species of Pythium. The taxonomy of the Pythium
species is involved, depending on the interpretation of the earlier names using the
specific epithet ‘‘destruens’’ (62). However, following the description of sexual
reproduction (27), the nomenclature has stabilized as given above. References
to names such as ‘‘Pythium gracile’’ should be regarded as ‘‘form names,’’ not
taxonomic entities, and much work still needs to be done before there is a fully
reliable antigenic (128) or molecular biological framework (21) in which this
pathogen can be placed.
The most recent, comprehensive review of the pathobiology and bibliogra-
phy will be found in Ref. 127, although the mycology is not always accurate.
As indicated by the disease names listed above, there is a granuloma with an
extensive lesion. This may include the production of coralloid ‘‘kunkers’’ that
contain viable hyphae. The disease is not confined to ungulates; a number of
other mammals are known to be capable of infection, including man. Mammal-
to-mammal transfer has only been achieved by using rabbits.
The fungus is thermophilic, as would be expected for a parasite of mam-
mals. Its survival outside mammals presumably depends on existence in warm,
stagnant water, but extensive ecological surveys for its presence have not been
carried out. It is therefore regarded as being restricted to the tropics and warm
temperate zones, and is particularly prevalent in southeast Asia (including Japan)
and Australasia, and Central and South America. Surprisingly, it has not been
reported from Africa. It is not regarded as indigenous in Europe.
4. The Tropical Amphibian Pathogen

Olpidium/Rhizophydium new genus (129). Illustrations: (129,130).
A disease of amphibians (rain forest frogs) in Australia and Central America has
recently been noted (129–132). The disease, termed chytridiomycosis, is caused
by monocentric, rhizoidal chytrids, which are endobiotic within and utilize the
keratinized epidermal cells. Thickening of the skin results, and it is suspected
that interference with water and gaseous transport results in mortality. There is
insufficient evidence as yet, however, to justify a causal relationship between the
disease and the epidemics reported (132,133).
These reports have stimulated a resurgence of interest in the infection of
amphibian eggs by Saprolegnia and Achlya (Jeffries, personal communication).
It has been known for many decades that frog spawn is often damaged by sapro-
legniaceous fungi, but it has not been established whether parasitism rather than
wound damage is the cause. Saprolegnia parasitica can be isolated from such
48 Dick
substrata, a factor which adds to the complexity of the pathobiology of this fun-
gus, discussed in other sections of this chapter. The diversity of the egg-laying
behavior of the amphibian may affect the potential for pathogenicity (134).
5. The Mosquito and Other Insect Pathogens

Lagenidium giganteum, Leptolegnia chapmanii, Crypticola spp., Couchia
circumplexa, Aphanomycopsis sexualis, Coelomomyces spp, and Cate-
naria spp.
The fungi grouped under this heading are from diverse flagellate families: Lageni-
dium giganteum (Pythiaceae), Leptolegnia chapmanii (Leptolegniaceae), Cou-
chia circumplexa (Saprolegniaceae), Aphanomycopsis sexualis (Leptolegniella-
ceae), Crypticola clavulifera and C. entomophaga (Crypticolaceae), Catenaria
spinosa, C. uncinata, C. ramosa (Catenariaceae, Blastocladiales), and Coelomo-
myces spp. (Coelomomycetaceae, Blastocladiales (135)).
6. The Biflagellate Taxa

Lagenidium giganteum is cosmopolitan, having been found in Europe, North and
South America, and Australia. Leptolegnia chapmanii has so far only been re-
ported from the eastern states of North America and Argentina. Couchia circum-
plexa, Aphanomycopisis sexualis, and Crypticola entomophaga are known only
from eastern North America, while Crypticola entomophaga has been recorded
only from tropical Australasia.
Lagenidium giganteum (136). Illustrations: (136–138, 140, 141).
Thallus of hyphae, initially nonseptate, becoming septate to give allantoid
segments 7–10 µm in diam. and 50–300 µm long. Zoosporangia devel-
oped from allantoid segments, 20–40 µm ⫻ 25–60 µm. Zoosporogenesis
with a homohylic vesicle, 20–50 µm diam. Zoospores of the principal
form, 8–9 µm ⫻ 9–10 µm. Zoospore cysts 10–14 µm diam. Oogonia
(26)38–42(63) µm diam. Antheridia with various origins. Oospores sin-
gle, 18–30 µm diam.
Leptolegnia chapmanii (141). Illustrations: (141).
Thallus extensive, of slender, nonseptate hyphae, gemmae abundant, usu-
ally lateral; variable in shape, large swollen, sometimes papillate. Zoo-
sporangia 70–240 µm ⫻ 15–40 µm. Zoospores dimorphic. Zoospore
cysts (first-formed) 13–15 µm diam., (from principal-form zoospores)
10–14 µm diam. Oogonia unpitted, with short papillate projections,
(26)38–42(63) µm diam. Antheridia with various origins. Oospores 1
(3), rarely maturing, subeccentric or eccentric, (18)36–40(52)9 µm diam.
Couchia circumplexa (142). Illustrations: (142).
Thallus of extramatrical hyphae, forming appressoria. Zoosporangia termi-
nal, ellipsoidal to clavate, 53–166 (⫺344) µm ⫻ 32–95 µm. Zoospores

dimorphic. Zoospore cysts 14–22 µm diam. Oogonia spherical, unpitted,
30–84 µm diam. Antheridia diclinous. Oospores 1–5, 23–47 µm diam.
Aphanomycopsis sexualis (143). Illustrations: (143).
Thallus endobiotic, richly branched, nonseptate except to delimit gametan-
gia. Zoosporangia developed from thallus, 206–280 µm ⫻ 88–120 µm,
with 1–10 discharge tubes. Zoospores dimorphic, first-formed zoospores
lacking flagella and encysting immediately, principal-form zoospores
5.4–8.3 µm ⫻ 4.2–6.3 µm. Zoospore cysts 6–7.3 µm diam., with short
spines. Oogonia swollen portions of the thallus, 82–240 µm ⫻ 44–72
µm. Antheridia present, septate. Oospores single, ovoid, 52–170 µm ⫻
44–72 µm; oospore wall 2.2–4.3 µm diam.
Crypticola clavulifera (144). Illustrations: (144).
Thallus limited, becoming septate to give allantoid segments 50–60 µm
long. Zoosporangia developed unilaterally from allantoid segments, 35–
40 µm ⫻ 12–13 µm. Zoosporogenesis intrasporangial. Zoospores of the
principal form, 8–10 ⫻ 5–6 µm. Zoospore cysts 10–14 µm diam. Oo-
gonia unknown.
Crypticola entomophaga (14, 36). Illustrations: (145).
Thallus ellipsoidal or irregular, nonseptate, up to 215–280 µm ⫻ 175–245
µm in caddis fly eggs. Zoosporangia developed from thallus, with 1–4
discharge tubes up to 3.7 mm long. Zoospores of the principal form
(dimorphic), polyplanetic, (8.5⫺)11(⫺16) µm ⫻ (6⫺)6.9(⫺8.5) µm.
Zoospore cysts (9⫺)10.5(⫺13.5) µm diameter. Oogonia unknown.
7. The Uniflagellate Taxa

Catenaria spinosa (146). Illustrations: (146).
Thallus simple or branched, with irregular catenulate swellings 3.8–17 µm
diam., and a few blunt rhizoids, becoming septate with one-celled isth-
muses. Zoosporangia of two types: thin walled and resistant: thin walled
zoosporangia 25–69 µm ⫻ 24–66 µm. Zoospores 7–11 ⫻ 4.5–7.5 µm.
Resistant sporangia golden brown, 30–62 µm ⫻ 25–61 µm, wall 1.3–
2.5 µm thick with short, spinelike appendages.
Catenaria uncinata (147). Illustrations: (147).
diam., and a few blunt rhizoids, becoming septate with equidistant swell-
ings separated by one-celled isthmuses. Zoosporangia of two types: thin
walled and resistant; thin-walled zoosporangia 22–62 µm ⫻ 18–56 µm.
Zoospores 3.1–5.6 ⫻ 2.1–3.4 µm. Resistant sporangia golden brown,
34–57 µm ⫻ 32–54 µm, with elongate, sometimes curved, spinelike
appendages tapering to an uncinate tip.
50 Dick
Catenaria ramosa (147). Illustrations: (147).

diam., and a few blunt rhizoids, becoming septate with one-celled isth-
muses. Zoosporangia of two types: thin walled and resistant; thinwalled
zoosporangia 28–54(⫺70) ⫻ 25–49 µm. Zoospores 3.5–6.8 ⫻ 2.4–4.8
µm. Resistant sporangia golden brown, 20–66(-76) µm ⫻ (8-)13–48
µm, appendages sparce, often branched.
Species of Catenaria parasitic in animals appear to be confined to insect

(Diptera) eggs in unidentified species of Chironomus, in Glyptotendipes lobiferus
Say., and in Dicrotendipes modestus Say., respectively. Host and habitat differ-
ences between these species have been recorded (147), but further work on their
biology and potential for control of the insects is needed.
The genus Coelomomyces contains over 60 species and varieties
(37,135,148–150), the species often being partly based on host. The species are
listed in alphabetic order after the type species in Table 5, with Index of Fungi
(IF) references.
Generic description for Coelomomyces (adapted from 149): Thallus simple

or branched, without rhizoids or cell walls, giving rise to ‘‘hyphal’’ seg-
ments by division or attenuation and fracture by host movement. Thin-
walled zoosporangia: apparently absent. (See Ref. 151; check alternate
host.) Resistant sporangia: formed as ‘‘hyphal’’ segments, developing
a wall after detachment, golden brown with a very thin outer membrane
and a smooth, pitted, striated, or ridged wall. (See Ref. 152.) Zoospores:
as for the order, except that they lack a well-defined nuclear cap. (See
Ref. 153.)
8. Commentary
As a result of the search for methods of biological control of mosquitoes there
has been a significant amount of research into the basic biology and biochemistry
of these fungi, particularly Lagenidium giganteum (16–19), for which over 150
references have been cited (36), and Coelomomyces (148). Habitats for agents
of biological control have also been screened, and tree-hole and leaf reservoir
(phytotelemata) (142,154) mosquito habitats have yielded a number of new taxa.
Insect pathogens are reported from dipteran eggs (142–147), but with few corrob-
orative collections and little data on pathobiology.
It is characteristic that most of these pathogens infect the larval stages,
rapidly gaining access to the haemocoel, with extensive intramatrical growth re-
sulting in death followed by extramatrical sporogenesis. An extensive review
(148) of Coelomomyces summarized much of the information on this genus. Its
suitability for biological control was severely compromised by four major factors:
Table 5 The Genus Coelomomyces
Species IF References
C. stegomyiae Keilin (1921) IF3: 5, 213; IF5: 527

C. africanus AJ Walker (1938) IF5: 526
C. angolensis H Ribeiro (1992) IF6(6): 329
C. anophelesicus MOP Iyengar (1935) IF3: 213
C. arcellaneus Couch & Lum (1985) IF5:526
C. arsenjevii Koval & ES Kuprian. (1981) IF5: 115
C. ascariformis Van Thiel (1962) IF3: 5, 213
C. azerbajdzanicus ES Kuprian. & Koval (1981) IF5: 526
C. beirnei Weiser & McCauley (1971) IF4: 129, 208
C. bisymmetricus Couch & HR Dodge (1947) IF3: 213
C. borealis Couch & Service (1985) IF5: 526
C. cairnsensis Laird (1962) IF3: 213
C. canadensis (Weiser & McCauley) Nolan (1978) IF4: 560
(as canadense)
C. carolinianus Couch et al. (1985) IF5: 526
C. celatus Couch & Hembree (1985) IF5: 526
C. chironomi Rǎsı́n (1929) IF4: 129, 208
C. ciferrii Arêa Leao & Carlota Pedroso (1965) IF3: 351
C. couchii Nolan & B. Taylor (1979) IF5: 7
C. cribrosus Couch & HR Dodge (1947) IF3: 213
C. dentialatus Couch & Rajap. (1985) IF5: 526
C. dodgei Couch (1945) IF3: 213
C. dubitskii Couch & Bland (1985) IF5: 526
C. elegans Couch & Rajap. (1985) IF5: 526
C. fasciatus Couch & MOP Iyengar (1985) IF5: 526
C. finlayae Laird (1962) IF3: 5, 213
C. grassei Rioux & Pech (1962) IF3: 213
C. iliensis Dubitskiı́ et al. (1973) IF4: 208; IF5: 334,
384, 526
C. indianus MOP Iyengar (1935) IF3: 213
C. iranii Weiser, Zaim & Saebi (1991) IF6(4): 200
C. neotropicus Lichtw. & LD Gómez (1993) IF6(10): 540
C. iyengarii Couch (1985) IF5: 526
C. keilinii Couch & HR Dodge (1947) IF3: 213
C. lacunosus Couch & OE Sousa (1985) IF5: 526
C. lairdii Maffi & Nolan (1977) (as lairdi) IF4: 623
C. lativittatus Couch & HR Dodge (1947) IF3: 213
C. maclayae Laird (1962) IF3: 5, 213
C. madagascaricus Couch & Grjebine (1985) IF5: 526
C. milkoi Dudka & Koval (1973) IF4: 242
C. musprattii Couch (1985) IF5: 526
C. notonectae Bogoyavl. (1922) IF5: 873
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Table 5 The Genus Coelomomyces
Species IF References
C. omorii Laird et al. (1975) IF4: 404

C. opifexi Pillai & JMB Sm. (1968) IF4: 208
C. orbicularis Couch & Muspratt (1985) IF5: 526
C. orbiculostriatus Couch & Pras. (1985) IF5: 526
C. pentangulatus Couch (1945) IF3: 213
C. ponticulus Nolan & Mogi (1980) IF5: 526
C. psorophorae Couch (1945) IF3: 213; IF5: 526
C. punctatus Couch & HR Dodge (1947) IF3: 213
C. quadrangulatus Couch (1945) IF3: 213
C. rafaelei Coluzzi & Rioux (1962) IF3: 299; IF4: 404
C. reticulatus Couch & AJ Walker (1985) IF5: 527
C. rugosus Couch & Service (1985) IF5: 527
C. sculptosporus Couch & HR Dodge (1947) IF3: 213
C. seriostriatus Couch & JB Davies (1985) IF5: 527
C. solomonis Laird (1962) IF3: 213
C. sulcatus Couch & MOP Iyengar (1985) IF5: 527
C. tasmaniensis Laird (1962) IF3: 214
C. thailandensis Couch et al. (1985) IF5: 527
C. triangulatus JN Couch & WW Martin (1985) IF5: 527
C. tuberculatus Couch & Rodhain (1985) IF5: 527
C. tuzetiae Manier et al. (1970) IF4: 30
C. utahensis Romney et al. (1985) IF5: 527
C. uranotaeniae Couch (1945) IF3: 214
C. walkeri Van Thiel (1962) IF3: 5, 314
Note: Species, with authorities in recommended form, listed in alphabetic order after the type species,
with Index of Fungi (IF) references (up to vol. 6, part 17).
Source: Refs. 37, 135, 148–150.
the species appeared to be strongly host-specific (149,150); the life history of at

least some species was discovered to be heteroceous, involving resistant sporan-
gial production confined to the alternate host, usually a microcrustacean (155);
it was observed (156) that the accumulation of fungal material in the mosquito
ovaries did not always result in the death of the host; and the economic aspects
of mass fungal inoculum preparation and its failure to regenerate were noted—
heavy inoculum resulted in early instar death, while lighter inoculum loads failed
to provide sufficient decrease in the population levels (157).
Lagenidium, in contrast, is much more amenable to manipulation and has
a much wider host range. There remain problems in formulating protocols for
biological control, but a full discussion is not appropriate here.
9. The Parasites of Marine Crustacea and Mollusks:

(Blastulidium Salilagenidium spp., Haliphthoros spp.,
Atkinsiella dubia, and Halodaphnea spp.)
The fungal parasites of marine littoral crabs and prawns have been described
from the northern hemisphere Atlantic and Pacific shores, starting with the de-
scriptions by Atkins (158–161) and Couch (43).
Egg masses are often the prime sites of parasitism. In most cases of juvenile
and adult infections, the fungus develops in the haemocoel and can completely
fill the swimmerets of shrimps and prawns. Epidemics have been reported (13)
from the mariculture centers in the Asian Pacific where the crustacean hosts form
an important part of the protein diet. The occurrence of these fungi on crabs and
barnacles does not appear to have the same economic impact (11).
Since all of these species are placed in a single order, the Salilagenidiales
(previously part of the polyphyletic Lagenidiales) (36), the distinctions between
the species, including hosts and any cultural information, are most concisely pre-
sented in the form of a fully descriptive key. Note that the Haliphthoraceae was
specifically erected for species without sexual reproduction (162) and that a new
generic name (Halodaphnea) has become necessary for Halicrusticida, to which
a number of Atkinsiella species were assigned. (For further taxonomic references,
see Refs. 14.)
Comparative molecular biological data are not yet available, and relation-
ships within the order and with other orders are still conjectural.
Another biflagellate fungal parasite of crabs was described and named as
Pythium thallasium (161), but Plaats-Niterink (163) regarded the generic place-
ment as doubtful because of the mode of sporangial renewal. Until this parasite is
rediscovered and redescribed its classification must be regarded as incerta sedis.
Three species of the Labyrinthista could also be mentioned here. Laby-
rinthuloides haliotidis is the only member of this genus reported as a parasite of
marine animals (164,165)—in this case the abalone of the north-east Pacific litto-
ral. The molecular biology of this organism has been documented (166), and
although straminipilous, its fungal status and generic attribution are in doubt.
Labyrinthula thaidis B. A. Cox & J. G. Mackin (as L. thaisi) (167) and L. jere-
marina L. Rolf (nomen nudum) (167) have been described from the gill tissue
of gateropod mollusks (Thais and Littorina, respectively). Again the descriptions
lack salient features for satisfactory placement in a classification. Both Laby-
rinthuloides (Thraustochytriales) and Labyrinthula (Labyrinthista) are genera pri-
marily inhabitants of plant material and detritus.
Blastulidium paedophthorum (Synonym: Blastulidiopsis chattonii). A. Sigot

(169). Illustrations (168).
The parasite of Daphnia from freshwater lagoons could be mentioned here.

54 Dick
It has rarely been found, and its affinities are unclear (36) in spite of ultrastructural
and taxonomic work that has been carried out (170).
10. The Parasites of Aschelminthes (Myzocytiopsis spp., Gonimochaete

spp., Chlamydomyzium spp., and Other Flagellate Fungi)
Table 3 lists the flagellate fungal parasites of the Aschelminthes (nematodes and
rotifers). Many of these taxa are known only from the original descriptions (cited
in Table 3) and a few subsequent recorded collections. The most commonly en-
countered flagellate fungal parasites of the Aschelminthes are placed in the Myzo-
cytiopsidales (171), and a key to these species is given in Refs. 36 and 55. How-
ever, the one flagellate fungal pathogen of nematodes that has received much
attention is Nematophthora gynophila (28), placed in the Leptolegniellaceae be-
cause of its distinctive oospore wall construction. Chytrid parasites, particularly
of nematode eggs, also occur where nematodes are abundant. Three species of
Catenaria (Blastocladiales) have been described as parasites of nematodes; C.
allomycis is also reported as occurring in nematodes (172).
Much of the research effort in the biological control of nematodes has been
directed to the facultatively parasitic ascomycetes because these fungi are more
easily cultured. Control of nematode infestation by Nematophthora is effective
eventually, but may cause commercial loss in the interim (28).
Unfortunately, little attempt has been made to understand the ecology of
any of these parasites in the complex soil ecosystem. Given the number of species
already described, it is probable that there is considerable niche specificity. Host
specificity of these obligate parasites is probably not critical since transfer in
laboratory conditions between nematodes, and nematodes and rotifers, is easily
achieved. The same situation appears to be true for the Haptoglossales. Future
studies may show that the critical features are the habitat of the nematode in
relation to air–water interfaces and the precise mechanism of fungal spore attach-
ment to the host; thus in different species of Myzocytiopsis the zoospores encyst
either at body orifices or at random over the cuticle. In Haptoglossa the shape
of the cyst containing the injection apparatus and the prevalence of a zoosporic
phase differ between species. Multiple infections by one species are common,
but the size of each thallus may be reduced with multiple infections. It is much
rarer for a host to contain more than one fungal species, but this situation has
been recorded (9). Usually only one species of a given genus is found in an
individual. Nematodes and rotifers infected by Myzocytiopsidales or Haptoglos-
sales remain active for a long time after much of the body space has been taken
up by the parasites; fungal spore release may be by thallus conversion into sporan-
gia or zoosporangia, which develop exit tubes through the skin (36,55), or by
sporangial disintegration within the body cavity and release by the total disinte-
gration of the host (44).
Nothing is known of the biochemical progress of disease in aschelminths

caused by flagellate fungi. No molecular biological data have yet been published
for any of these flagellate fungi, although such studies are underway (G. W.
Beakes and S. L. Glockling, personal communication).
Endosphaerium funiculatum (173). Illustrations: (173).
Endosphaerium is a monotypic genus (species E. funiculatum) erected for a

parasite on Aschelminthes in the mantle cavity of a freshwater pelecypod bivalve
mollusk. It is known only from the original report. Unfortunately, the description
of the fungus lacks almost all the diagnostic criteria necessary for classification.
The author placed the fungus in the Pythiaceae, but it is difficult to see why. The
fungus must be regarded as doubtful until rediscovered and redescribed.
11. Parasites of Noncellular Protoctista

The records for fungal pathogens of protozoan parasites are meager. Table 4 lists
the hosts and cites the original descriptions, most of which are rather old records.
Few subsequent supportive collections have been reported for any of these spe-
cies. Diagnostic criteria that would now be considered essential are mostly lack-
ing for species placed in Sphaerita and Pseudosphaerita (174), and so the taxon-
omy itself must be considered highly dubious. The comparable algal/fungal and
algal/protozoan relationships have been reviewed (42), and more details from
the original descriptions are available. (See Ref. 175.) Very little ecological re-
search has been carried out on the occurrence, virulence, and distribution of these
parasites, and further generalizations would be premature.
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3
Zygomycetes
The Order Mucorales
M. A. A. Schipper and J. A. Stalpers

Centraalbureau voor Schimmelcultures, Institute of the Royal
Netherlands Academy of Arts and Sciences, Utrecht, The Netherlands
I. INTRODUCTION
This chapter deals with the members of the Mucorales that cause mycotic diseases
in mammals and birds, with emphasis on identification of the species involved.
As the species are generally only identifiable when cultured, special attention has
been given to cultural characters, growth conditions, and mating, while the num-
ber of clinical data is limited, as these are treated in detail in various modern
handbooks (1–4).
Mycotic diseases caused by Mucorales are referred to as mucormycoses.
Two other terms occur in recent literature: zygomycoses, a broader term, which
also covers diseases caused by Entomophthorales (e.g., Basidiobolus, Conidiobo-
lus), the only other order of the Zygomycetes causing diseases in warm-blooded
vertebrates (see Chap. 4), and phycomycoses, which even include diseases caused
by Pythium and related species, which are currently classified in a different king-
dom, the Chromista (See Chap. 2.) Since the diseases caused by Mucorales are
fairly uniform with respect to epidemiology, clinical aspects, and pathology, and
are with very few exceptions clearly distinct from those caused by Entomophthor-
ales, the term mucormycoses is preferred here.
Mucormycoses cover a wide range of diseases, including cutaneous, gastro-
intestinal, pulmonary, rhinocerebral, and disseminated types. The causal agents
belong to the genera Absidia, Apophysomyces, Cokeromyces, Cunninghamella,
Mortierella, Mucor, Rhizomucor, Rhizopus, Saksenaea, Syncephalastrum, and
Thermomucor.
67
68 Schipper and Stalpers
Prompt identification is essential, because mucormycoses belong to the

most rapidly progressing mycoses. Identification at the genus and species level
is usually only possible in culture. A correct identification also requires some
knowledge of related species as well as of the more generally occurring sapro-
phytes.
The current keys are not restricted to accepted pathogens only, but build
according to the ‘‘positive key’’ principle. The first key starts at class level and
only those orders containing pathogenic organisms are considered further. This
is repeated at family, genus, and species levels. The pathogenic species are de-
scribed in some detail.
II. HISTORY
Fürbringer (5) was the first to report on the pathogenic behavior of Mucorales.
Lichtheim (6) isolated two species of Mucorineae, which he suspected to be
pathogenic, and he inoculated both strains into rabbits to test this hypothesis.
The strains were identified as Mucor rhizopodiformis (⫽ Rhizopus microsporus
var. rhizopodiformis) and Mucor (Absidia) corymbifera. Barthelat (7) published
a review of all known pathogenic Mucorineae, including clinical reports, and
after testing the reaction of various animals, such as rabbits, guinea pigs, and
chickens, accepted the following species as pathogenic: Mucor corymbifer, M.
ramosus, M. truchisi, M. regnieri (all Absidia corymbifera), Mucor pusillus, Rhi-
zomucor parasiticus (both Rhizomucor pusillus), and Rhizopus cohnii (⫽ Rhizo-
pus microsporus). No pathogenicity could be reported for Mucor mucedo, M.
racemosus, M. alternans (M. circinelloides), and Rhizopus nigricans (⫽ Rh. sto-
lonifer).
Ainsworth and Austwick (8) reviewed the fungal diseases of animals, while
Ader and Dodd (9) reviewed the literature on mucormycoses up to 1978.
III. METHODS
A. Direct Examination
Material obtained from sputum, skin scraping, tissue preparation, or sinus aspi-
rates may give a fast clue to the presence of a mucormycosis, because in such
cases hyphae are often—but not necessarily—abundantly present, and these
broad (5–15 µm), irregularly branched, aseptate, or rarely septate hyphae are
quite diagnostic. They are only characteristic for mucormycosis, however; identi-
fication at family, genus, or species level from human material is generally impos-
Zygomycetes 69
sible, as sporogenous structures are typically absent. Several methods can be

applied, and these are discussed below.
1. Direct Smear
Direct observation can be made of the material mounted in 10% KOH. The slide
is gently heated. This method is fast, but not always sufficient (10).
2. Methenamine Silver Reaction

The methenamine silver reaction of Grocott and Gomori is based on a mixture
of chromic acid, methamine-silvernitrate, gold chloride, and sodium thiosulphate.
The method takes about 1 hr.
3. Fluorescence
Uvitex 2B (Fungiqual, CIBA-Geigy, Basal, Switzerland). Uvitex 2B is
described by Kuyper et al. (11). It binds specifically to chitin of the cell walls
of fungi. The preparation takes about 30 min. The slide has to be examined at
400 nm.
Blankophor P (Bayer, Germany). Blankophor P is described by Monod
et al. (12). The preparation takes about 20 min, and observation is at 420 nm. A
similar stain is Fungi-Fluor (Polysciences).
B. Cultivation of the Organisms

Mucorales generally grow well on artificial media and without special effort iso-
lates from more than 90% of the material diagnosed as mucormycosis by direct
examination were successful (11). In particular, malt agar (MA), oatmeal agar
(OA), or cornmeal agar (CMA) are generally applicable, while the popular Sa-
bouraud agar ranks among the less recommendable media. The optimal condi-
tions for the production of sporangia or zygospores vary for the different species;
they are summarized in Table 1. Especially in Apophysomyces, Mortierella, and
Saksenaea sporulation may be poor or absent when the conditions are not optimal.
Mixed or contaminated cultures can be purified by monosporangium iso-
lates. Dip a hot inoculation needle in agar, touch a sporangium with the now
coated needle, and inoculate a new petri dish.
Because of the invasive growth of Mucorales it is advisable to examine
and isolate from attacked but not already necrotic tissue or from sinus aspirates.
In case of isolation from superficial smears false positives are more likely to
occur, as the spores of zygomycetes occur abundantly in the air. Grinding of
Table 1 Summary of Culture Conditions for the Pathogenic Taxa of the Mucorales
Name Media Temp. (°C) Storage (°C) Remark
Absidia MA4, PDA, OA, 20, 36 5

CMA
Apophysomyces YPPS, MA4 33 16
Cokeromyces MA4, PDA, OA, 20–22 5
YPPS
Cunninghamella MA2, PDA, 20–22 5
PCA, OA
Mortierella PDA, CMA, 22 5 See also under
MA2 M. wolfii
M. wolfii 35 16
Mucor MA4, CMA, 20 5
PDA
M. amphibiorum 16
M. indicus 25–35 16
Rhizomucor MA4, OA, PDA 36 5
Rhizopus MA4, CMA, OA 16
Rh. oryzae 27
Rh. microsporus 36
Saksenaea Hay infusion agar 25 See also under
(double layer) Saksenaea
Syncephalastrum MA4, PDA, OA 36 5
Thermomucor MA4 5
Note: For medium formulae see Section III.C.
material can give negative results, because the mycelium is coenocytic and the
methods may result in only damaged (dead) cells.
The viability of vegetative spores is often limited; subculturing should in-
clude transfer of pieces of substrate mycelium. Many species sporulate poorly
when oxygen concentrations are low. (Beware of closely fitting petri dishes.)
Mucorales are generally heterothallic and the occurrence of zygospores is
quite rare. Matings can assist in confirming a supposed identity, however. (See
Sec. V.B.)
The morphological study includes both macroscopical cultural characters
and microscopic morphology. Water mounts are preferred over other mounting
fluids, which may produce structural changes (swelling or shrinkage). The charac-
teristics of sporangiophores, sporangia (size), stolons, and rhizoids should be
studied in situ in undisturbed colonies with a stereomicroscope.
Zygomycetes 71
To prevent delay in identification cultures should be incubated on at least

two different media at various temperature; for example, 25, 30, 36, and 40°C.
For the suppression of bacterial growth, see Sec. III.C.
C. Preparation of Media
Media are sterilized by autoclaving at 121°C for 15 min, unless stated otherwise.
Formulae for extracts and additives (when complex) are given separately. All
media are for 1 liter and contain 15 g agar.
1. CMA (Cornmeal Agar). Add 60 g freshly ground cornmeal agar to
1 liter water, heat to boiling, and simmer gently for 1 hr. Squeeze and
filter through cloth. Fill up to 1 liter and sterilize.
2. Hay (hay-infusion agar). Sterilize 50 g hay for 30 min at 121°C,
filter, fill bottles, adjust to 1 liter and adjust pH to 6.2 with KH 2 PO 4.
Sterilize.
3. MA2 (Malt Agar). Dilute brewery malt with water to 10% sugar solu-
tion (level 10 on Brix saccharose meter). Sterilize. Fill 200 ml (2%)
or 400 ml (4%) up to 1 liter. Sterilize. There are also good commercial
MAs available.
4. OA (Oatmeal Agar). Wrap 30 g oatmeal flakes in cloth and hang in
pan. Bring to the boil and simmer gently for 2 hr. Squeeze and filter
through cloth. Sterilize.
5. PCA (potato–carrot agar). Add 20 g pealed and chopped carrots and
20 g scrubbed and diced potatoes to 1 liter water and boil and simmer
for 1 hr. Boil again for 5 min and filter. Sterilize.
6. PDA (potato-dextrose agar). Add 200 g scrubbed and diced potatoes
to 1 liter water and boil for 1 hr. Let it pass through a fine sieve, add
20 g glucose, and boil until dissolved. Sterilize. Avoid new potatoes.
7. SNA (synthetischer nährstoffarmer agar, Synthetic poor medi-
um). Add 1 g K 2 HPO 4 , 1 g KNO 3 , 0.5 g MgSO 4.7H 2 O, 0.5 g KCl,
0.2 g glucose, and 0.2 g saccharose to 1 liter distilled water. Sterilize.
Pieces of filter paper may be added as carbon source.
8. YPSS (yeast powder-soluble starch agar). Boil 1 g K 2 HPO 4, 0.5 g
MgSO 4.7H 2 O, 15 g soluble starch, and 4 g yeast extract (Difco) in 1
liter of water until the ingredients are dissolved. Fill up to 1 liter.
For the suppression of bacterial growth, 1 ml of one of the following antibiotic
solutions is added to the petri dishes before pouring out the agar, to give final
concentrations (in parentheses) of: penicillin-G (50 ppm); streptomycin (30–50
ppm); aureomycin (10–50 ppm) neomycin (100 ppm); and novobiocin (100 ppm)
or vanomycin (10 ppm). Chloramphenicol (50 ppm) is resistant to autoclaving
and can be added before sterilization.
IV. MUCORMYCOSES
Generally, the immune system prevents infections with the Mucorales, but in
immunocompromised patients (e.g., AIDS, diabetes, hepatitis, after trans-
plantations and immunosuppression, intravenous drug abuse, leukemia), infec-
tions can be acute and serious (13,14). Characteristic is vascular invasion by
hyphae, leading to thrombosis, infarction, and necrosis of tissue.
Invasion by the fungus generally occurs through
Inhalation of the spores
Ingestion
Traumatic implantation
Surgery
Contamination of burn wounds
Traumatized skin
Ears, nose, nails, and eyes
The frequent occurrence of the spores in the air (especially Mucor and Rhizopus)
explains reported laboratory infection; for example, from the use of contaminated
ectoplast bandages, postoperative wound infection, and infected protheses.
In particular the use of deferoxamine incurs an increased risk for mucor-
mycosis. Dialysis patients having an iron overload based on a history of frequent
transfusions and treated with deferoxamine frequently developed severe mucor-
mycoses (15). Rhizoferrin, a siderophore isolated from Rh. microsporus, may
mediate the infection (16), while Seeverens et al. (17) stressed the role of oxygen
radicals. Boelaert et al. (18) advised against the prolonged use of deferoxamine
therapy because of the increased risk of mucormycosis.
The following main types of mucormycoses are recognized (3,19):

Cutaneous mucormycosis: Infection generally occurs by injection or by
implantation in wounds, especially burns, but it may also result from
ingrowing mycelium from other organs. Most known mucoralean patho-
gens have been reported from cutaneous or subcutaneous infections:
Apophysomyces, Cunninghamella, Rhizopus, Saksenaea, and Syncepha-
lastrum.
Rhinocerebral mucormycosis: Infection is generally by inhalation and ger-
mination of the sporangiospores in the nose or paranasal sinuses. Devel-
opment can be fast, especially when arterial walls are infected. Most
reports concern Rhizopus spp., but cases caused by Saksenaea may also
occur.
Pulmonary mucormycosis: Infection originates generally from inhaled
sporangiospores or from disseminated mucormycosis. It may be acute,
resulting in relatively large amounts of mycelium, or subacute, by
Zygomycetes 73
spreading through the airways. Because of the oxygen-rich environment,

growth can be very fast, often with lethal consequences. Most cases are
caused by Absidia or Cunninghamella, some by Mucor spp.
Gastrointestinal mucormycosis: The infection is generally caused by indi-
gestion or following surgery in the abdomen. Cases have been reported
involving Absidia, Corymbifera, and Rhizopus spp.
Disseminated mucormycosis: Infection generally starts in the lungs or para-
nasal cavities, and may spread into the central nervous system, the vascu-
lar system, or the brain. Severely immunocompromised patients may de-
velop mucormycosis in virtually any organ. Causal agents are:
Rhizomucor spp. and Saksenaea. vasiforme.
V. IDENTIFICATION
A. Morphological Characters
Characters observed in microscopical slides include the following.
1. Vegetative Characters
Mycelium: total of aerial and submerged hyphae produced. In the Zygomy-
cetes the hyphae are in principle aseptate, except in two cases: to separate
reproductive structures and to separate dead or damaged parts from the
active mycelium.
Stolons: creeping aerial hyphae from which rhizoids and sporangiophores
are produced (Fig. 1).
Rhizoids: short, rootlike hyphae, which can be simple or branched (Fig. 1).
2. Reproductive Characters
Sporangiophores: branched (Fig. 2) or unbranched structures bearing spo-
rangia. When branched, the development is sequential. Sporangiophores
can be hyphoid or specialized, and then often terminating with a swollen
structure on which the sporangia are more or less simultaneously pro-
duced (Figs. 3, 4). The branches can be monopodial (a persistent axis
produces more fertile branches) or sympodial (continued growth after
the main axis has produced a terminal sporangium; Fig. 2). Sporulation
is indeterminate when the conidiophore length increases with continuing
sporulation, or determinate when new sporangia are produced below the
older ones.
Sporangia: collective term for a reproductive cell whose contents become
transformed into asexual spore(s). An axial part of the mature sporan-
gium can remain sterile and is called a columella (Fig. 5). The apophysis
Figure 1 Schematic representation of Rhizopus groups; (a) Rh. stolonifer group, (b) Rh.
oryzae group, (c) Rh. microsporus group.
Figure 2 Branching patterns; (a) sympodial, (b) monopodial.

Zygomycetes 75
Figure 3 Syncephalastrum racemosum—sporangiophore and merosporangia.
Figure 4 Cokeromyces recurvatus—young and mature sporangiophore.

Figure 5 Columella types (from Ref. (84): (a) with distinct collar (M. ramosissimus),
(b) globose (M. hiemalis, (c) ovoid (M. silvaticus), (d) pyriform (M. mucedo), (e) hemis-
phaerical (Zygorhynchus vuilleminii), (f ) hemisphaerical with apophysis and apical projec-
tion, (g) hemisphaerical with broad apophysis (Rhizopus sp.).
is the basal section of the columella, which is not surrounded by the

sporangium wall (Fig. 6). The sporangial wall can be smooth, spiny,
transparent, or dark. At maturity the wall can be deliquescent (dissolving
in a drop of liquid) or persistent (breaking). After spore liberation rem-
nants of the original sporangial wall may form a collar (Fig. 7).
The term sporangium is used here somewhat restrictedly for those cells
producing numerous (more than 30, and often more than 100) spores.
Two additional terms are used for special forms: sporangioles and meros-
porangia.
Sporangioles: small, usually globose sporangia, containing few (1–30)
spores. The wall is usually persistent. The monospored sporangioles
(sometimes called conidia) are formed on pedicels that are often borne
on vesicles (Fig. 8).
Merosporangia: cylindrical sporangia in which the spores are produced in
a single row (Figs. 8).
Chlamydospores: thick-walled, terminal, or intercalary cells that are func-
tional resting spores.
Zygospores: diploid cells resulting from the fusion of two haploid cells
(Figs. 9, 10).
B. Mating
As a rule Zygomycetes produce hyphae without regular septation, resulting in
tubes filled with cytoplasm in which the nuclei can move freely. Conditions for
mating species of Mucorales are given in Table 2. Septa are only produced near
Zygomycetes 77
Figure 6 Sporangiophore of Absidia; (a) sporangium, (b) columella, (c) apophysis.
reproductive structures and to separate damaged or ‘‘aged’’ parts of the myce-

lium. Zygomycetes are mainly characterized by their sexual reproduction: fusion
of coenocytic gametangia resulting in a thick-walled zygospore (Figs. 9, 10).
Three types are distinguished: heterothallic, with gametangia originating from
different mycelia; homothallic, gametangia produced by a single mycelium; and
azygosporic, when the ‘‘zygospore’’ is produced by a single gametangium only
(parthenogenic zygospores).
Blakeslee (20) showed that the majority of the Zygomycetes are heterothal-
lic and unable to produce zygospores unless two genetically different mycelia
(mating types) were present. He designated such strains ⫹ and ⫺. Burgeff (21)
and Plempel (22) discovered that ⫹ and ⫺ mycelia produced mating type-specific
substances (sex hormones), that act complementarily to produce pheromones.
Figure 7 Mucor ramosissimus: (a–b) sporangiophores, (c–d) columellae with collar,

(e) sporangiospores.
Figure 8 Cunninghamella bertholletiae (from Ref. 71): (a) conidiophore, (b) conidia,
(c–d) zygospores.
Figure 9 Various stages in the development of zygospores in Mucor hiemalis.

Zygomycetes 79
Figure 10 Morphological variation in zygospore formation from heterothallic to homo-

thallic azygospores; a-c heterothallic with different types of suspensors; (a) Rhizomucor
pusillus, (b) Mucor hiemalis, (c) Mucor genevensis; d-g homothallic with variously shaped
suspensors and decreasing distances of the origin of participating hyphae: (d) Rhizopus
sexualis, (e) Rh. homothallicus, (f) Zygorhynchus moelleri, (g) Z. japonicus, (h) azygo-
spore, Mucor bainieri.
These pheromones induce zygophore formation and act as chemotropic agents

between mating partners. The initial stages of the mating process depend on the
mating type only, and may occur between ⫹ and ⫺ strains of quite different
taxa, thus allowing an identification and a consistent use of the designations ⫹
and ⫺. The completion of the process, starting with the lysis of the fusion wall,
is considered species-specific.
A confusing element is the production of azygospores in both legitimate
Table 2 Mating Conditions for Pathogenic Species of Mucorales
Taxa Medium a Temperature
Absidia corymbifera MYA 33°C

Cunninghamella bertholletiae MYA 35°C
Mucor amphibiorum MEA 25°C
M. circinelloides f. circinnelloides Whey agar 25°C
M. circinelloides f. lusitanicus MEA 25°C
M. hiemalis f. hiemalis MEA 20°C
M. hiemalis f. luteus MEA 20°C
M. racemosus f. racemosus Cherry decoction agar 10°C
M. racemosus f. sphaerosporus Cherry decoction agar 10°C
Rhizomucor pusillus MEA (YPSS, YEA) 27°C
Rhizopus microsporus group YEA (YPSS, MEA) 30°C
Rh. oryzae YEA 30°C
Syncephalastrum racemosum YEA 25°C
a
Abbreviations: MYA, malt yeast extract agar; MEA, malt extract agar; YPSS, yeast powder-soluble
starch agar; YEA, yeast extract agar.
matings, which also yield normal zygospores, and in interspecific pairings, in

which normal zygospores do not occur. Zygospores result from the interaction
between different hyphae or parts of hyphae, each secreting its own sex-specific
substance. In heterothallic species the ⫹ and ⫺ sites are located on hyphae from
different thalli. In homothallic species the copulating hyphae are connected either
in the substrate or on the aerial mycelium, but the ⫹ and ⫺ sites are distinctly
separate. Azygospores are produced from one single site only, which therefore
might be ⫹/⫺ in genetic constitution or where agglomerations of ⫹ and ⫺ are
close together. Following a very careful study of the mating processes of Mucori-
neae, Ling Young (23) suggested that azygospore formation in intraspecific mat-
ings may result from weak sexual potency of the partners leading to anomalies
of the fusion processes and the prevention of actual zygospore formation.
Blakeslee and others accepted the occurrence of such azygospores in interspecific
contrasts as anomalies caused by a ‘‘strong sexual ability of the partners.’’
It seems that azygospores, whether formed in intraspecific or interspecific
crosses, are produced through similar processes. They are certainly not rare. In
mating experiments with strains of 19 different species, Schipper (24) found zy-
gospore-like bodies in 150 interspecific pairings. These were often pale, small,
sometimes slightly misshapen, and were produced rather late in comparison with
most intraspecific matings. They were never really abundant and were generally
produced within a wall of interwoven hyphae and incomplete conjugations. After
careful observation of their development it was found that the fusion wall did not
Zygomycetes 81
disintegrate and that only one suspensor was fully developed. Under the scanning
electron microscope the azygospores in interspecific matings resembled normal
zygospores arrested at an early stage of development (25).
Although zygospores are a characteristic of Zygomycetes, they are not
known from many species. There are several reasons for this situation: the cir-
cumstances for zygospore production are more critical than those for sporangio-
spore production, homothallic strains are relatively rare, and the mating types of
heterothallic species are only rarely isolated from the same locality. The germina-
tion of zygospores is slow (often after a resting period), and zygospores are thus
an unlikely source of contamination.
C. Zygospore Production in Pathogenic Species

All types of zygospores mentioned above occur in the spectrum of pathogenic
Mucorales, but for a number of species zygospores are unknown. As the condi-
tions for the production of zygospores are usually critical, Table 2 specifies them
for the species concerned.
Isolates of Rhizopus azygosporus show a striking morphological resemblance
to azygosporic strains in the progeny of Rh. microsporus x Rh. rhizopodiformis.
Schipper et al. (26) found that they produced apparently normal zygospores. Germi-
nating zygospores formed germsporangia. A part of the progeny were azygosporic
strains, with morphological characteristics intermediate between those of the par-
ents. In Rh. microsporus var. chinensis, which also has features intermediate be-
tween var. microsporus and var. rhizopodiformis, the mating ability was found to
be rather poor or absent. In old slants azygospores may occur.
The close relationship between taxa of the Rh. microsporus group was also
confirmed by antigenic studies on vars. microsporus, rhizopodiformis, progeny
of microsporus x rhizopodiformis, and var. chinensis. Rh. microsporus var. chi-
nensis, Rh. azygosporus, and azygosporic isolates obtained from matings between
microsporus x rhizopodiformis show similarity in general morphology, which is
intermediate between those of the parents. Azygosporic strains resulting from
Rhizopus microsporus x rhizopodiformis may lose the ability to produce azygo-
spores or produce them in reduced numbers. Rhizopus azygosporus retains azygo-
spores through careful subculturing with azygosporic material. From these obser-
vations it could be concluded that Rh. microsporus var. chinensis and Rh.
azygosporus may be the results of spontaneous matings of Rh. microsporus var.
microsporus with var. rhizopodiformis.
Absidia blakesleeana, A. corymbifera, Rhizomucor pusillus, and Rh. mi-
crosporus not only share a number of physiological characters, but also morpho-
logical ones; they are all thermotolerant, potentially pathogenic, have an optimal
zygospore production at 30–33°C on malt yeast extract agar (MYA), are capable
of good growth at high sugar concentrations (20–40%), share antigens (27), and
produce azygospores in interspecific matings (A. blakesleeana with A. corymbif-

era and Rh. pusillus, Rh. microsporus with Rh. pusillus).
VI. TAXONOMY
A. Zygomycetes
The division Zygomycota contains two classes: the Trichomycetes with obligate
parasites on arthropods (not considered here) and the Zygomycetes. They are
mainly characterized by their sexual reproduction; the fusion of coenocytic gam-
etangia (zygogamy) results in a thick-walled zygote, the zygospore.
The Zygomycetes include two orders that are of interest to medical mycol-
ogy, Mucorales and Entomophthorales, which differ mainly in asexual, but to some
extent also sexual reproduction. In the Mucorales, nonmotile sporangiospores are
formed in sporangia, or else in the subdivisions of sporangia usually called meros-
porangia when they are elongate and contain sporangiospores in one row, and spor-
angioles when they are globose and contain clusters of a few (or a single) sporangi-
ospores: Single-spored sporangioles occurring in some families are recognized as
conidia when they are discharged in toto and when their membrane is coalescent
with the wall of the spore. The zygospores develop directly from the fused gametan-
gia. The ornamentation and color of the zygospore wall, the equality or inequality
of the size of the suspensors, and the presence or absence of projections originating
from the suspensors may contribute to the definition of families and genera. The
pathogenic Entomophthorales are treated in Chap. 4.
Relevant literature includes refs. 28 and 29.
Key to the Orders of the Zygomycetes

1a. Mycelium more or less regularly septate 2
1b. Mycelium aseptate, except near propagative structures 3
2a. Merosporangia with two spores Dimargaritales
2b. Merosporangia with a single spore Kickxellales
3a. Spores forcibly discharged (see Chap. 4)
Entomophthorales
3b. Spores not forcibly discharged (but compare Pilobolus,
where the sporangium is forcibly discharged) 4
4a. Endomycorrhizal Glomales
4b. Not endomycorrhizal 5
5a. Asexual spores absent Endogonales
5b. Asexual spores present 6
6a. Obligate parasites on invertebrates (except Piptocephali-
daceae) Zoopagales
6b. Not parasitic on invertebrates Mucorales
Zygomycetes 83
B. Mucorales
The hyphae are thin-walled, nonseptate (septa only occurring near reproductive
organs or in old, necrotic hyphae), coenocytic, and 3 to 12 µm wide. The sporan-
giospores are produced in sporangia or sporangioles.
The order Mucorales contains 13 families. Many species are not known to
form zygospores, but their systematic position is easily established by their simi-
lar asexual characteristics.
Most of the Mucorales grow rapidly on culture media. Characteristics are
generally optimally produced on MA, OA, or potato-dextrose agar; they may be
less expressed on common medical media such as Sabouraud dextrose agar
(SDA), maltose agar (MA), or synthetic mucor agar (SMA). The members of
most families are free-living saprophytes in soil, on decaying vegetable matter
such as stale bread and other food, manure from various mammals, and so on,
whereas those of only a few families can be facultative parasites on other fungi
or higher plants. They are not aquatic. Many species, including those capable of
producing disease in humans or mammals, are widely distributed, and their asex-
ual spores are prevalent in the air. Pathogenicity to vertebrates is an exceptional
feature in the life of these fungi and is determined essentially by predispositions
of the host (diabetes, malignant hemopathia, immunosuppression, etc.). In the
host tissues, only a vegetative mycelium is usually formed, the morphology of
which is characteristic of the Mucorales and indistinguishable among all the spe-
cies of the order.
Key to the Families of the Mucorales

1a. Cylindrical multispored merosporangia simulta-
neously produced on terminal swellings; spores in a
single row Syncephalastraceae
1b. Cylindrical multispored merosporangia absent 2
2a. Sporangia present, sporangioles absent 3
2b. Sporangioles present, sporangia present or absent 8
3a. Sporangiophores with greenish metallic luster, tall
(up to 15 cm), unbranched Phycomycetaceae
3b. Sporangiophores without metallic luster 4
4a. Sporangia becoming more or less intactly separated
from sporangiophore (either violently or passively);
sporangial walls apically cutinized Pilobolaceae
4b. Sporangial wall deliquescent or breaking on the spo-
rangiophore 5
5a. Sporangial wall persistent, fracturing into 2–4 seg-
ments along preformed suture(s) Gilbertella
5b. Sporangial wall deliquescent or breaking into numer-
ous small parts, leaving a collar; preformed sutures
absent 6
6a. Sporangia lacking a columella (rudiments some-

times present) Mortierellaceae
6b. Sporangia with a distinct columella 7
7a. Sporangia flask-shaped, with a long neck Saksenaeaceae
7b. Sporangia globose to pyriform Mucoraceae
8a. Sporangia and sporangioles present 9
8b. Sporangia absent 10
9a. Sporangia and sporangioles borne on separate and
distinct sporangiophores; sporangiospores striate Choanephoraceae
9b. Sporangia and sporangioles borne on the same or
morphologically similar sporangiophores; sporangi-
ospores smooth Thamnidiaceae
10a. Sporangioles on sporangiophores Thamnidiaceae
10b. Sporangioles produced on vesicles 11
11a. Sporangioles always monospored 12
11b. At least a number of the sporangioles multispored 13
12a. Sporangiophores without sterile apical branch; spor-
angioles on distinct vesicle Cunninghamellaceae
12b. Sporangiophores terminating with a sterile spine,
sporangioles sympodial, or produced on small vesi-
cles. Chaetocladiaceae
13a. Sporangioles borne on secondary vesicles, which are
borne on branches arising from large, primary vesi-
cles; sporangioles with persistent appendaged walls Radiomycetaceae
13b. Sporangioles borne on primary vesicles; sporangi-
oles dehiscing by fracture from a preformed zone Mycotyphaceae
1. Mucoraceae
All sporangia are globose or pyriform, rarely dumbbell-shaped, contain few to
many sporangiospores, and are provided with a columella. The columella may
or may not show an apophysis. The sporangium membrane is usually not cu-
tinized and is persistent or diffluent; in the latter case it may leave a ‘‘collar.’’
Merosporangia, sporangioles, or conidia are always absent. The morphology of
the sporangium is important for the definition of genera.
Key to the Genera of the Mucoraceae

1a. Homothallic zygospores with extremely unequal suspen-
sors formed on the same aerial hypha. Zygorhynchus
1b. Zygospores when present not formed on a single aerial
hypha. (Be aware of azygospores with one suspensor
only.) 2
2a. Parasitic on fungi. 3
2b. Not growing on fungi. 5
3a. On Zygomycetes, forming galls (facultative parasite). In
Zygomycetes 85
pure culture narrow sporangiophores are produced on

the apices of substrate hyphae, on the natural substrate
on aerial hyphae. (Compare Mucor hiemalis.) Parasitella
3b. On Hymenomycetes. 4
4a. On Agaricales, preferably Mycena. Aerial hyphae with
short, lateral spinelike branchlets. Sporangia
multispored. Spinellus
4b. On Boletales, preferably Boletus. Sporangiophores di-
chotomously branched, producing few-spored sporangio-
les. Syzygites
5a. Sporangiophores always on distinct stolons and rhizoids,
hardly ever on aerial or substrate hyphae. 6
5b. Sporangiophores originating from substrate hyphae or
aerial hyphae. Stolons or rhizoids may also be present. 12
6a. Sporangia globose. 7
6b. Sporangia pyriform or dumbbell-shaped. 8
7a. Apophysis absent. Dark-colored sporangia on un-
branched sporangiophores arising from well-developed
stolons opposite distinct rhizoids.
(Note: Poorly sporulating strains with abundant chlam- Rhizopus
ydospores in the mycelium are sometimes recognized as
Amylomyces.)
7b. Apophysis present, hemispherical. Gongronella
8a. Sporangia dumbbell-shaped. Halteromyces
8b. Sporangia pyriform. 9
9a. Sporangia arising from substrate hyphae. Protomycocladus
9b. Sporangia borne on aerial hyphae. 10
10a. Besides sporangiospores also dark angular spores (co-
nidia) borne singly on branches of aerial mycelium. Chlamydoabsidia
10b. Spores never borne singly. 11
11a. Sporangiophores may arise opposite rhizoids; columella
funnel- or bell-shaped. Apophysomyces
11b. Sporangiophores never opposite rhizoids. Columella glo-
bose to conical, sometimes with projection. Absidia
12a. Species thermophilic, growing at 36°C. Sporangia dark. 13
12b. Species not growing at 36°C. 14
13a. Apophysis present, conspicuous. Thermomucor
13b. Apophysis absent. Sporangia globose, dark, with distinct
columella. Sporangiophores branched, originating from
short aerial hyphae or from stolons with simple or
sparsely branched rhizoids. Rhizomucor
14a. Sporangia hyaline. Actinomucor
14b. Sporangia dark. 15
15a. Sporangiophores arising from aerial hyphae. Apophysis
present or absent. Hyphomucor
Figure 11 Indeterminate circinate sporangiophores of Circinella umbellata.
15b. Sporangiophores arising from the substrate. 16

16a. Sporangiophores circinate, branching below sporangia
(Fig. 11). Apophysis present, short. Sporangia globose,
with persistent wall, breaking at maturity. Collars pres-
ent, long. Columella cylindrical to conical Circinella
16b. Sporangiophores branched or unbranched. Main pattern
not circinate, although short, circinate branches occasion-
ally may occur (Fig. 12). Apophysis absent Mucor (Table 3).
Figure 12 Mucor circinelloides: (a) sporangiophores, (b) columellae, (c) sporangio-

spores.
Table 3 Differential Characteristics of the Genera of Mucoraceae That Include Pathogenic Species
Rhizoids Zygospores
Genus and stolons Sporangiophores Sporangia Columellae Apophysis Sporangiospores when present Figure
Zygomycetes
Rhizopus Present Single or in Globose; gray, Subglobose to Present Angular to glo- Rough; mostly 1, 22
tufts; usually or brown slightly elon- bose orna- between un-
unbranched; gated or pyri- mented; stri- equal sus-
mostly brown form ate to globose pensors
Absidia Present Branched; often Pyriform Hemispherical; Present; conspic- Globose to cy- Smooth to 3, 9
in corymbs; often with uous; conical lindrical; slightly
almost hya- projection(s) smooth roughened;
line with equato-
rial ridges;
suspensors al-
most equal
Rhizomucor Present Monopodially or Globose; gray; Subglobose to Absent Globose to sub- Rough; between 17, 18
sympodially opaque and slightly pyri- globose; equal sus-
branched; glittering form; brown small; smooth pensors
dark brown
Mucor Absent Branched or un- Globose Various forms Absent Globose to cy- Rough; between 4a–d, 7,
branched; (e.g., glo- lindrical equal sus- 13–16
mostly hya- bose, de- pensors
line pressed, pyri-
form,
elongated)
Thermomucor Present Monopodially or Globose; gray; Subglobose to Present Globose to sub- Smooth; suspen- 20, 21
sympodially opaque and slightly pyri- globose; sors unequal
branched; glittering form; brown small; smooth
dark brown
87
Absidia. All sporangia are pyriform. The columella, which is always pro-
vided with a conspicuous, conical apophysis, is usually hemispherical on its top
and often bears one or more projections. The sporangiophores may originate from
stolons in the intervals between rhizoids or from a finely branched aerial myce-
lium. (The latter is the case for the pathogenic species.) In the zygosporogenic
species the suspensors may carry projections enveloping the zygospore, but these
are not found in the pathogenic species, A. corymbifera.
Type species: A. repens
Relevant literature: Hesseltine and Ellis (30–32); Ellis and Hesseltine.
(33,34); Scholer and Müller (35); and Nottebrock et al. (36).
Key to the Species of Absidia [After Schipper (37)]

1a. Colonies 40 mm or less in diameter, in a month at
25°C; suckerlike branches in the substrate myce-
lium (species of uncertain position) 2
1b. Colonies usually filling petri dish (90 mm diame-
ter) in a few days; suckerlike substrate hyphae ab-
sent 3
2a. Homothallic, zygospores present at 25°C A. parricida
2b. Zygospores absent (unknown) A. zychae
3a. Determinate growth of the fertile aerial hyphae,
generally ending in a large pyriform sporangium;
good growth at 36°C 4
3b. Indeterminate growth of the fertile aerial hyphae;
typically no growth at 36°C (subgenus Absidia) 6
4a. Stolons and rhizoids absent; sporangiophores aris-
ing from the substrate (excluded from Absidia)
4b. Stolons and rhizoids present; sporangiophores ar-
ranged in random fashion on the stolons; whorls
or verticils not obvious (subgenus Mycocladus) 5
5a. Sporangiospores subglobose, partly with rough-
ened walls; at 45°C growth insignificant to absent A. blakesleeana
5b. Sporangiospores subglobose to ellipsoid or ellip-
soid-cylindrical, smooth; at 45°C rather good
growth A. corymbifera
6a. Sporangiospores globose or short ellipsoidal 7
6b. Sporangiospores cylindrical or lacrymoid-cuneate 11
7a. Sporangiospores globose-short ellipsoidal or
slightly angular; sporangiophores both of the usual
Absidia-type and single, short sporangiophores in
series along stolons A. repens
7b. Sporangiospores globose; sporangiophores of one
type only 8
Zygomycetes 89
8a. Columellae with projections of intricate shape A. macrospora

8b. Columellae with a single apical projection 9
9a. Sporangia up to 35 µm diam A. californica
9b. Sporangia up to 50 µm diam 10
10a. Young colonies bluish A. coerulea
10b. Young colonies greenish A. glauca
11a. Sporangiospores lacrymoid-cuneate A. cuneospora
11b. Sporangiospores cylindrical with rounded ends 12
12a. Sporangiospores variable in size, 3–6 ⫻ 2–3.5 µm A. heterospora
12b. Sporangiospores up to 5 ⫻ 2.5 µm 13
13a. At 30°C no growth; optimal at 15°C A. psychrophila
13b. At 30°C growth; optimal at 20–24°C
14a. Homothallic zygospores present
(Note: A. anomala may differ in unequal, monoap- A. spinosa
pendiculate suspensors and sporangia larger than
70 µm.)
14b. Zygospores absent 15
15a. Whorls of 5 or more sporangiophores quite
common A. cylindrospora
15b. Whorls of more than 3 sporangiophores rare 16
16a. Sporangiophores unequal in length A. fusca
16b. Sporangiophores equal in length A. pseudocylindrospora
The pathogenic species of Absidia are:
Absidia corymbifera (Cohn) Saccardo & Trotter (Fig. 13)

Syn.: Absidia ramosa (Lindt) Lendner
Mycelium is rapid-growing, very high, and light dirty gray to almost white in
some strains, but comparatively low (3–4 mm), whitish to dark grayish-brown,
depending on the spore production. The complicated pattern with many superim-
posed ramifications bearing sporangia is basically the same in all strains. Stolons
(distinct horizontal hyphae) are narrow, producing radially diffusing hyphae (rhi-
zoids) for adherence to the substratum. Sporangiophores are finely branched, bot-
ryose (grapelike) to racemose (corymb), usually without a septum beneath the
sporangium, and subterminally slightly brownish. Sporangia pyriform, which are
10–120(⫺150) µm in diameter, are transparent and almost colorless when young,
becoming opaque (sometimes glittering) and grayish-brown to greenish-beige;
the sporangium membrane is diffluent. Apophysis is long, and conical, often bear-
ing a collar. Columella are hemispherical to short-ovoidal above the apophysis
and 5–70(⫺85) µm in diameter, often with one to two mamilliform projections.
Sporangiospores are smooth, slightly yellow to greenish, and globose to long-
ellipsoid. The mean diameters in typical ‘‘globose strains’’ are 3.2–4.0 ⫻ 3–3.5
Figure 13 Absidia corymbifera: (a) branching pattern of sporangiophores on stolon,

(b) columellae, (c) sporangiospores.
µm, in ‘‘intermediate strains’’ 3.5–5.0 ⫻ 3.0–3.8 µm, and in ‘‘elongate strains’’

4.0–5.0 ⫻ 2.3–3 µm. Heterothallic. Zygospores are short, ellipsoid, 60–100 ⫻
45–80 µm, thick-walled with very flat projections approximately 10 µm in diame-
ter and one to three equatorial ridges, and bright reddish-brown. The suspensors
are devoid of projections, equal or slightly unequal, the thicker suspensors or
both often with a slightly roughened wall. The maximum temperature of growth
is 48–52°C.
Absidia corymbifera was probably responsible for the first reported case
of human mucor-mycosis. It has been considered the third most frequent agent
after Rh. oryzae and Rh. rhizopodiformis, but Kwon-Chung and Bennett (3) con-
sider it to be very infrequent. The species has been isolated from brain, pulmo-
nary, kidney, and cutaneous infections (e.g., Ref. 38). It has been reported to be
the most frequent agent of mammalian mucormycosis (including bovine mycotic
abortion caused by Mucorales) and was also repeatedly isolated from mucor-
mycoses in birds.
A. hyalospora (Saito) Lendner has been shown to be pathogenic to mice
in axenic experiments (39). Schipper (37) treated this species as a synonym of
A. blakesleeana, but in the neotype (CBS 173.67) the sporangiospores are up to
7(⫺10) µm in diameter, while in typical A. blakesleeana the sporangiospores are
4 to 6 µm (average 5 µm) in diameter.
Apophysomyces. The genus is closely related to Absidia. The sporangio-
phores may arise opposite the rhizoids, however. The pyriform sporangia show
well-defined funnel- to bell-shaped apophysis. There is only one species, A. eleg-
ans Misra et al. (Fig. 14).
Zygomycetes 91
Figure 14 Funnel- to bell-shaped apophyses of Apophysomyces elegans.
The colony is brownish, and the aerial mycelium is sparse. Sporangiophores

develop on the stolons with rhizoids below, straight or slightly curved, un-
branched, brown, 150–250(⫺500) ⫻ 3.5–6 µm. The sporangia are terminal, 20–
60 µm in diameter. The apophysis is funnel- to bell-shaped. The columella are
hemispherical. The sporangiospores are ellipsoid to oblong, (3⫺)5.5–8 ⫻ 4–5
µm. Optimal development is at 36°C. For relevant literature; see Misra et al.
(40).
A. elegans has been reported to cause mucormucosis after contamination
of burn wounds.
Mucor. Sporangiophores are simple or more often branched, originating
from substrate hyphae (stolons absent), and the rhizoids are absent. Sporangia
are globose, and without apophysis. The zygospores are thick-walled and reddish-
brown to dark brown or black, with warty to stellate ornamentation. The suspen-
sors are as a rule opposed and without projections.
Type species: M. caninus

Relevant literature: Schipper (24,41,42)
Members of the genus Mucor were identified as agents of mucormycoses less
frequently than those of Rhizopus, Absidia, and Rhizomucor. Not counting the
obvious errors in diagnosis the names reported are concentrated on M. circinel-
loides, M. hiemalis, and M. ramosissimus. The reports on M. racemosus and M.
indicus are uncertain.
Key to the Species of Mucor [Based on Schipper (24)]

1a. Tallest sporangiophores usually repeat-
edly sympodially branched; sporangia
usually less than 100 µm in diameter;
zygospores or azygospores, when pres-
ent, reddish-brown to dark brown 2
1b. Tallest sporangiophores unbranched or
weakly sympodially branched or at least
some sporangia with a diameter of
more than 100 µm; zygospores or azy-
gospores, when present, dark brown to
black 14
2a. Growth and sporulation at 37°C, growth
at 40°C M. indicus
2b. Poor or no growth at 37°C, no growth
at 40°C 3
3a. Reddish-brown azygospores borne termi-
nally and subterminally on branched
azygophores present in monosporangial
cultures M. bainieri
3b. Reddish-brown azygospores on
branched azygophores absent in mo-
nosporangial cultures 4
4a. Spores hyaline, ellipsoid, 1.5–2 ⫻ 1
µm M. nanus
4b. Spores at least 4 µm long 5
5a. Sporangiospores ellipsoid (1.3 ⬍ Q ⬍
1.6) or subglobose and approximately 4
to 6 µm in diameter, smooth; chlamydo-
spores in sporangia uncommon 6
5b. Sporangiospores broadly ellipsoid
(1.15 ⬍ Q ⬍ 1.3) or globose and up to
8 to 10 µm in diameter, smooth or ver-
rucose; chlamydospores in sporangio-
phores abundant or rare 11
Zygomycetes 93
6a. Colonies low, velvety; recurved sporan-

giophores absent 7
6b. Colonies otherwise; recurved sporangio-
phores present or absent 8
7a. Sporangia blackish, with extremely per-
sistent walls; columellae applanate; spo-
rangiophores with swollen regions M. ramosissimus
7b. Sporangia grayish-brown, with diffluent
walls; columellae mostly subglobose;
sporangiophores of regular width M. zonatus
8a. Colonies grayish; sporangiophores up to
7(⫺10) µm in diameter; sporangia
blackish 9
8b. Colonies brownish; sporangiophores up
to 14(⫺17) µm in diameter; sporangia
brownish to brownish-gray, rarely dark
brown 10
9a. Sporangiospores subglobose M. circinelloides f. janssenii
9b. Sporangiospores ellipsoid M. circinelloides f. griseocyanus
10a. Columellae globose M. circinelloides f. lusitanicus
10b. Columellae obovoid M. circinelloides f. circinelloides
11a. Sporangiophores repeatedly branched in
a sympodial fashion, with long branches
originating just below the preceding spo-
rangium and at acute angles (except oc-
casionally at the top) M. fuscus
11b. Sporangiophores branched in a mixed
sympodial and monopodial fashion, not
as described above 12
12a. Columellae usually with apical projec-
tions; sporangiospores globose to subglo-
bose, brownish, punctate; chlamydo-
spores in sporangiophores rare M. plumbeus
12b. Columellae usually smooth; sporangio-
spores subglobose or broadly ellipsoid,
grayish, generally smooth; chlamydo-
spores in sporangiophores usually abun-
dant 13
13a. Sporangiospores mainly subglobose M. racemosus f. sphaerosporus
13b. Sporangiospores broadly ellipsoid 14
14a. Columellae obovoid to broadly pyri-
form, often 50 µm and more in height;
monopodial branches short M. racemosus f. racemosus
14b. Columellae subglobose, obovoid or ellip-

soid, mostly 20 µm or less in height,
rarely up to 40 µm; monopodial
branches long M. racemosus f. chibinensis
15a. Tall sporangiophores slightly sympodi-
ally branched, branches occurring rather
late; sporangia with a diameter of less
than 80 µm, sporangiospores ellipsoid 16
15b. Tall sporangiophores unbranched except
for an infrequent single sympodial
branch or at least some sporangia with
a diameter of more than 80 µm or spor-
angiospores not ellipsoid 20
16a. Zygospores present in monosporangial
cultures M. genevensis
16b. Zygospores absent in monosporangial
cultures 17
17a. Branches arising just below the black-
ish-brown sporangia, often swollen at
the base; columellae globose; sporangio-
spores cylindrical M. hiemalis f. silvaticus
17b. Branches arising at a longer distance be-
low the yellowish to dark-brown spo-
rangia 18
18a. Sporangia yellowish; columellae glo-
bose; sporangiospores narrowly ellip-
soid M. hiemalis f. luteus
18b. Sporangia brownish to dark brown; colu-
mellae globose and ellipsoid; sporangio-
spores ellipsoid to cylindrical-ellipsoid 19
19a. Sporangiospores mainly cylindrical ellip-
soid M. hiemalis f. corticola
19b. Sporangiospores ellipsoid, sometimes
flattened at one side M. hiemalis f. hiemalis
20a. Young sporangiophores erect 21
20b. Young sporangiophores transitorily re-
curved 46
21a. Tall sporangiophores sympodially
branched 23
21b. Tall sporangiophores unbranched or in-
frequently with a single branch 22
22a. Sporangia often more than 150 µm in di-
ameter 27
22b. Sporangia usually not exceeding 150
µm in diameter 30
Zygomycetes 95
23a. Colonies approximately 1 mm in height;

at least some sporangia over 100 µm in
diameter and having persistent walls;
sporangiospores approximately 8 ⫻ 6
µm M. algariensis
23b. Colonies more than 1 mm in height 24
24a. Sporangiophores of two types: tall and
short, sympodially branched; sporangia
on tall sporangiophores with deliques-
cent walls, sporangia on short sporangio-
phores with persistent walls; sporangi-
ospores ellipsoid mostly 7 ⫻ 4 mm M. saturninus
24b. Sporangiophores either of one type only
or of two types; sporangiospores ellip-
soid, over 7 µm in length or subglobose 25
25a. Sporangiospores ellipsoid M. flavus
25b. Sporangiospores globose or subglobose 26
26a. Sporangiospores globose or subglobose,
4 to 5 µm in diameter, both at 5°C and
20°C M. minutus
26b. Sporangiospores globose or subglobose,
3.5 to 7 µm in diameter, when grown at
20°C, ellipsoid, 5.5–8 ⫻ 3–4 µm when
grown at 5°C M. strictus
27a. Columellae ovoid or conical 28
27b. Columellae obovoid, ellipsoid or pyri-
form 29
28a. Mature sporangia dorsiventrally flat-
tened, often 400 µm and over in diame-
ter; sporangiospores 25 µm and over in
average length; mesophilic; aerial myce-
lium abundant M. plasmaticus
28b. Mature sporangia slightly dorsiventrally
flattened, rarely exceeding 200 µm in di-
ameter; sporangiospores usually 7.5 to
10 µm in length; psychrophilic: poor
sporulation at 20°C; slow superficial
growth at all temperatures M. psychrophilus
29a. Sporangiospores usually ellipsoid, up to
10 µm in length; rapid growth on
cherry agar or acid beerwort agar M. piriformis
29b. Sporangiospores typically cylindrical-
ellipsoid, often over 10 µm in length; re-
stricted growth on cherry agar or acid
beerwort agar M. mucedo
30a. Zygospores or azygospores present in

monosporangial cultures 31
30b. Zygospores or azygospores absent in
monosporangial cultures 32
31a. Sporangiospores bacilliform M. bacilliformis
31b. Sporangiospores globose or broadly el-
lipsoid M. azygosporus
32a. Sporangiospores ellipsoid or cylindrical-
ellipsoid, 8 µm or less in length 33
32b. Sporangiospores ellipsoid, over 12 µm
in length and/or globose 37
33a. Sporangiospores narrow ellipsoid, with
a granule (globule) at each end 34
33b. Sporangiospores ellipsoid, without gran-
ules at the ends 35
34a. Columellae mostly obclavate M. guilliermondii
34b. Columellae globose to subglobose M. subtilissimus
35a. Sporangia less than 100 µm in diameter M. microsporus
35b. Sporangia over 100 µm in diameter
present 36
36a. Colony very dark gray; sporangia blu-
ish-black; columellae mouse-gray or
brownish M. mousanensis
36b. Colony gray; sporangia pale brown or
dark gray; columellae hyaline, with or
without brownish contents M. ucrainicus
37a. Sporangia 75 µm or less in diameter 38
37b. Sporangia often 100 µm or more in di-
ameter 43
38a. Sporangia up to 75 µm in diameter; spo-
rangiophores often 15 µm or more in
width 39
38b. Sporangia not exceeding 60 µm in diam-
eter; sporangiophores not exceeding 10
µm in width 40
39a. Sporangiospores globose, 3.5 to 5.5 µm
in diameter, nonagglutinate M. amphibiorum
39b. Sporangiospores ellipsoid or plano-
convex-ellipsoid, up to approximately
18 ⫻ 10 µm, agglutinate M. odoratus
40a. Sporangiophores 8 µm or less in width;
globose giant cells present 41
40b. Sporangiophores up to 10(⫺15) µm in
width; giant cells absent 42
Zygomycetes 97
41a. Sporangiospores ellipsoid M. zychae var. zychae

41b. Sporangiospores (sub)globose M. zychae var. linnemanniae
42a. Sporangiospores ellipsoid or flattened at
one side M. prayagensis
42b. Sporangiospores subglobose M. sinensis
43a. Sporangiospores extremely variable in
shape and size: ellipsoid, less than 10
µm in length, mixed with much larger,
ellipsoid to globose sporangiospores up
to 30 µm in length M. inaequisporus
43b. Sporangiospores less variable, either el-
lipsoid or (sub)globose 44
44a. Sporangiospores (sub)globose; sporan-
giophores with a sterile, sickle-shaped
branch at the base M. falcatus
44b. Sporangiospores ellipsoid; sporangio-
phores unbranched 45
45a. Columellae ellipsoid-obovoid or glo-
bose; no growth at 37°C M. variosporus
45b. Columellae applanate to elongated ap-
planate or conical; growth at 37°C M. variabilis
46a. Sporangia up to 250 µm or more in di-
ameter 47
46b. Sporangia 200 µm or less in diameter 49
47a. Sporangiospores oblong ellipsoid, up to
40 µm in length M. oblongiellipticus
47b. Sporangiospores ellipsoid, up to 21 µm
long 48
48a. Spores 11.5–16.5 ⫻ 6.8–8.4 µm M. oblongisporus
48b. Spores 12.5–21 ⫻ 7.5–12 µm M. grandis
49a. Sporangia 60 µm or less in diameter M. guilliermondii
49b. Sporangia up to 125 µm and over in di-
ameter 50
50a. Sporangiospores irregularly polyhedral M. tuberculisporus
50b. Sporangiospores ellipsoid or oblong-
ellipsoid 51
51a. Sporangiola absent M. recurvus var. recurvus
51b. A few short, permanently recurved
branches bearing sporangiola with per-
sistent spiny walls present in aging cul-
tures M. recurvus var. indicus
The pathogenic species of Mucor:

Mucor circinelloides Tieghem (with the formae circinelloides, lusitanicus,
griseocyanus, and janssenii; Fig. 12. Growth fast, reaching 7 cm within
2 days. Sporangiophores repeatedly sympodially branched, sometimes
recurved, up to 6 mm tall and 20 µm wide. Sporangia brown or blackish,
usually less than 100 µm in diameter. Sporangiospores subglobose, 4–
6 µm diameter, or broadly ellipsoid to ellipsoid, 4.3–6.8 ⫻ 3.7–4.7 µm,
mostly 5.4 ⫻ 4 µm. Temperature: from 5–30°C good growth and sporu-
lation, at 37°C poor growth and sporulation. M. circinelloides has been
reported from subcutaneous skin lesions and from urine without any evi-
dence of infection. The species was isolated from peritoneal granulomata
of cattle and swine, in fowl, and in ganders. Occasionally identification
may be handicapped by atypical features. The distance between the spo-
rangium and the next lateral branch can be so reduced that the sporangia
seem to be sessile, and furthermore the sporangia often abort and devel-
opment fails. Thin-walled sterile sporangia, terminal swellings, and
swellings in the sporangiophore frequently occur. The sporangia in such
cultures are small, the columellae subglobose to applanate, and the spor-
angiospores tend to be subglobose and variable in size.
M. ramosissimus Samutsevitsch (Fig. 7). Growth relatively slow, reaching
7 cm in 7 days. Sporangiophores repeatedly sympodially branched, up
to 2 mm tall and 18 µm wide. Sporangia yellowish to blackish, globose
to dorsiventrally flattened, up to 75 µm in diameter, with extremely per-
sistent walls. Sporangiospores subglobose to broadly ellipsoid, 4–7(⫺8)
µm in diameter or 5–8 ⫻ 4.5–6 µm. Zygospores unknown. Temperature:
from 5–30°C good growth and sporulation, at 36°C growth extremely
restricted. M. ramosissimus has been isolated from cerebral mucor-
mucosis and chronic mucormucosis of the face (43).
M. indicus Lendner (Fig. 15): Growth fast, reaching 7 cm within 2 days.
Sporangiophores repeatedly sympodially branched, up to 10 mm tall and
14 µm wide. Sporangia brown, up to 75 µm wide. Sporangiospores sub-
globose to ellipsoid, 5.4–6.4 ⫻ 3.8–4.8 µm (at 30°). Zygospores black,
up to 100 µm in diameter, with pointed-stellate ornamentation and un-
equal suspensors. Temperature: from 10–15°C poor growth and no spor-
ulation, from 25–37°C good growth and sporulation, at 40°C growth but
no sporulation. Occasionally strains of M. indicus and M. circinelloides
are rather similar. A second region of profusely branched short sporan-
giophores was never observed in M. indicus, whereas it is often present
in M. circinelloides. Globose columellae predominate in M. indicus,
while in M. circinelloides obovoid columellae are most common. In case
of doubt, the temperature relations can generally be decisive. M. indicus
has been reported from gastric mucormycosis.
Zygomycetes 99
Figure 15 Mucor indicus: (a) sporangiophore, (b) columellae, (c) sporangiospores.
M. hiemalis Wehmer f. luteus (Fig. 16): Growth fast, reaching 7 cm within

2 days. Sporangiophores unbranched at first, then with few branches, up
to 15 mm tall and 14 µm wide. Sporangia yellowish to dark brown, up
to 70 µm wide, walls deliquescent. Sporangiospores narrowly ellipsoid
to cylindrical, 5.7–8.7 ⫻ 2.7–5.4 µm. Zygospores blackish-brown, up to
70 µm in diameter, with low warted ornamentation and equal suspensors.
Temperature: from 5–25°C good growth and sporulation, at 30°C stunted
growth, at 37°C no growth. Mucor hiemalis has been isolated from sub-
cutaneous infections.
M. racemosus Fres. f. racemosus (Fig. 17): Growth fast, reaching 7 cm
within 2 days. Sporangiophores monopodially or sympodially branched,
up to 20 mm tall and 17 µm wide. Chlamydospores abundant to rare in
the sporangiophores. Sporangia hyaline, becoming brownish to brownish
gray, globose, up to 80(⫺90) µm in diameter. Sporangiospores subglo-
bose to broadly ellipsoid 5.5–8.5(⫺10) ⫻ 4–7 µm in diameter or 5.5 to
8 µm in diameter. Zygospores reddish-brown to bright brown, with low
warts and equal suspensors. Temperature: from 5–30°C good growth
and sporulation; maximum temperature 32°C. Considered doubtful by
the maximum temperature of 32°C. Some reports on natural infections
are nevertheless quite convincing: mucormycose in horse, chicken, and
even human infections (pulmonary) in a renal transplant recipient (Table
4).
Rhizomucor. Aerial mycelium is relatively low (up to 2 mm), and is
brownish. Sporangiophores are branched, with simple or weakly branched rhi-
Figure 16 Mucor hiemalis f. hiemalis: (a) sporangiophores, (b) columellae, (c) sporan-
giospores M. hiemalis f. luteus, (d) sporangiophores, (e) columellae, (f) sporangiospores.
Figure 17 Mucor racemosus; (a) sporangiophores, (b) columellae, (c) sporangiospores,

(d) chlamydospores.
Table 4 Differential Characteristics of the Pathogenic Species of Mucor
Maximum
temperature
Species of growth (°C) Sporangiophores Sporangia Columellae Sporangiospores
Zygomycetes
M. circinelloides 36 Sympodially Diameter varying Globose, in larger Ellipsoid; length up

branched; long from 20–80 µm; sporangia ellipsoid to 6–7 µm or sub-
branches erect, membrane dif- globose; 4–6 µm
and short branches fluent in larger but diameter
sometimes circi- rupturing or persis-
nate tent in small spor-
angia
M. ramosissimus 36 Sympodially Up to 75 µm in di- Alpanate; absent in Ellipsoid; length up
branched; erect ameter but mostly in small sporangia to 7–8 µm
with swollen re- smaller; mem-
gions brane extremely
persistent
M. indicus 42 Sympodially up to 75 µm in diam- Subglobose to apla- Subglobose to ellip-
branched; erect eter; membrane nate, rarely elon- soid ⫾ 5.5 ⫻ 4.5
diffluent gate µm
M. racemosus 32 Sympodially or Up to 80–90 µm in Subglobose to pyri- Subglobose or
monopodially diameter; mem- form, often with broadly ellipsoid;
branched; (mixed) brane mostly dif- truncate basis 8–10 µm long;
typically with fluent but persis- smooth or verru-
abundant chlam- tent in smaller culose
ydospores sporangia
M. hiemalis f. luteus 30 Sympodially erect Up to 60(-70) µm in Globose Narrow ellipsoid to
diameter; diffluent nearly fusiform;
3.3–9.5 ⫻ 1.4–4
101
µm
zoids, with comparatively small sporangia (diameter up to 100 µm) with opaque,
glittering walls. Columellae are small, subglobose to slightly pyriform, without
or with a minute apophysis. Sporangiospores are subglobose. Zygospores are
globose, covered with blunt projections, formed in the aerial mycelium between
smooth, isogamous opposite suspensors. Growth starts at 22–24°C, optimal tem-
perature is 37–40°C, and the maximum temperature ⬎50°C.
Type species: Rhizomucor parasiticus (Lucet & Cost.) Schipper
Relevant literature: Schipper (44)
[Note: not included are: Rh. nanitalensis Joshi (45) with deformed sporangio-
spores and Rh. variabilis R.Y. Zheng & G.Q. Chen (46), which does not belong
in Rhizomucor, because it is not thermophilic, has pale colored sporangia, and
the sporangiophores have a different branching pattern.]
Key to the Species of Rhizomucor [on MEA, 36°C, After Schipper (44)]
1a. Poor growth, hardly any sporulation, sporangiophores up to 35
µm wide; osmophilic Rh. tauricus
1b. Good growth and sporulation; sporangiophores up to 15 µm
wide; osmotolerant 2
2a. Zygospores absent or when present more than 50 µm in diam-
eter Rh. pusillus
2b. Zygospores present, less than 50 µm in diameter Rh. miehei
The pathogenic species of Rhizomucor are:

Rhizomucor pusillus (Lindt) Schipper (Fig. 18)
Syn.: Rhizomucor parasiticus (Lucet & Costantin) Schipper
The colony is 1(⫺2) mm high, and brownish. Stolons are up to 17 µm in diameter,
with roughened walls, and are brown. The rhizoids are weakly developed. The
sporangiophores branch in a mixed monopodial-sympodial fashion, are mostly
subterminal, and are up to 11(⫺15) µm in diameter. They are brownish, with
slightly encrusted walls. The sporangia are gray and glittering. They are up to
80(⫺100) µm in diameter, with encrusted walls that rupture at maturity. The
columellae are ovoid to pyriform, up to 45 ⫻ 38 µm, light brown to mouse-gray,
smooth, and rarely with very small protrusions. The collars are poorly defined
to undefined. The sporangiospores are subglobose. A few are ellipsoid, typically
3 to 4 µm in diameter. The zygospores are formed in aerial mycelium. They are
globose to slightly compressed, up to 70 ⫻ 63 µm (including blunt spines). They
are dark brown to black. The suspensors are equal, elongated, and conical. They
are heterothallic and rarely homothallic.
Zygomycetes 103
Figure 18 Rhizomucor pusillus: (a) sporangiophores, (b) columellae, (c) sporangio-

spores, (d) zygospore between suspensors.
Rhizomucor pusillus is a common causative agent of animal mycoses (39).

Artificially invoked mortal mycoses were described as early as 1886 by Lindt
(47). Scholer (39) observed macroscopical changes in the kidneys. The species
was frequently isolated from diseased mammals such as cattle, horses, swine,
and seals (8), causing, for example, bovine mucormycotic abortion. There are
only a few reports on human mucormycosis [lung (48), endocarditis (49), cutane-
ous infections (50,51)].
Rhizomucor miehei (Cooney & Emerson) Schipper (Fig. 19): Differentiat-
ing characters from Rh. pusillus: colony color dirty gray to deep olive,
branching of sporangiophores mostly sympodially, homothallic, zygo-
spores up to 50 µm in diameter. Assimilation of sucrose is negative.
Reports of animal mucormycosis caused by ‘‘zygosporic M. pusillus’’
may actually be Rh. miehei. All available information points to a similar
pathogenic behavior as reported under Rh. pusillus.
C. Rhizomucor tauricus (Milko & Schkurenko) Schipper (Fig. 20): The
species differs from Rh. pusillus in the olivaceous color and weak
branching (due to poor sporulation) and the requirement a of a high (10–
50%) concentration of sucrose (osmophilic). Zygospores are unknown.
In view of its growth at 37°C and above (up to 55°C), the species may
be considered potentially pathogenic (Table 5).
Figure 19 Rhizomucor miehei: (a) sporangiophores, (b) rhizoids, (c) columellae, (d)
sporangiospores, (e) zygospore between suspensors.
Thermomucor Subrahamanyam, Mehrotra & Thirumalachar (Figs. 21, 22).

Colonies are up to 2 mm high. They are grayish-brown to brown, with stolons
up to 20 µm in diameter. The rhizoids are generally simple, up to 200 µm long
and 10 µm wide. The sporangiophores are brownish, up to 40 µm wide, and with
roughened walls. They are repeatedly sympodially branched. The sporangia are
dark brown or gray and glittering, and are up to 125 µm in diameter. Collars are
absent. Columellae are dark brown, hemispherical, up to 85 ⫻ 100(⫺150) µm,
and subtended by a dark brown apophysis. The sporangiospores are subglobose,
hyaline to slightly brown, and smooth. They are 3.5–6(⫺6.5) µm. The zygospores
are borne near the substrate. They are reddishbrown to dark brown, smooth, and
up to 57 ⫻ 51 µm. The suspensors are equal. They are homothallic, with a growth
range of 24–53°C.
Type (and only) species: Thermomucor indicae-seudaticae Subrahama-
nyam, Mehrotra & Thirumalachar
Zygomycetes 105
Figure 20 Rhizomucor tauricus: (a) sporangiophores, (b) columellae, (c) sporangio-

spores.
Relevant literature: Subrahamanyan et al. (52), Schipper (53)

Thermomucor resembles Rhizomucor in regard to the stolons, rhizoids, repeated
branching of the sporangiophores, dark pigment, and high maximum temperature
of growth (55°C), but differs by the presence of a conspicuous apophysis and
by smooth zygospores. Thermomucor is pathogenic to mice, but less so than
Rhizomucor spp. (H. J. Scholer, unpublished data). The species is considered a
potential pathogen.
Rhizopus. The sporangiophores are unbranched, single or usually in clus-
ters, and formed on stolons opposite rhizoids. The sporangia are globose, dis-
tinctly columellate, apophysate, and grayish to brownish. The sporangiospores
Table 5 Differential Characteristics of the Species of Rhizomucor

Species
Rm. pusillus Rm. miehei Rm. tauricus
Maximum tempera- 54–58 54–58 55

ture of growth (°C)
Colony color Hair-brown Dirty gray to deep Gray-olive
olive
Sporangiophores Branched monopodi- Branched Unbranched or
ally (often with branched subter-
subterminal false minally
umbels) or sympo-
dially (mixed)
Columellae Subglobose to Subglobose to Globose to obovoid
slightly pyriform slightly pyriform
Zygospores Almost only in In monosporic cul- Not known
crosses; up to 70 ture; up to 50 µm
µm in diameter in diameter
Thiamine requirement Independent Dependent (stimu- Not tested
lated)
Osmophily No No Marked
are (sub)globose, ellipsoid, or angular. If produced, the zygospores usually have

unequal, more or less globose suspensors. The following three distinct groups
are distinguished (Fig. 1):
1. Rh. stolonifer group: rhizoids relatively long, branched. Sporangio-
phores up to 2000 ⫻ 25 µm; sporangia up to 250 µm in diameter,
mouse-gray to brownish; columellae conical-cylindrical, up to 130 µm
high; sporangiospores angular-subglobose to ellipsoid, with distinct
ridges. No growth at 33°C.
2. Rh. oryzae group: rhizoids relatively long, unbranched. Sporangio-
phores up to 1500(⫺2000) ⫻ 15(⫺20) µm; sporangia up to 200 µm
in diameter, mouse-gray to brownish; columellae conical-cylindrical;
sporangiospores angular-subglobose to ellipsoid, with distinct ridges,
not typical at higher temperatures. Growth at 40°C; no growth at 45°C.
3. Rh. microsporus group: rhizoids small, unbranched. Sporangiophores
up to 500(⫺800) long; sporangia up to 80(⫺100) µm in diameter,
mouse-gray to brownish; columellae variable (Fig. 23); sporangio-
spores angular-subglobose to ellipsoid, with or without ridges, warted
to echinulate. Growth at 45°C.
Type species: Mucor stolonifer Ehrenb.
Relevant literature: Schipper (54); Schipper and Stalpers (55)
Zygomycetes 107
Figure 21 Thermomucor indicae-seudaticae: (a) young sporangiophore, (1) on short

aerial hypha, (2) on stolon with rhizoids; (b) rhizoids; (c) branched sporangiophore; (d)
sporangium with columella; (e) columellae; (f ) sporangiospores.
Figure 22 Thermomucor indicae-seudaticae: smooth zygospores.

Figure 23 Shapes of columellae in Rhizopus microsporus: (a) pyriform, (b) pyriform-

ellipsoid, (c) ellipsoid, (d) (sub)globose to subglobose-conical.
Key to the Species [after Schipper (54); Schipper and Stalpers (55)]
1a. Sporangiophores often more than 1
mm long; rhizoids relatively long,
branched or not; sporangia over 100
µm in diameter; no growth at 45°C 2
1b. Sporangiophores up to 0.5(⫺0.8) mm;
rhizoids short, simple; sporangia up to
100 µm in diameter; usually growth at
45°C; Rh. microsporus group 3
2a. Rhizoids usually branched; sporangia
up to 275 µm in diameter; no growth
at 36°C Rh. stolonifer
2b. Rhizoids simple; sporangia up to
200(⫺240) µm in diameter; growth at
36°C Rh. oryzae
3a. Zygospores or azygospores present in
single-spore isolates 4
3b. No zygospores produced in single-
spore isolates 5
4a. Zygospores present Rh. homothallicus
4b. Azygospores present Rh. azygosporus
5a. Sporangiophores in clusters of up to
9(⫺10) 6
Zygomycetes 109
5b. Sporangiophores in groups of 1–

3(⫺4). Rh. microsporus group 7
6a. Sporangiospores smooth, angular to
broadly ellipsoid, up to 8 µm long Rh. caespitosus
6b. Sporangiospores striate, subglobose to
ovoid, 5.8 to 6.8 µm long Rh. schipperae
7a. Sporangiospores striate, distinctly
angular-ellipsoid Rh. microsporus var. microsporus
7b. Sporangiospores not distinctly striate,
(sub)globose to broadly ellipsoid, but
sometimes angular 8
8a. Sporangiospores at least on average
smaller than 5 µm; radial growth at
50°C (120 hr) at least 35 mm Rh. microsporus var. rhizopodiformis
8b. Sporangiospores larger; radial growth
at 50°C (120 hr) up to 10 mm 9
9a. Sporangiospores of two kinds:
(sub)globose, up to 9 µm in diameter,
and irregular, larger ones Rh. microsporus var. oligosporus
9b. Sporangiospores globose to ellipsoid
or slightly angular, up to 7.5 µm in
diameter Rh. microsporus var. chinensis
The pathogenic species of Rhizopus are:

Rhizopus oryzae Went & Prinsen Geerligs
Syn.: Rhizopus arrhizus Fischer ss auct.
Mycelium is high grayish-yellow to grayish-brown or slightly olive. The rhizoids
are almost hyaline to dark brown, often with a reddish shade. The sporangio-
phores are single or in tufts. They are essentially unbranched, (500⫺)1000–
1500(⫺2000) ⫻ 15–25 µm, yellowish-brown to dark brown, occasionally with
local swellings up to 50 µm wide or bifurcate near the apex. The sporangia are
100–200 µm in diameter. The apophysis is 3–12 µm high. The sporangiospores
are variable, roughly rhomboid, sometimes nearly ellipsoid or subglobose, longi-
tudinally striate, (4⫺)6–8(⫺12) ⫻ (3⫺)4.5–6(⫺8) µm. The zygospores are glo-
bose or laterally flattened, brown to slightly red-brown, (60⫺)80–100(⫺140) µm,
with stellate ornamentation, with very unequal suspensors. Sporangia produced in
the higher temperature range are often atypical.
Rhizopus oryzae is the most common agent of human mucormycosis, and
is responsible for nearly 90% of the cranial (rhinocerebral) form (56) (Table 6).
Rhizopus microsporus van Tieghem: Mycelium yellowish-brown to gray

to dark grayish-brown; rhizoids almost hyaline to pale brown; sporangio-
Table 6 Summary of Diagnostic Characters of Rhizopus Groups

Stolonifer-group oryzae microsporus-group
Rhizoids Complex, well devel- Medium Simple

oped
Sporangiophores 1–3(-4) mm Up to 1(-2.5) mm Up to 0.5(-1) mm
(length)
Sporangia (diameter) Up to 250(-300) µm Up to 160(-240) µm Up to 100 µm
Zygospores (diame- Black, up to 225 µm Brown, up to 140 µm Reddish-brown, up to
ter) 90(-100) µm
Suspensors Approximately equal Unequal Unequal
Maximum growth 33°C 45°C Over 45°C
temperature
phores single or groups of 2 to 4, unbranched, up to 500(⫺800) ⫻ 8–

15 µm, brownish; sporangia up to 80(⫺100) µm in diameter; apophysis
indistinct; sporangiospores variable, roughly rhomboid, and longitudi-
nally striate or irregular or regularly subglobose to ellipsoid, minutely
spinulose, 3 to 9 µm long; zygospores globose or laterally flattened, red-
dish-brown, up to 90(⫺100) µm in diameter, with stellate ornamentation,
with very unequal suspensors.
The species is treated after Schipper (54), thus including Rh. rhizopodi-
formis (Cohn) Zopf, Rh. oligosporus Saito, and Rh. chinensis Saito as
varieties, which are often recognized at the species level in the medical
literature. For differences see Table 7. Rhizopus azygosporus Yuan &
Jong, isolated from tempeh in Indonesia, is considered an azygospore-
producing intermediate between Rh. microsporus var. rhizopodiformis
and var. microsporus.
Rhizopus microsporus ranks second after Rh. oryzae as an agent of human
mucormycosis, being especially responsible for the cutaneous and gas-
trointestinal mycoses forms of the disease (14,57). It is only exception-
ally found in cranial (rhinocerebral) mycoses (58). It is also known to
be hospital-acquired (59). Polonelli et al. (60) conducted antigenic stud-
ies with the Rh. microsporus complex. The azygosporic Rh. azygosporus
has been reported from lethal mucormycosis in premature babies (61).
Rh. microsporus also occurs often in mammals, particularly pigs and
cattle.
Rh. schipperae Weitzman et al.: The species is very close to Rh. mi-
crosporus var. microsporus, mainly differing in the larger groups (up to
10) of sporangiophores per stolon, the abundant production of chlamydo-
spores (globose to ellipsoid, thin- to thick-walled, up to 20 µm in diame-
Zygomycetes 111
ter) on all media, the poor production of sporangia (and thus a paler
color) and negative mating results with the mating types of Rh. mi-
crosporus. The species was isolated from bronchial wash and lung speci-
mens from a patient with myeloma (62) (Table 7).
2. Mortierellaceae
The sporangia are from one- to many-spored and globose. The columella are
absent or very small, and never protrude into the sporangium. The mycelium is
often unusually fine.
Key to the Genera of the Mortierellaceae

1a. Sporangiospores with two vermiform appendages Aquamortierella
1b. Sporangiospores without vermiform appendages 2
2a. Thin-walled sporangia borne in sporocarps Modicella
2b. Sporangia not in sporocarps 3
3a. Sporangia globose 4
3b. Sporangia not globose 6
4a. Sporangiophores arising progressively on specialized fer-
tile hyphae of indeterminate growth Dissophora
4b. Sporangiospores arising from normal vegetative hyphae 5
5a. Colonies velvety, not exceeding 3 mm in height; sporan-
gia mostly ochraceous or vinaceous; odor absent or indis-
tinct Micromucor
5b. Colonies cottony to arachnoid with longer aerial myce-
lium, ascendent or prostrate; sporangia not pigmented;
odor often garliclike Mortierella
6a. Sporangia transversely elongate, cylindrical or sausage-
shaped, with 1–5 apical spines; sporangiophores dichoto-
mously branched Echinosporangium
6b. Sporangia ovoid to fusiform, without apical spines. Spo-
rangiophores with terminal swelling on which a number
of sporangioles develop simultaneously Umbellopsis
Mortierella species rank among the commonest of soil fungi. They are fast grow-
ing and freely sporulating in nature. On 2% MA they often produce abundant
aerial mycelium but few sporangia. On poorer media, such as Soil Extract Agar
or SNA, sporulation is enhanced and much more differentiated. These media,
however, are not suitable to isolate the fungus.
Type species:
Relevant literature: Gams (63), Domsch et al. (64)
112
Table 7 Summary of the Characteristics of the Rhizopus microsporus Group

Rhizopus
microsporus group Colony Sporangiophores Sporangia Collumellae Sporangiospores Temperature
Rhizopus mi- Pale brownish-gray Up to 400 ⫻ 10 Grayish-black; up (Sub)globose to Angular to broadly 46°C: good growth;
crosporus var. µm (mostly swol- to 80 µm conical ellipsoid, up to 50°C: no growth
microsporus len); brownish; 6–5 (7–5) µm;
often in pairs distinctly striate
var. rhizopodi- Dark grayish- Up to 500 ⫻ 8 Bluish to grayish- Distinctly pyriform (Sub)globose; up to 50°C: good growth
formis brown µm; brownish; black; up to 100 columella seem 5(-6) µm in diam-
1–4 together µm indicative for rhi- eter; minutely
zopodiformis but spinulose
absence is not
conclusive
var. oligosporus Pale yellowish- Up to 300 ⫻ 15 Blackish, up to 80– (Sub)globose to (Sub)globose; up to At 40°C: good
brown to gray µm; brownish; 100 µm (av. 50– conical 9 µm in diameter growth and spor-
1–3 together 60 µm) ulating; at 45°C:
restricted growth
var. chinensis Pale brownish-gray Up to 500(-700) ⫻ Blackish; up to Subglobose-conical Globose-ellipsoid; At 45°C good
10(-12) µm; 1–3 75(-80) µm some slightly an- growth, but im-
together gular; up to 7–5 mature; at 50°C:
µm diam no growth
Schipper and Stalpers
Rhizopus homothal- Dark brownish- Up to 850 ⫻ 15 Blackish-gray; up Enlarged conical Angular globose; 46°C: good growth;
licus gray; zygospores µm; brownish; to 100(-125) µm up to 7–5(-8) 50°C: no growth
present 1–3 together µm
Rhizopus azy- Morphologically in-
gosporus termediate be-
Zygomycetes
tween ‘‘mi-
crosporus’’ and
‘‘rhizopodi-
formis’’; azygo-
spores present in
single sporan-
gium isolates
Rhizopus caespito- Gray Up to 200–500 µm Brownish-black up (Sub)globose to Angular to broadly 15–36°C: sporula-
sus ⫻ 10 µm; brown- to 60(-75) µm in conical; ellipsoid; up to tion; 45–50°C:
ish; 1–9 together diameter brownish 6–8 µm growth, no sporu-
lation
Rhizopus schip- Grayish-white 100–460 ⫻ 5–15 Grayish-black; up Subglobose to coni- Subglobose to 23–45°C: good
perae µm; brown on to 80 µm cal; hyaline to ovoid; 5.8–6.8 growth
stolons; single or pale tan ⫻ 4.8–5.8 µm;
in pairs on rhi- (faintly) striate
zoids; up to 10
113
Key to the Sections and Relevant Species of Mortierella [After Gams (63)]
1a. Colonies velvety, not exceeding 3 mm in height;
sporangia mostly ochraceous or vinaceous, often
with a small columella; without distinctive odor. subgen. Micromucor
1b. Aerial mycelium consisting of longer, ascendent,
or prostrate hyphae, white, cottony, or arachnoid;
sporangia usually not pigmented, without or with
a rudimentary columella; mostly with a garliclike
odor. subgen. Mortierella 2
2a. Only chlamydospores present (recognizable as
Mortierella by colony habit and odor) sect. Stylospora
2b. Sporangiophores and sporangia (sporangioles)
present 3
3a. Sporangiophores always unbranched 4
3b. Sporangiophores branched (at least sometimes) 6
4a. Sporangiophores usually exceeding 200 µm in
length Sect. Simplex
4b. Sporangiophores less than 150 µm in length 5
5a. Sporangia, at least partly, many-spored; sporangio-
phores with distinctly widening base Sect. Alpina
5b. Sporangia one-spored; very slender sporangio-
phores arising in dense rows from the aerial hyphae Sect. Schmuckeri
6a. Branches arising mainly from the lower part of the
sporangiophore (basitonous) 7
6b. Branches arising from the middle or upper part of
the sporangiophore (mesotonous or acrotonous) 10
7a. Sporangia containing many, or at least several,
smooth or ornamented spores Sect. Hygrophila
7b. Sporangia one-spored, often ornamented Sect. Stylospora
8a. Sporangia with 1 to 2 spores; sporangiophores
short, with broad base, strongly tapered in the mid-
dle part and arising in dense rows from the aerial
hyphae Sect. Haplosporangium
8b. Sporangia many-spored 12
9a. Branches arising from the uppermost part of the
sporangiophore in clusters from an inflated region,
chlamydospores absent (when chlamydospores
present; Compare M. wolfii under 24a) Sect. Actinomortierella
9b. Branching in another way 13
10a. Sporangiophores racemosely branched with a thick
main stem and thin, short branches; Sect. Mortier-
ella 11
10b. Sporangiophores often bent upwards above an as-
cendent basal part and with a minute columella;
Sect. Spinosa 14
Zygomycetes 115
11a. Spores with reticulate walls M. reticulata

11b. Spores with smooth or granulate walls 12
12a. Sporangia up to five-spored; spores finely warty,
11 to 16 µm in diameter; irregularly lobate chlam-
ydospores present in the agar M. oligospora
12b. Sporangia many-spored; aerial chlamydospores reg-
ularly spinulose 13
13a. Spores smooth-walled, 10 to 12 µm in diameter M. polycephala
13b. Spores finely echinulate, 12 to 15 µm in diameter M. echinulata
14a. Sporangiophores generally not exceeding 200 µm
in length 15
14b. Sporangiophores typically much longer 17
15a. Spores globose, 18 to 25 µm in diameter, with a
double membrane M. acrotona
15b. Spores much smaller, with a single membrane 16
16a. Chlamydospores constantly absent; spores 3 to 4
µm in diameter M. pulchella
16b. Chlamydospores scarcely produced, lemon-shaped,
about 6 µm in diameter; spores 4–7(⫺10) µm in
diameter M. epicladia
17a. Sporangiophores with umbellate branches arising
from the same level; spores planoconvex, 12–14
⫻ 5–7 µm M. umbellata
17b. Sporangiophores with branches inserted at differ-
ent levels; spores different 18
18a. Spores globose to subglobose 19
18b. Spores ellipsoidal to cylindrical 21
19a. Spores not exceeding 4 µm in diameter M. parvispora
19b. Spores larger 20
20a. Sporangiophores often exceeding 1 cm in length;
spores more than 10 µm in diameter M. nantahalensis
20b. Sporangiophores 200 to 800 µm long; spores less
than 10 µm in diameter M. gamsii
21a. Chlamydospores densely covered with blunt
spines; spores 4.0–5.5 ⫻ 2.0–3.0 µm M. fimbricystis
21b. If present, chlamydospores smooth-walled or irreg-
ularly lobate 22
22a. Spores not exceeding 5.5 µm in length; chlamydo-
spores regularly globose, lemon-shaped, or absent 23
22b. Spores larger; chlamydospores bearing irregularly
lobate appendages 24
23a. Chlamydospores absent or tardily produced and
lemon-shaped; sporangiophores 400 to more than
1500 µm long; spores 3.5–4.0(⫺5.0) ⫻ 2.0–2.5
µm M. jenkinii
23b. Chlamydospores abundantly produced, globose, 20

to 60 µm in diameter, thick-walled; sporangio-
phores usually not exceeding 300 µm; spores 3–
4 ⫻ 1.2–2.0 µm M. cystojenkinii
24a. Sporangia leaving a pronounced collarette; spores
with a double membrane M. wolfii
24b. Sporangia not leaving a collarette but with a trace
of a columella; spores with a single membrane M. exigua
Pathogenic Species of Mortierella. Mortierella wolfii Mehrotra & Baijal

(Fig. 24) is characterized by sporangiophores 80 to 250 µm long, branched imme-
diately below the apex, leaving a conspicuous collar after the dehiscence of the
sporangia. The sporangiospores are short-cylindrical, 6–10 ⫻ 3–5 µm. Charac-
teristic chlamydospores are up to 35 µm in diameter, with short, dichotomously
branched outgrowths (amoeboid, stylospores) may be present; The odor is garlic-
like. According to Crisan (65), the maximum temperature of growth is 48°C.
Sporulation is fostered on soil extract agar incubated at temperatures above 25°C,
but some strains are still difficult to identify because of the paucity of spore
production.
Figure 24 Mortierella wolfii (from Gams in Ref. 64): sporangiophores with acrotonous
branching, sporangiospores, and chlamydospores, smooth or with pseudopodiform orna-
mentation.
Zygomycetes 117
M. wolfii is not known with certainty as a human pathogen but was proved
to be a common agent of bovine mycotic abortion in New Zealand (66–70). A
significant number of the cows were also affected by pneumonia caused by the
same fungus. Previously reported cases from New Zealand, which were first at-
tributed to M. alpina and M. zychae, are probably also due to M. wolfii. More
recently, the latter species was also found as an agent of bovine mycotic abortion
in Great Britain and the United States.
M. polycephala has been mentioned as the causal agent of lung mycosis
and mycotic abortion of cattle (64), but as the maximum temperature of growth
of this species is 26°C, this is probably a misidentification; it most likely is M.
wolfii.
3. Cunninghamellaceae
The family contains only one genus, Cunninghamella Matr. It is characterized by
monosporous sporangioles on short pedicels, arising simultaneously on swollen
globose to obovoid vesicles, borne apically on branched or unbranched sporan-
giophores.
Key to the Species of Cunninghamella (71)

1a. Homothallic; branching of conidiophores never verticillate C. homothallica
1b. Heterothallic; branching of conidiophores simple or verti-
cillate 2
2a. Mature colonies grayish due to dark conidia 3
2b. Colonies whitish to yellowish 6
3a. Conidia always globose, echinulate, brown C. phaeospora
3b. At least some conidia not globose, smooth, or echinulate 4
4a. Conidia smooth, globose to ovoid or lacrymoid C. polymorpha
4b. Conidia echinulate, globose to ellipsoid or ovoid 5
5a. Good growth at 42°C C. bertholettiae
5b. No growth at 40°C C. elegans
6a. Colonies powdery because of abundant sporulation; conid-
iophores irregular, pseudoverticillately branched C. echinulata
6b. Colonies not powdery 7
7a. Colonies remaining white; conidia with long echinulae; co-
nidiophores irregularly to cymosely branched C. vesiculosa
7b. Colonies becoming yellowish; conidia with short echinu-
lae; conidiophores simple or verticillately branched C. blakesleeana
Pathogenic Species of Cunninghamella. There has been an ongoing de-

bate if C. elegans Lendner is the correct name for the pathogenic species, which
in a more restricted sense is named C. bertholletiae Stadel (72). Samson (71)
considered them identical (based on morphology only). Weitzman and Crist (73)
reported that clinical isolates and saprophytic strains were morphologically simi-
lar, but incompatible mating partners. Moreover, the clinical isolates grew well
at 42°C, while the other strains did not. They concluded that C. elegans and
C. bertholletiae are distinct species. Elders (unpublished, 1986) confirmed the
morphological similarity. She also found close serological relationships. She dis-
covered that saprophytic isolates did not grow at 40°C, while the clinical isolates
displayed good growth. Their maximum growth temperature was 46°C. Mating
incompatibility was found as indicated by Weitzman and Crist, with one excep-
tion: C. elegans CBS 657.85 (origin unknown), which could be successfully
mated with various strains of both C. bertholletiae and C. elegans.
Cunninghamella bertholletiae Stadel (Fig. 28). Growth is rapid. Myce-
lium is high, light gray to brownish. Hyphae are up to 20 µm wide. Conidiophores
have verticillate or solitary branches. The vesicles are subglobose to pyriform.
The apical ones are up to 40 µm in diameter, and those on the lateral branches
are 10 to 30 µm in diameter. Conidia are globose, ovoid, or ellipsoid, 7 to 11
µm in diameter, or 6–10 ⫻ 9–13 µm, short echinulate, subhyaline, and brownish
in mass. The zygospores, are brown, tuberculate, 18–28 ⫻ 25–35 µm, and hetero-
thallic. C. bertholletiae has been isolated from several cases of mucormycosis
in man.
4. Saksenaeaceae
The family contains only a single genus, Saksenaea Saksena, with only one spe-
cies, Saksenaea vasiformis Saksena (Fig. 25).
The Mycelium is well developed and fast-growing. The sporangia are sin-
gle or more rarely in groups of two, with basal, dichotomously branched rhizoids.
The sporangiophores are erect, 24–65 ⫻ 6.5–9.5 µm. The sporangia are terminal.
They are flask-shaped with a spherical venter, 22–52 ⫻ 16–44 µm, with a long
neck, 54–200 ⫻ 6.5–11 µm, with expands apically, 8–14.5 µm in diameter. The
columellae are distinct, and hemispherical. The sporangiospores are subcylindri-
cal, smooth, and 2.8–4.2 ⫻ 1.5–2 µm.
Maximum temperature: 44°C
Relevant literature: Saksena (74)
When isolated, S. vasiformis does not sporulate on routine mycological media
and thus is likely to be discarded as a contaminant. Sporulation can be obtained
by growing the species on hay infusion agar (20 ml per petri dish) and 28–30°C
in the dark. Padhye and Ajello (75) placed blocks of SDA–agar containing the
fungus on a plate with 20 ml sterile distilled water with 0.2 ml of filter-sterilized
10% yeast extract solution in the dark at 37°C. After 7 days abundant sporulation
was obtained.
The species was recorded to cause cutaneous and subcutaneous infections,
necrotizing cellulitis, osteomyelitis, cranial and rhinocerebral infections, and dis-
Zygomycetes 119
Figure 25 Saksenaea vasiformis (from Ref. 4): sporangia liberating sporangiospores.
seminated infections (3,76,77). In most cases the infection was acquired through
traumatized skin (78).
5. Syncephalastraceae
The family contains one genus with one species and is characterized by the forma-
tion of numerous cylindrical merosporangia with 3 to 18 spores in a single row
on a globose vesicle. The sporangial wall is deliquescent.
Syncephalastrum racemosum Cohn (Fig. 3). The colony is up to 15 mm
high, and is olive gray. The sporangiophores are typically racemosely branched.
They are up to 15 µm in diameter; with terminal vesicles up to 40 µm in diameter.
They are brownish, often subtended by septa and bearing numerous merosporan-
gia and are up to 80 µm in diameter. The merosporangia are cylindrical-clavate,
about 25 ⫻ 5 µm, with 3 to 18 spores (average 7). The sporangiospores are
globose to subglobose, verruculose, and 3 to 5 µm in diameter. The maximum
temperature is 40°C. For relevant literature see Schipper and Stalpers (79).
S. racemosum has once been reported from a cutaneous infection (80) and
as causal agent of bovine mycotic abortion (81).
6. Thamnidiaceae
Sporangia and sporangioles are borne on the same or separate (but morphologi-
cally similar) sporangiospores (or only sporangioles present). Sporangiospores
are thin-walled and smooth.
Key to the Genera [after Benny and Kimbrough (82)]

1a. Sporangia and sporangioles present 2
1b. Sporangia absent, only sporangioles (containing
up to 10 spores) present 9
2a. Sporangioles pyriform to ellipsoid 3
2b. Sporangioles globose to subglobose 4
3a. Rhizoids and stolons present; columella of sporan-
gioles subglobose, ovoid, or ellipsoid Thamnostylum
3b. Rhizoids and stolons absent; columella of sporan-
gioles often slightly constricted or apically broad-
ened (obpyriform) Pirella
4a. Sporangia subtended by a clavate to obovoid
swelling Fennellomyces
4b. Subsporangial swelling absent 5
5a. Both unispored and multispored sporangioles pres-
ent; sporangiole wall verrucose to minutely spinu-
lose Backusella
5b. Unispored sporangioles absent 6
6a. Sporangiole wall distinctly spinulose Kirkomyces
6b. Sporangiole wall smooth to verrucose 7
7a. Sporangioles borne on well-developed vesicles
arising from main axis of sporangiophore Thamnostylum nigricans
7b. Sporangioles not borne on vesicles, arising di-
rectly from the sporangiophore or on lateral
branchlets 8
8a. Sporangioles arising directly from sporangio-
phores or on verticillate branchlets Helicostylum
8b. Sporangioles borne terminally on dichotomously
branched lateral branchlets Thamnidium
9a. Sporangioles obpyriform Thamnostylum
9b. Sporangioles globose to subglobose 10
10a. Sporangioles arising from the tips of the ultimate
branches of the sporangiophores, not subtended
by vesicles Ellisomyces
10b. Sporangioles arising from vesicles 11
11a. Sporangiophores terminally condensed dichoto-
mously branched (appearing lobate); sporangiole
pedicels up to 60 µm long Zychaea
Zygomycetes 121
11b. Sporangiophores terminally unbranched; vesicles

subglobose to broadly clavate; sporangiole pedi-
cels up to 120 µm long Cokeromyces
Pathogenic species. Cokeromyces contains only one species, C. recurva-

tus Poitras (Fig. 4). Growth is rather slow. Sporangioles are produced at the tip
of long, recurved stalks arising from a terminal vesicle of usually unbranched
sporangiophores. Vesicles are 12 to 32 µm wide, with 5 to 30 sporangioles. The
sporangiole pedicel is elongated, becoming up to 120 µm long. The sporangioles
are globose, 8.5 to 12.5 µm, with 12 to 20 spores. The spores are ovoid to irregu-
lar, smooth, and about 4.5 ⫻ 2.5 µm. The zygospores are abundant, globose,
brown, ornamented, and 33 to 55 µm in diameter. They are homothallic.
The species occurs commonly on the dung of small animals, but was once
reported from a case of chronic cystisis (83).
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4
Zygomycetes
The Order Entomophthorales
Shung-Chang Jong
American Type Culture Collection, Manassas, Virginia, U.S.A.
Frank M. Dugan
USDA-ARS Western Regional Plant Introduction Station, USDA,
Pullman, Washington, U.S.A.
I. INTRODUCTION
Numerous species of Entomophthorales cause disease in animals, including in-

sects and other lower animals, but few are documented as causing disease in
humans. Basidiobolus ranarum Eidam (⫽ B. haptosporus Drechsler), B. incon-
gruus Drechsler, and Conidiobolus coronatus (Costanin) Batko are the recorded
agents of disease in humans (1–3).
Consensus on nomenclature and identity of these fungi is not universal. De
Hoog and Guarro utilize Delacroxia coronata as the preferred name for C. coro-
natus and imply a distinction between B. ranarum and B. haptosporus (4). King
avoided applying the name B. ranarum to human-derived isolates (5,6), and Evans
and Richardson applied the name B. meristosporus to human-derived strains (7).
Diseases induced by these agents have received various names, the most
accepted of which are entomophthoromycosis basidiobolae and entomophthoro-
mycosis conidiobolae, or simply basidiobolomycosis and conidiobolomycosis,
for the agents in Basidiobolus and Conidiobolus, respectively (8,1). Infection by
Conidiobolus incongruus is rare (6,9), but is now known from immunocomprom-
ised patients (10). Basidiobolus ranarum occasionally infects humans, and very
rarely other mammals; most cases are confined to tropical regions (11). Children
are the principal victims; infection typically occurs in the body trunk or extremi-
This chapter is based on that by Douglas S. King in the first edition.
127
128 Jong and Dugan
ties (12). Conidiobolus coronatus afflicts humans and other higher mammals,
especially in the tropics. Infection of the nasal passages (rhinoentomophthoro-
mycosis) is typical (4). The majority of case reports are from tropical rainforest
areas in west Africa. Adult agricultural and outdoor workers are the usual victims
(13). Conidiobolus coronatus typically inhabits decaying vegetation. C. incon-
gruus has also been recovered from that substrate. Basidiobolus ranarum is most
commonly isolated from gastrointestinal tracts or dung of amphibians or other
animals, but also from soil or decayed vegetation (1,10).
II. MORPHOLOGY AND CYTOLOGY

OF THE ENTOMOPHTHORALES
A. Propagative and Vegetative Structures
Conidia (asexual spores) are produced singly at the apices of simple or branched
conidiophores. Most species produce two types of conidia, although C. coronatus
can produce four types. Each type of conidium can germinate to produce vegeta-
tive mycelium, or germination may result in production of another conidium.
Except in the genus Massospora, which lacks forcibly discharged conidia, pri-
mary conidia are forcibly discharged from conidiophores arising from vegetative
elements. In Conidiobolus, such discharge is typically effected by pressure devel-
oped within the conidium; release of the conidium is accompanied by eversion
of the conidial wall at the point of attachment to the conidiophore, resulting in
a prominent basal papillum. In Basidiobolus, discharge is effected via pressure
developed within the conidiophore, which is ruptured in the discharge process.
Most genera are uniform with respect to conidial shape, nuclear number, and
mode of discharge of the primary conidia. Primary conidia are phototropic; they
frequently germinate to produce smaller, secondary conidia, which are also pho-
totropic. Some species of Entomophthora and Conidiobolus produce two types
of secondary conidia: One type resembles the primary conidia, while the other
has a different shape (6).
In several species of Entomophthora and Conidiobolus and most species
of Basidiobolus, primary and secondary conidia may also produce capillospores.
These latter conidia, borne atop very long, thin conidiophores, are elongated and
passively discharged. Multiplicative conidia (microconidia) of various sorts typ-
ify some species (e.g., B. microsporous and C. incongruus); villous conidia are
produced by C. coronatus.
Conidia function primarily as disseminating propagules. Chlamydospores
and zygospores serve as resting spores for surviving unfavorable environmental
conditions. Chlamydospores develop from pre-existing vegetative structures by
formation of a thick wall. Zygospores are the result of sexual reproduction; they
are formed after the conjugation of equal or unequal gametangia. Homothallism
The Order Entomophthorales 129
(both gametangia from the same individual) is typical of the order. Azygospores
are formed in some species of Entomophthora; these spores are formed without
the prior production of gametangia (6).
The mycelium (vegetative hyphae) of entomophthorales may be composed
of short segments, which Thaxter termed ‘‘hyphal bodies’’ (14); or true hyphae
may be present. In some species of Conidiobolus, vegetative growth is restricted
to coenocytic mycelium (15).
B. Characters of Modern Families

Classic treatments of the Entomophthorales utilized only one family (Ento-
mophthoraceae), with up to 10 genera (6,16). Humber presented a revised classi-
fication with six families: Entomophthoraceae, Completoriaceae, Ancylistaceae,
Meristacraceae, Neozygitaceae, and Basidiobolaceae (17). Characters of the pri-
mary conidia were foundational for assignment to family: morphology of the
conidium and its papillar region, the number of nuclei and number of wall layers
in the conidium, its mode of discharge, and morphology of conidiogenous cells
and conidiophores. Table 1 provides a synopsis of the principal characters and
genera for each family. Humber provides a more extensive synopsis (17); Hawks-
worth et al. provide a key to families (18).
III. PATHOGENIC GENERA AND SPECIES

A. Basidiobolus
1. Biology
Members of the genus Basidiobolus (Fig. 1) are distinguished primarily by the
conidiophores and manner of discharge of the conidia and the development of the
gametangia for production of zygospores. Globose conidia (Fig. 1a) frequently
produce replicative conidia (Fig. 1b). Forcible discharge results when the swollen
subconidial vesicle of the conidiophore ruptures and contracts. The conidiophore
apex, discharged along with the conidium, usually separates in flight (Fig. 1c).
This discharge of the apex of the vesicle, attached (albeit temporarily) to the
conidium, is among the entomophthorales unique to Basidiobolus (5,6).
Another distinctive character of Basidiobolus is the ‘‘beaked’’ zygospore
(Fig. 1d). Conjugation typically occurs between contiguous segments of the same
hypha (even between cells of a recently divided conidium), but may also occur
between different hyphae. During conjugation, each gametangium produces a
protuberance; after the contents of one gametangium flow into the other, the
zygospore is formed, leaving the beak-shaped protuberances attached to the side
of the spore.
130 Jong and Dugan
Table 1 Families of the Entomophthorales
Entomophthoraceae
Conidiophores simple to dichotomously or digitately branched, with conidiogenous
cells apical on each branch.
Primary conidia unitunicate or bitunicate, forcibly discharged (except Massospora)
by papillar eversion, or by propulsion of conidiophore contents.
Nuclei typically 5–12 µm, staining readily with aceto-orcein or bismark brown; no
prominent nucleolus.
Pathogens of insects or other arthropods.
Batkoa, Entomophaga, Entomophthora, Erynia, Eryniopsis, Furia, Massospora, Pan-
dora, Strongwellsea, Tarichium, Zoophthora.
Completoriaceae
Conidiophores simple, short, with no separate conidiogenous cells.
Conidia unitunicate, forcibly discharged by papillar eversion.
Nuclei large, with condensed, ‘‘granular’’ chromatin at interphase.
Obligate intercellular parasites of fern gametophytes.
Completoria
Ancylistaceae
Conidiophores simple or infrequently branched, bearing a single terminal conidium
per branch.
Primary conidia unitunicate, forcibly discharged by papillar eversion.
Nuclei small (3–5 µm), not condensed (‘‘granular’’) at interphase, not staining
strongly with aceto-orcein or bismark brown.
Saprobes in soil or pathogens of insects or other invertebrates, or facultative para-
sites of vertebrates.
Ancylistes, Conidiobolus, Macrobiotophthora
Meristcraceae
Conidiophores unbranched, each bearing several conidia.
Primary conidia unitunicate, forcibly discharged by papillar eversion or passively dis-
charged.
Nuclei small (3–5 µm), with prominent nucleolus, no significant ‘‘granular’’ nucleo-
plasm in interphase, not staining strongly with aceto-orcein or Bismark brown.
Obligate pathogens of soil invertebrates, especially nematodes and tardigrades.
Ballocephala, Meristacrum, Zygonemomyces.
Neozygitaceae
Conidiophores typically simple, cylindrical to clavate, with apical conidiogenous
cell.
Conidia unitunicate, melanized, multinucleate, with small or truncate papilla, dis-
charged by papillar eversion.
Nuclei small (3–5 µm), condensed chromatin inconspicuous, staining poorly with
aceto-orcein or other nuclear stains.
Obligate pathogens of mites and insects, especially homopterans.
Neozygites, Thaxterosporium.
Table 1 Continued
Basidiobolaceae
Conidiophores simple or seldom branched, with a swollen apex.
Primary conidia unitunicate and uninucleate forcibly discharged with circumsessile
rupture of conidiophore.
Nuclei large (typically ⬎10 µm), with no condensed ‘‘granular’’ chromatin during
interphase, not staining strongly with aceto-orcein or Bismark brown.
Saprobes in soil, colonizers of the guts of amphibians or reptiles, or facultative para-
sites in vertebrates.
Basidiobolus.
With the exception of B. microsporus, species of Basidiobolus produce

capillospores. Capillospores of Basidiobolus are distinguished by a mucilaginous
apex (Fig. 1e). The microspores of B. microsporus are morphologically similar
to capillospores, but differ both developmentally and in size, and they lack an
adhesive apex.
In nature, species of the genus are characteristically associated with the gut
contents of reptiles and amphibians, but most have also been isolated from plant
detritus and can grow on common mycological media (19,20–21). B. microsporus
is associated with rodent dung; B. magnus with decayed plant material (22).
Strains of Basidiobolus exhibit a tendency for loss of diagnostic characters
(conidia and zygospores) when the strains are maintained in culture. Strains
should be preserved in liquid nitrogen vapor as quickly as possible after isolation.
2. Taxonomy
Modern literature on basidiobolomycocis most frequently uses the names Basidi-
obolus ranarum or B. haptosporus. We prefer B. ranarum, with synonyms as
follows (23):
Basidiobolus ranarum Eidam, 1887
⫽ B. meristosporus Drechser, 1955
⫽ B. heterosporus Srinivasan & Thirumalachar, 1965
⫽ B. haptosporus Drechsler, 1947
⫽ B. haptosporus var. minor Srinivasan & Thirumalachar, 1965
Basidiobolus is the sole genus of the Basidiobolaceae (Table 1). In addition
to characters listed in Table 1, the production of ‘‘beaked’’ zygospores (Fig. 1d)
is diagnostic for the genus. The large nuclei, with prominent nucleoli, are also
distinctive (17,24,25,26). King (6) found the traditional morphological and other
criteria used for species separation less than satisfactory: odor and the texture
of the zygospore wall, both primary characters for species separation, were not
132 Jong and Dugan
Figure 1 Basidiobolus ranarum. Bars ⫽ 20 µm. (a) Primary conidium on conidiophore;

(b) replicative conidium on conidiophore arising from another conidium; (c) discharged
conidium and conidiophore apex; (d) mature zygospore with ‘‘beak’’; (e) capillospore
with adhesive material at apex. (Source: Ref. 6.)
consistently reliable, nor was optimum temperature for growth. Kwon-Chung and
Bennett reviewed attempts at species separation via antigenic studies, restriction
analysis of rDNA, and isozyme banding (1). They concluded that ‘‘the problem
of speciation of Basidiobolus is still unresolved,’’ but that ‘‘it is reasonable to
accept B. ranarum as the pathogen that infects man.’’ Species other than those
in the above synonymy are B. magnus, B. philippinensis, and B. microsporus.
King (6) considered B. microsporus—but not B. philippinensis—distinct from
B. haptosporus. He did not place B. ranarum or B. magnus into synonymy with
B. haptosporus (6). Natural infection of mammals other than humans is rare (6).
3. Infection, Diagnosis, and Treatment

Basidiobolus ranarum most probably gains entrance to human tissues via insect
bites and minor trauma (1). Basidiobolomycosis occurs almost exclusively in
children. An enlarged subcutaneous mass is the primary symptom, with the upper
limbs and buttocks most frequently involved (7). Tissue for biopsy should be
recovered from the growing nodules on the border of the subcutaneous masses.
Tissue for biopsy should be inoculated onto culture media without delay; it should
not be kept for any extended time in a refrigerator (27). Biopsy tissue can be
mounted in potassium hydroxide, teased apart, and gently heated prior to micro-
scopic examination; an eosinophilic, sleevelike coating typically surrounds broad
hyphae embedded in the tissue. Sabouraud glucose agar with antibacterial antibi-
otics but without cycloheximide is normally used for isolation. Fast-growing col-
onies are initially whitish, later becoming buff or gray, with a waxy, radiate
surface. Some colonies have a Streptomyces-like odor (1,3,7). Immunofluores-
cence techniques are of growing importance in diagnosis (28,29). Treatment is
with saturated solutions of potassium iodide, or more rarely with ketoconazole
(1,30).
B. Conidiobolus
1. Biology
King extensively described the taxonomy of the genus Conidiobolus (15,31,32).
All species display forcible discharge of conidia via pressure developed within
the conidium. Conidia are produced atop unbranched conidiophores (Figs. 2a,
2b). After discharge, each conidium displays a papillum (Figs. 3c, 3b) because
of eversion of the wall adjacent to the condiophore. Multiplicative conidia (Fig.
3c) are common to several species, including C. incongruus. As many as 40
are produced from a single conidium. They are forcibly discharged but are not
phototropic. Only Conidiobolus coronatus produces villous conidia (Fig. 2d).
These conidia may be produced from primary or replicative conidia by the pro-
duction of the thick wall and villous appendages. Chlamydospores (Fig. 2e) are
134 Jong and Dugan
Figure 2 Conidiobolus coronatus. Bars ⫽ 20 µm. (a) Primary conidium on conidio-

phore arising from vegetative mycelium. Arrows indicate dome-shaped septum separating
conidium and conidiophore that splits during discharge of the conidium; (b) replicative
conidium on conidiophore arising from a globose (either primary or replicative) conidium;
(c) discharged globose conidium; note rounded shape of papillum (see Fig. 3b); (d) rela-
tively thin-walled villose conidium; (e) chlamydospore arising from hyphae in agar.
(Source: Ref. 6.)
Figure 3 Conidiobolus incongruus. Bars ⫽ 20 µm. (a) Almost mature primary conid-
ium on conidiophore arising from vegetative mycelium; (b) globose discharged conidium;
note pointed shaped of papillum (see Fig. 2c); (c) Multiplicative conidia on radially proj-
ecting sterigmata (⫽ microconidiophores) arising from a discharged primary conidium;
(d) mature zygospore. The zygospore lies within the persistent wall of one of the gametan-
gia. The other gametangium is not visible. (Source: Ref. 6.)
reported from several species, including some strains of C. coronatus. Zygo-

spores (Fig. 3d) are known in several species, including C. incongruus, but not
C. coronatus (6,7)
C. coronatus and other members of the genus are consistently isolated from
plant debris, although some, including C. coronatus, are also isolated from the
fruiting bodies of higher fungi or from insects. C. coronatus is cosmopolitan and
frequently isolated, but C. incongruus is infrequently isolated. The tendency to
loss of sporulation ability in culture is less than that of Basidiobolus strains, but
is still a problem: Some human-derived isolates fail to produce villose conidia
(5).
136 Jong and Dugan
2. Taxonomy
There is general consensus on the identity of the agents of conidiobolomycosis
in humans: Conidiobolus incongruus and the more common C. coronatus. Our
nomenclator for these agents follows King (5):
Conidiobolus incongruus Drechsler, 1960
⫽ Conidiobolus gonimodes Drechsler, 1961
Conidiobolus coronatus (Costantin) Bakto, 1964
⫽ Boudierella coronata Costantin, 1897
⫽ Delacroixia coronata (Costantin) Saccardo & Sydow, 1899
⫽ Entomophthora coronata (Costantin) Kevorkian, 1937
⫽ Conidiobolus coronatus (Costantin) Srinivasan & Thirumalachar,
1964
⫽ C. villosus Martin, 1925
Conidiobolomycosis in horses usually results from infection by C. coro-
nata, but C. lamprauges has also been implicated in infection of horses:
Conidiobolus lamprauges Drechsler, 1953
⫽ C. nanodes Drechler, 1955
In classic taxonomy, Conidiobolus was separated from Entomophthora by
the saprophytic habit of the former and the insect-pathogenic habit of the latter.
C. coronata, being at least occasionally isolated from insects, was placed in Ento-
mophthora, but more species of Entomophthora have been cultured, and more
species of Conidiobolus have been isolated from insects. Although characters of
the primary conidiophore (macronematous for Entomophthora versus micronem-
atous for Conidiobolus) were proposed as useful for separation of these genera
(33), King regarded the criteria as unreliable (26). Humber proposed cytological
critera (Table 1) (17). Recent investigations of sterol content suggest separation
of C. coronatus (as Delacroixia coronatus) from other fungi in Entomophthora,
Basidiobolus, and Conidiobolus (34).
Conidiobolus coronatus is a common saprophyte in plant debris, an occa-
sional pathogen of insects, and a well-known pathogen of horses and humans,
especially in the tropics (1,35,36). Although most reports address infections of
horses or humans, C. coronatus has also caused infection in chimpanzees (37,38).
There is evidence that C. coronatus can sporulate in host tissue (39). Recent
research has focused on proteases and lipases, which may be of importance with
regard to pathogenesis (13,40,41).
Conidiobolus incongruus, also a saprophyte in plant debris, is a pathogen
of humans, and has now been diagnosed as causing rhinocerebral and nasal zygo-
mycosis in sheep (42). Infections in humans are very rare (9,43,44). C. lamp-
rauges Dreschler, found in plant debris and occasionally isolated from insects,
has been isolated from a nasopharyngeal nodule of a horse (45). King provided
a key to 26 species of Conidiobolus (32).
3. Infection, Diagnosis, and Treatment

Infection of nasal mucosa and sinuses is typical for conidiobolomycosis; disease
is frequently fatal in immunocompromised individuals (3). The method of entry
is uncertain, although traumatized nasal mucosa is a possibility. Obstruction of
nasal passages, rhinorrhea, and nosebleeds are common symptoms. Edema may
produce conspicuously swollen areas in or adjacent to sinuses: ‘‘Relentless pro-
gression over many years is the rule’’ (1). Culture of tissue from biopsy should
be inoculated onto Sabouraud glucose agar without cycloheximide. Colonies are
fast-growing, glabrous or powdery, white becoming beige to brown, flat, with a
pale reverse (2,7). Immunodiffusion has been successfully employed for serodi-
agnosis (46). Although C. coronatus is susceptible to amphotericin B, and al-
though iodides, ketoconazole, fluconazole, or surgery have resulted in instances
of improvement in individual patients, there is no consistently satisfactory treat-
ment for conidiobolomycosis (1,47,48). Occasional instances of successful ther-
apy include amphoteracin B plus terbinafine (49), fluconazole (50), and ketocona-
zole plus potassium iodide (51).
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of the Fungi. 8th ed. Wallingford, Oxon, UK: CAB International, 1995.
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5
Onygenales
Arthrodermataceae
Dexter H. Howard
UCLA School of Medicine, Los Angeles, California, U.S.A.
Irene Weitzman
Columbia University College of Physicians and Surgeons, New York,
New York, and Arizona State University, Tempe, Arizona, U.S.A.
Arvind A. Padhye
National Center for Infectious Disease, Centers for Disease Control
and Prevention, Atlanta, Georgia, U.S.A.
I. INTRODUCTION
The family Arthrodermataceae of the order Onygenales comprises human and

animal pathogens and a number of nonpathogenic forms isolated from soil and
various keratin-containing substrates such as birds’ nests and animal fur that are
not known to cause infections. The pathogens are referred to as dermatophytes,
a term that designates a large, closely related group of keratinophilic fungi that
cause infections of the skin, hair, and nails. Most of these fungi were originally
described as Hyphomycetes (anamorphs), but several are now known to have a
perfect state (teleomorph) placed in the genus Arthroderma in the family Arthro-
dermataceae of the order Onygenales.
The infections produced by the dermatophytes, called dermatophytoses
(syn. tinea, ringworm), are generally restricted to the nonliving, cornified layers
of the skin and its appendages (1, 1a, 2), but occasionally the dermis and subcuta-
neous tissue may be involved in a form of the disease known as Majocchi’s
granuloma, and pseudomycetomas have been described (1–5). Although the pen-
141
142 Howard et al.
etration of tissue by the dermatophytes is usually quite superficial, adsorption of

products from the fungi leads to sensitization of the host, an event that is mani-
fested by specific delayed hypersensitivity and other sorts of allergic responses.
Generalized cutaneous infections (6, 7), primary invasive cutaneous infections
(8, 9), and more rarely disseminated systemic infections have been reported in
immunocompromised patients (10).
II. HISTORICAL ANTECEDENTS
The development of knowledge about the ringworm fungi parallels that of medi-
cal mycology in general, for these microorganisms were among the first to be
identified as human and animal pathogens (11). The story, with variations, has
been told on several occasions (11–13). Briefly, recognition of the dermatophytes
began in 1837 with the work of Robert Remak, who found arthroconidia and
hyphae in the hair shafts of a child with favus. Remak did not publish his findings
at this time but allowed them to be incorporated into the thesis of a close friend,
Xavier Hube (11, 12). It has been said that Remak did not recognize the impor-
tance of his discovery (12) and that it remained for Lucas Schoenlein to claim
favus as a mycotic disease (14). In doing this Schoenlein was strongly influenced
by Bassi’s classic work on the fungal nature of the muscardine disease of silk-
worms (12). In 1840, Remak published his observations on the ‘‘plant-like na-
ture’’ of the structures he had observed in the hair shafts of patients with favus
(15), and he subsequently showed that the infection was contagious by success-
fully inoculating himself (16). In 1845, he described the fungus of favus more
completely and named it in honor of Schoenlein (12, 17). Some have interpreted
this as an effort to curry favor with Schoenlein in order to obtain a university
chair, an appointment which at that time was generally denied to Jews (18). If
this were the motivation for the somewhat unusual behavior of Robert Remak,
the effort was not totally successful, for he was appointed ‘‘extraordinary profes-
sor’’ (but not full professor, which he wanted) at the University of Berlin. To
his credit he eschewed the obvious alternative of a certificate of baptism to
achieve his goal (12).
David Gruby also refused to buy a university chair with a certificate of
baptism (12). Gruby published his thesis in Vienna and then settled down in
Paris. From 1841 to 1844 he published a series of papers that are generally ac-
knowledged to have established him as the founder of medical mycology (11,
13, 19). In the first two papers of this series Gruby described, independently of
Remak and Schoenlein, the fungal nature of favus (20, 21). The next year he
gave an account of an ectothrix dermatophytosis of the beard (22), and although
he did not name the fungus, it was subsequently described as Microsporon (Tri-
chophyton) mentagrophtes by Charles Robin (23). In 1843, Gruby (24) published
Onygenales: Arthrodermataceae 143
his account of classic epidemic ringworm of the scalp and named the etiologic
agent Microsporum audouinii in honor of Victor Audouin, director of the Paris
Natural History Museum. His final published work on dermatophytes was one
in which he established the etiologic relation of T. tonsurans Malmsten, 1848 to
endothrix dermatophytosis (25). Although extraneous to the subject of this chap-
ter, it should be noted that Gruby also described the fungal nature of thrush (26).
Interestingly, Gruby made no further mycological observations after 1844, but
concentrated on his medical practice in Paris and attended many notables, includ-
ing George Sand, Alexander Dumas (both father and son), Frederic Chopin, Franz
Liszt, and Heinrich Heine (27).
In the middle of the nineteenth century there arose a concept that caused
considerable confusion for a number of years. The work of Anton De Bary (28)
and that of the Tulasne brothers (29) had established the fact of polymorphism
among certain fungi, notably the parasites of cereals. The term polymorphism
denoted that one fungus could appear in different forms in the various stages of
its life cycle. This concept came to be widely and often inappropriately applied
with the disastrous consequences that all fungi and bacteria were thought to be
much the same and simply developmental stages of one another (28).
The result of this extension of the concept of polymorphism to those fungi
in which it played no role for a time obscured the specific etiology of infectious
diseases (30). The one person who was important in rescuing the idea of the
specificity of cause in the mycoses was Raimond Sabouraud, who insisted that
there were different species of dermatophytes, which he placed in four genera:
Achorion, Epidermophyton, Microsporum, and Trichophyton (31). In spite of the
firm foundation laid by Sabouraud, however, the literature on dermatophytes sub-
sequently became confused by investigators who went too far in the opposite
direction and based species identification on small variations in the clinical ap-
pearance of lesions or on slight differences in colonial morphology. The situation
became so muddled that in 1935 some 118 species of dermatophytes were de-
scribed by C. W. Dodge in his monographic consideration of medical mycology
(32).
Emmons proposed the basis for the current taxonomic treatment of the
dermatophytes (33). He adopted a rigorous approach, based on accepted rules of
nomenclature, to the classification of the ringworm fungi and established three
genera (Microsporum, Trichophyton, and Epidermophyton) to contain all the spe-
cies considered legitimate. Emmons’s proposal is widely accepted as valid (1,
2), although alternative schemes are suggested from time to time.
The existence of a sexual phase of growth among certain dermatophytes
was indicated by the observations of a number of early workers. (See Ref. 34
for a review of the literature.) Nannizzi (35) described cleistothecia in a strain
of Sabouraudites (Microsporum) gypseum. He named the isolate Gymnoascus
gypseus. Nannizzi’s work was generally discredited because it was thought that
144 Howard et al.
he dealt with contaminated substrates. His observations were proven to be correct

by Griffin (36) and Stockdale (34), however, and the existence of a sexual form
of growth was also demonstrated for species in the genus Trichophyton by the
results of studies by Dawson and Gentles (37, 38).
The rediscovery of a sexual phase of growth among the dermatophytes led
to a flurry of taxonomic and genetic studies. Few other groups of medically im-
portant fungi have been subjected to review as often as have the dermatophytes
(39, 40). Accordingly, in this chapter the major emphasis is placed on directing
the reader to appropriate reviews in which more comprehensive coverage of a
given topic is presented.
III. DERMATOPHYTE MEMBERS OF THE FAMILY

ARTHRODERMATACEAE
A. General Considerations
1. Definitions
The dermatophytes comprise a large, closely related group of fungi that cause
infections of the hair, skin, and nails (1, 2). The word tinea is used to designate
these infections, known colloquially as ringworm, and an adjective is appended
to that word to indicate the primary area of involvement. For example, tinea
capitis is ringworm of the scalp, tinea barbae is ringworm of the beard, tinea
corporis is ringworm of the general body surfaces, tinea cruris is ringworm of
the groin, tinea pedis is ringworm of the feet, and tinea unguium is ringworm of
the nails (1, 2, 40, 41). The diseases may also be termed collectively dermato-
phytoses (1, 2, 40).
The species of dermatophytes covered in this chapter are listed in Table 1.
2. Identification
The dermatophytes are identified primarily on the basis of their appearance in
materials from the host and in culture (1, 40, 45–47).
The Appearance of Dermatophytes in the Tissues of the Host. Direct ex-
amination of materials from a host infected with a dermatophyte not infrequently
reveals the fungus. Specimens of skin, nails, and hair from suspicious lesions
are placed on a slide in a drop of 10–20% potassium hydroxide (KOH) or in KOH
with Calcofluor white (47). A coverslip is placed over the drop. The preparation is
heated gently over a low flame and flattened before being examined microscopi-
cally. The various species of dermatophytes look alike in skin scales and nail
scrapings (1). The appearance is that of septate, branching hyphae that may break
up into barrel-shaped or rounded arthroconidia (1). Invasion of the hair may be
Table 1 Anamorph-Teleomorph States of the Dermatophytes
Anamorphs Teleomorphs
Epidermophyton Unknown
E. floccosum Unknown
Microsporum Arthroderma
M. audouinii Unknown
M. canis var. canis A. otae
M. canis var. distortum A. otae
M. cookei A. cajetani
M. equinum Unknown
M. ferrugineum Unknown
M. fulvum A. fulva
M. gallinae Unknown
M. gypseum A. gypseum
M. gypseum A. incurvatum
M. nanum A. obtusum
M. persicolor A. persicolor
M. praecox Unknown
M. racemosum A. racemosum
M. vanbreuseghemii A. grubyi
Trichophyton a Arthroderma
T. concentricum Unknown
T. equinum var. equinum Unknown
T. equinum var. autotrophicum Unknown
T. gourvilii Unknown
T. megninii Unknown
T. mentagrophytes var. erinaceae A. benhamiae
T. mentagrophytes var. interdigitale A. benhamiae, A. vanbreuseghemii
T. mentagrophytes var. mentagrophytes A. benhamiae
T. mentagrophytes var. quinckeanum A. benhamiae
T. rubrum Unknown
T. schoenleinii Unknown
T. simii A. simii
T. soudanense Unknown
T. tonsurans var. tonsurans Unknown
T. tonsurans var. sulfureum Unknown
T. verrucosum var. album Unknown
T. verrucosum var. ochraceum Unknown
T. verrucosum var. discoides Unknown
T. violaceum Unknown
T. yaoundei b Unknown
a
T. raubitschekii (42) and T. kanei (43) would appear to be variants of T. rubrum which ‘‘is a highly
variable species’’ (1). T. krajdenii (44) is excluded because some consider it to be T. mentagrophytes
var. nodulare. This variety of T. mentagrophytes has not been validated, but isolates of T. krajdenii
mate with A. benhamiae (A. A. Padhye, unpublished data).
b
Not validly described.
146 Howard et al.
either of the ectothrix or endothrix sort (1). Details of the appearance of different
species of dermatophytes in and on the surface of hair will be given with the
description of individual species. (For further details on direct microscopic exam-
inations see Refs. 40, 45–47.)
Wood’s Light. A Wood’s lamp emits ultraviolet light at approximately
365 nm. Hairs invaded by some species of Microsporum emit a greenish-yellow
fluorescence. Hairs involved by most species of Trichophyton do not fluoresce,
but T. schoenleinii-infected hairs emit a dull bluish-white fluorescence. Epider-
mophyton spp. ordinarily do not invade hair. (See discussion of E. floccosum,
Section IB1a.) Infected skin scales and nails do not fluoresce.
Cultures. Specimens from lesions suspected of being tinea are cultured.
Several recipes for suitable media are available (40). Some variation of glucose
peptone agar with inhibitors is generally employed. One such recipe that is com-
monly used is Sabouraud peptone glucose agar (Emmons’s modification) with
cycloheximide and chloramphenicol (40). The colonial appearance will form a
part of the description to be given for each species considered. (For other useful
culture media see Refs. 40, 45–48.)
Generally the dermatophytes are identified solely on the basis of their colo-
nial and microscopic morphology, and this will be the approach taken in the
description of taxonomy to follow. Physiological tests may also be of great value
in their identification, however. For example, it was recognized early on that the
inability of M. audouinii to grow on rice grains served to distinguish it from M.
canis and M. gypseum (49). The classic work of Lucille Georg and her colleagues
led eventually to the development of a series of nutritional tests that could be
used to augment species recognition among the Trichophyton spp. (40, 41, 46,
47). A series of Trichophyton agars for distinguishing among Trichophyton spp.
is available. (For a discussion see Refs. 40, 47.) Ancillary diagnostic approaches
include (1) the ability to perforate hair in vitro (40), (2) urease production (46),
and (3) growth at various temperatures (e.g., T. verrucosum grows optimally at
37°C). More recently the techniques of molecular biology have been applied to
taxonomic decisions. (For a review see ref. 40.)
3. Epidemiology
The natural occurrence of dermatophytes varies among the species. Typical asso-
ciations are generally recognized, although it is not always easy to describe a
species as belonging to only one category (1, 2). Anthropophilic species are obli-
gate parasites of humans. Zoophilic species infect humans but are predominantly
found on other animals (cattle, cats, dogs, etc). Geophilic species are commonly
found in soil (1, 2). Among the geophilic species are those of Microsporum,
Epidermophyton, and Trichophyton, which have not been found to cause disease.
Table 2 Anamorph-Teleomorph States

of the Saprophytic Arthrodermataceae
Anamorphs Teleomorphs
Epidermophyton Unknown
E. stockdaleae Unknown
Microsporum Arthroderma
M. amazonicus A. borellii
M. boullardii A. corniculatum
M. magellanicum Unknown
M. ripariae Unknown
Microsporum sp. A. cookiellum
Trichophyton Arthroderma
T. ajelloi a A. uncinatum
T. fischeri Unknown
T. flavescens A. flavescens
T. georgiae A. ciferrii
T. gloriae A. gloriae
T. longifusum Unknown
T. mariatii Unknown
T. phaseoliforme Unknown
T. terrestre A. insingulare,
A. lenticularum,
A. quadrifidum
T. vanbreuseghemii A. gertleri
a
There are reports of infections caused by T. ajelloi, but these
isolations remain doubtful.
Because of their morphological similarity to pathogens, they are members of the

Arthrodermataceae and are included in this chapter (Tables 1, 2).
Some of the dermatophytes have a very restricted geographic distribution,
whereas others are cosmopolitan. Examples of each are recorded in Table 3. An
excellent review of this topic has appeared recently (40).
4. Taxonomy
Prior to 1961 the taxonomy of the dermatophytes was based on the anamorphic
forms. The practical needs of the clinical laboratory also involve observations
on this stage for identification. The customary arrangement of a discussion of
the dermatophytes is thus based on the conidia of the asexual phase of growth
even when comprehensive coverage is also given the teleomorphic forms (1, 2,
40, 41, 45).
148 Howard et al.
Table 3 Geographic Distribution of Dermatophytes
Anthropophilic Zoophilic Geophilic
Cosmopolitan
E. floccosum M. canis var. canis M. cookei
M. audouinii M. equinum M. fulvum
T. mentagrophytes M. gallinae M. gypseum
var. interdigitale T. equinum M. nanum
T. rubrum T. mentagrophytes M. persicolor
T. tonsurans var. mentagrophytes T. ajelloi
var. tonsurans T. verrucosum
var. sulfureum var. album
var. ochraceum
var. discoides
Limited
T. concentricum M. canis var. distortum M. praecox
M. ferrugineum T. equinum M. racemosum
T. gourvilii var. autrophicum M. vanbreuseghemii
T. megninii T. mentagrophytes T. simii
T. schoenleinii var. erinacei
T. soudanense var. quinckeanum
T. violaceum
T. yaoundei
In 1934, Emmons proposed that the dermatophytes be classified in three

genera: Epidermophyton, Microsporum, and Trichophyton (33). These genera
were distinguished by the morphology of the large, multiseptate macroconidia
that each produced. A second type of conidia, the small unicellular, spherical,
oval, or pyriform microconidia, was used to some degree for differentiation of
species. The scheme was widely adopted and remains the dominant one in the
field today (1, 2, 40, 41, 44–46).
The morphological features that distinguish the three genera are shown in
Figure 1. The macroconidia of Epidermophyton are smooth-walled, clavate, and
borne in clusters of two or three; those of Microsporum are rough-walled, mostly
spindle-shaped, and usually borne singly; and those of Trichophyton are smooth-
walled, mostly cylindrical, and borne singly.
B. Introduction to the Arthrodermataceae

The morphology of the anamorphic (asexual) states of the dermatophytes
suggested to earlier investigators that these fungi were closely related to the
Gymnoascaceae. The discovery of the teleomorphic (sexual) state of the der-
Figure 1 Spore types in the three genera of dermatophytes. Source: From Ref. 33,
p. 337.
matophytes revealed them to be Ascomycetes, and the appearance of their per-

idial hyphae (hyphae making up the outer covering wall of the fruiting body)
reinforced their affiliation with the Gymnoascaceae. Currah’s thorough study of
the taxonomy of the Onygenales (51), however, resulted in his revision of this
order by creating four families—Onygenaceae, Arthrodermataceae, Myxotricha-
ceae, and Gymnoascaceae—on the basis of expanded characters rather than ear-
lier classification schemes that placed primary emphasis on the characteristics of
the peridium and its appendages. Currah’s major distinction between the families
in the Onygenales was based on ascospore morphology, the peridium and its
appendages, the nature of occurrence of the anamorphs, and the substrate of habi-
tat. The teleomorphic genera of the dermatophytes, Arthroderma and Nannizzia,
were placed in the Arthrodermataceae rather than the Gymnoascaceae.
The genus Nannizzia was created by Stockdale in 1961 (34) to accommo-
date N. incurvata, the newly described teleomorph of Microsporum gypseum.
Although she considered N. incurvata to most closely resemble Arthroderma
150 Howard et al.
curreyi, enough differences in the appearance of the peridium and its appendages
warranted creating a new genus. In 1986, Weitzman et al. (52) concluded after
a careful study of additional species within the genera Arthroderma and Nanniz-
zia that the two taxa were congeneric. The differences originally observed by
Stockdale now overlapped and represented a continuum and no longer warranted
two separate genera. Owing to priority, Nannizzia is a later synonym of Arthrod-
erma. Recent molecular studies by Kawasaki (53) supported the conclusion by
Weitzman et al. (52) that the two are congeneric.
Index to Species of the Arthrodermataceae

I. Pathogenic species
A. Telemorphs (anamorphs)
1. Arthroderma (Microsporum and Trichophyton)
a) A. benhamiae (T. mentagrophytes var. mentagro-
phytes) (T. mentagrophytes var. interdigitale) (T. men-
tagrophytes var. quinckeanum) (T. mentagrophytes
var. erinacei)
b) A. cajetani (M. cookei)
c) A. fulvum (M. fulvum)
d) A. grubyi (M. vanbreuseghemii)
e) A. gypseum (M. gypseum)
f) A. incurvatum (M. gypseum)
g) A. obtusum (M. nanum)
h) A. otae (M. canis var. canis; M. canis var. distortum)
i) A. persicolor (M. persicolor)
j) A. racemosum (M. racemosum)
k) A. simii (T. simii)
l) A. vanbreuseghamii (T. mentagrophytes var. interdigi-
tale)
B. Anamorphs (Teleomorph unknown)
1. Epidermophyton
a) E. floccosum
2. Microsporum
b) M. audouinii
c) M. equinum
d) M. ferrugineum
e) M. gallinae
f) M. praecox
3. Trichophyton
a) T. concentricum
b) T. equinum var. equinum; T. equinum var. autotro-
phicum
c) T. gourvilii
d) T. megninii
e) T. rubrum
f) T. schoenleinii
g) T. soudanense
h) T. tonsurans var. tonsurans; T. tonsurans var. sulfur-
eum
i) T. verrucosum var. ochraceum; T. verrucosum var. al-
bum; T. verrucosum var. discoides
j) T. violaceum
k) T. yaoundei
II. Saprophytic nonpathogens
A. Teleomorphs (anamorphs)
1. Arthroderma (Microsporum and Trichophyton)
a) A. borellii (M. amazonicum)
b) A. ciferrii (T. georgiae)
c) A. cookiellum (Microsporum sp.)
d) A. corniculatum (M. boullardii)
e) A. flavescens (T. flavescens)
f ) A. gertleri (T. vanbreuseghamii)
g) A. gloriae (T. gloriae)
h) A. insingulare (T. terrestre)
i) A. lenticularum (T. terrestre)
j) A. quadrifidum (T. terrestre)
k) A. uncinatum (T. ajelloi)
B. Anamorphs (teleomorph unknown)
a) E. stockdaleae
b) M. magellanicum
c) M. ripariae
d) T. fischeri
e) T. longifusum
f ) T. mariatii
g) T. phaseoliforme
C. Pathogenic Species: Teleomorphs

Arthroderma Currey ex Berkeley emend. Weitzman, McGinnis, Padhye
et Ajello. Mycotaxon 25:505, 1986. Type species: A. curreyi Berkeley.
Outlines of British Fungology, p. 357, 1860 ⫽ Nannizzia Stockdale, Sa-
bouraudia 1:45, 1961.
Ascocarp globose (Fig. 2a), whitish to pale yellow or buff; peridium (Fig.
2b) consisting of a densely packed network of interwoven hyphae; perid-
152 Howard et al.
Figure 2 Morphology of the telemorphic state of the Arthrodermataceae. The examples

shown are those formed by Arthroderma uncinatum (anamorph ⫽ Trichophyton ajelloi).
(a) Ascocarps on hair, ⫻25. (b) Single ascocarp, ⫻240. (c) Peridial hyphae, ⫻450. (d)
Asci (arrow) and ascospores, ⫻1400.
ial hyphae hyaline (Fig. 2c), pale yellow or buff, septate, usually unci-
nately, verticillately, or dichotomously branched; outer peridial hyphal
cell walls echinulate, densely asperulate, or verruculose, with 1–3 slight
to moderate constrictions, or deeply constricted in the middle, symmetri-
cally or asymmetrically dumbbell-shaped; peridial appendages consist
of tightly to loosely coiled spiral hyphae, which may be terminal or lat-
eral, some species producing additional terminal appendages consisting
of elongate, slender, tapered hyphae, or macroconidia; gymnothecial ini-
tials composed of a clavate antheridium surrounded by a coiled ascogo-
nium; asci globose, subglobose, or oval, evanescent, eight-spored (Fig.
2d), 3.9–8 ⫻ 3.5–7.5 µm; ascospores oval (Fig. 2d), lenticular, oblate,
smooth, hyaline, yellow in mass, 1.5–6 ⫻ 1.4–4 µm; homothallic or
heterothallic; Chrysosporium, Microsporum, or Trichophyton ana-
morphs.
I. Arthroderma benhamiae Ajello & Cheng, Sabouraudia 5:232, 1967
A. Conidial state
Trichophyton mentagrophytes (Robin) Blanchard, sensu lato,
Traite de pathologie general, vol. 2, Masson et Cie, Paris, 1896,
p. 811.
Distinctive varieties:
Trichophyton interdigitale Priestley, Med. J. Aust. 4: 417–475,
1917.
Trichophyton quinckeanum (Zopf) MacLeod & Muende, Prac-
tical Handbook of the Pathology of the Skin. 2nd ed. London:
H. K. Lewis & Co. Ltd., 1940, p. 383.
Trichophyton erinacei (Smith & Marples) Padhye & Carmi-
chael, Sabouraudia 7: 178–181, 1969.
B. Description
Heterothallic. Ascocarp globose, pale white, 250–450 µm in
diameter, excluding appendages. Peridial hyphae hyaline, sep-
tate, dichotomously branched, thin-walled. Cells dumbbell-
shaped, echinulate, asymmetrically constricted, 8–12 ⫻ 4.5–
5.2 µm. Appendages of two sorts: (1) elongated, smooth-walled
hyphae tapering at the apex, 60–200 µm long; (2) smooth-
walled, spiral hyphae. Asci globose to ovate, 4.2–7.2 ⫻ 3.6–
6.6 µm, thin-walled, evanescent, eight-spored. Ascospores
hyaline, smooth, oblate, 1.5–1.8 ⫻ 2.5–2.8 µm, yellow in
mass.
Colonies develop rapidly on glucose peptone agar; floc-
cose powdery to granular, cream, yellowish to peach-colored.
Reverse pale yellow to brown to reddish-brown. Microconidia
(Fig. 3a) clavate, borne laterally on undifferentiated hyphae in
154 Howard et al.
Figure 3 Micro- and macroconidia (a, ⫻850) and spiral hyphae (b, ⫻640) of Trichophy-
ton mentagrophytes.
floccose strains or nearly spherical on conidiophores, forming

grapelike clusters in granular strains. Macroconidia (Fig. 3a)
rare or abundant in granular forms, clavate, smooth-walled,
20–50 ⫻ 6–8 µm, with 3–5 septa. Spiral hyphae (Fig. 3b) and
nodular organs may be present.
C. Discussion
This species is a common cause of tinea pedis, tinea corporis,
and tinea unguium. It may also cause tinea barbae and tinea
capitis. The hairs show small-spored ectothrix involvement.
Arthroderma benhamiae also infects cattle, horses, dogs, cats,
sheep, pigs, rabbits, squirrels, monkeys, chinchillas, silver
foxes, laboratory rats, mice, and other animals (1, 2, 41). The
fungus, which is worldwide in distribution, has also been iso-
lated from soil (1, 2, 41). The microconidia of highly granular
cultures are infectious, and laboratory-acquired infections may
occur in humans and animals (54).
The discovery of the sexual phase of growth of T. menta-
grophytes has helped to sort out some of the confusion about
the status of species and varieties of the genus that had been
based on morphological variations. For example, the organism
causing mouse favus, T. quinckeanum, was shown to be con-

specific with T. mentagrophytes by Ajello et al. (55), and many
isolates of T. interdigitale are compatible with tester strains of
A. benhamiae (56). Weitzman (57) has summarized the work
that eventually led to recognition of T. erinacei as a variety of
T. mentagrophytes, and she emphasized that a large number of
tester strains of diverse origin may be necessary in order to
reveal true relationships. Weitzman et al. (58) made a detailed
cytological study of A. benhamiae and have determined the
chromosome number to be 4 (59).
Among the dermatophytes A. benhamiae is well suited
to genetic studies because: (1) under properly controlled condi-
tions it abundantly produces uninucleate microconidia that are
hardy enough for easy manipulation in the laboratory, (2) it is
an important human pathogen, (3) it is easily mated, and (4)
the ascospores are reasonably easy to manipulate and give a
good percentage of viability. Kwon-Chung has critically re-
viewed early reports of reversion of pleomorphic mutants of
dermatophytes and of a putative suppressor mutation in T. men-
tagrophytes (39). Rippon (60) and Rippon and Garber (61) re-
ported a difference in extracellular elastase production and vir-
ulence between one strain each of the (⫹) and (⫺) mating types
of A. benhamiae. Chu-Cheung and Maniotis showed that the
locus controlling elastase segregated independently of the mat-
ing type locus but was closely linked to that governing colonial
morphology (62). Regrettably, the authors did not assess viru-
lence in the segregants. In a recent study the usefulness of re-
striction enzyme analysis of mitDNA was assessed in taxon-
omy of T. interdigitale (63).
II. Arthroderma cajetani (Ajello) Ajello, Weitzman, McGinnis et Pad-
hye, comb. nov., Mycotaxon 25:505, 1986; basionym: Nannizzia
cajetani (as ‘‘cajetana’’) Ajello, Sabouraudia 1:175, 1961
A. Conidial state
Microsporum cookei Ajello, Mycologia 51:69–70, 1959.
B. Description
Heterothallic. Ascocarp globose, pale yellow, 368–686 µm in
diameter. Peridial hyphae hyaline, septate, verticillately
branched. Cells echinulate, thin-walled, and slightly constricted
at the site of septations. Appendages of two kinds: (1) elongate,
slender, tapered, smooth hyphae up to 480 µm in length, 3.6
µm in diameter at the base, 2.4 µm at midlength, and 1.2 µm
at the tip; (2) elongate, smooth-walled, slender hyphae coiled
156 Howard et al.
into spirals. Asci globose to ovate, 6–9 µm in diameter. Asco-

spores ovate, smooth-walled, and golden, 3–3.6 ⫻ 1.8 µm.
Colonies on glucose peptone agar grow rapidly and are
flat and spreading, with a rather powdery surface that is yellow-
ish, buff, or dark tan. The reverse is deep purplish-red. Macro-
conidia are oval to ellipsoidal, thick-walled, and echinulate,
31–50 ⫻ 10–15 µm with six septa. Microconidia obovate, are
produced abundantly.
C. Discussion
This species is geophilic and has a worldwide distribution. The
fungus may be isolated from dogs and monkeys (1, 2). Tinea
corporis has been reported rarely in humans (1, 2). Genetic
analysis of the incompatibility system in N. cajetani has been
accomplished by Kwon-Chung (64).
III. Arthroderma fulvum (Stockdale) Weitzman, McGinnis, Padhye et
Ajello, comb. nov., Mycotaxon 25:505, 1986; basionym: Nannizzia
fulva Stockdale, Sabouraudia 3:120, 1963; syn: N. gypsea (Nan-
nizzi) Stockdale var. fulva (Stockdale) Apinis, Mycol. Paper 96:33,
1964
A. Conidial state
Microsporum fulvum Uriburu, Argent Med I: 563–582, 1909
B. Description
Heterothallic. Ascocarp globose, pale buff, 500–1250 µm in
diameter, excluding appendages. Peridial hyphae are hyaline,
septate, branched hyphae with thin, densely verrucose walls.
Up to four branches arising in succession at the apex of the
same cell; the branches are straight or curved over toward the
ascocarp. Appendages of three kinds: (1) straight, slender,
smooth-walled, septate hyphae up to 250 µm long, 3–4.5 µm
in diameter at the base, tapering to 1.5–2 µm at the tip; (2)
slender, smooth-walled, septate, spiral hyphae, not branched
or much branched, each branch loosely to tightly coiled with
up to 15 turns; (3) moderately thick-walled, verrucose macro-
conidia, usually cylindrical, tapering toward the apex and the
base, or clavate, sometimes ellipsoid or fusiform, 30–50 ⫻
9–12 µm, with up to five septa. Asci subglobose, evanescent,
thin-walled, 5–7 µm in diameter, with eight ascospores. Asco-
spores smooth-walled, lenticular, 1.5–2 ⫻ 2–5.4 µm, yellow
in mass.
Colonies on glucose peptone agar are dense, downy to
granular, pale buff to rosy-buff. The reverse is rosy-buff to
amber. Macroconidia are predominantly cylindrical, slightly ta-
pering toward both ends, and with a rounded apex or clavate,

occasionally ellipsoid or fusiform, 25–28 ⫻ 5–12 µm, with up
to five septa and moderately thick, verrucose walls. Whiplike
appendages (see N. gypsea) are rare. Macroconidia borne singly
or on branched conidiophores. Microconidia 1.7–3.3 ⫻ 3.3–
8.3 µm, clavate, smooth-walled or slightly rough-walled, ses-
sile or on short pedicels, borne laterally along the hyphae. Spi-
ral hyphae similar to those seen on ascocarp are formed abun-
dantly on the vegetative mycelium.
C. Discussion
This species produces tinea corporis and tinea capitis in humans
(1). Hair involvement is of the ectothrix sort but there is no
fluorescence under a Wood’s lamp. The fungus is also reported
from dogs and occasionally from other animals (1, 2). Arthrod-
erma fulvum is a geophilic dermatophyte found throughout the
world.
Genetic studies on the relationship between the enzyme
elastase and the mating type of N. fulva have been reviewed
by Kwon-Chung (39).
IV. Arthroderma grubyi (Georg, Ajello, Friedman et Brinkman) Ajello,
Weitzman, McGinnis et Padhye, comb. nov., Mycotaxon 25:505,
1986; basionym: Nannizzia grubyi (as ‘‘grubyia’’) Georg, Ajello,
Friedman et Brinkman, Sabouraudia 1:194, 1962
A. Conidial state
Microsporum vanbreuseghemii Georg, Ajello, Friedman et
Brinkman, Sabouraudia 1:189–196, 1962.
B. Description
Heterothallic. Ascocarp globose, white to pale buff, 150–600
µm in diameter, exclusive of appendages. Peridial hyphae are
hyaline, septate, branching dichotomously and uncinately,
mostly curved to the outside of the main hypha. Cells are mod-
erately thick-walled, densely echinulate, and moderately con-
stricted in the center. Appendages terminate bluntly or taper
into smooth-walled, loosely coiled hyphae with two to three
turns or into elongate, thin, smooth-walled, loosely coiled, sep-
tate hyphae tapering from 3.0 µm in diameter at the base to
1.5 µm at the tip, with 30–50 tight coils. Macroconidia thick-
walled, cylindrofusiform, multiseptate, and densely echinulate,
borne laterally and terminally on the peridial hyphae. Asci are
globose, thin-walled, evanescent, 4.8–6.0 µm in diameter, with
eight ascospores. Ascospores hyaline, pale yellow, smooth-
walled, ovate, 2.4 ⫻ 3.0 µm, yellow in mass.
158 Howard et al.
Colonies on glucose peptone agar are rapid growing, flat

with powdery to fluffy surface. Surface color white to yellow-
ish; pink to deep rose colonies have also been described (65).
The reverse is light yellow to bright lemon yellow. (Some
strains are colorless.) Macroconidia are numerous, cylindrofus-
iform with thick, echinulated walls, and with 5–12 septa. The
size range is 58.8–61.7 ⫻ 10.4–10.6 µm. Macroconidia are
numerous, pyriform to obovate, 9.2 ⫻ 4.0 µm, borne laterally
on the hyphae, sessile, or on pedicels.
C. Discussion
This species was originally described from tinea lesions in a
Malabar squirrel, a dog, and two humans. The fungus has been
isolated from soil (1, 2). Experimental infections in guinea pigs
resulted in hyphal elements within the hair shafts and large
numbers of spores on the surface of the hair. The hairs did not
fluoresce under a Wood’s lamp. In a natural human infection,
however, the hairs did fluoresce (65).
V. Arthroderma gypseum (Nannizzi) Weitzman, McGinnis, Padhye et
Ajello, comb. nov., Mycotaxon 25:505, 1986; basionym: Gymnoas-
cus gypseus Nannizzi, Atti. Accad. Fisioscr. Siena Med.-fis. 2:93,
1927; syn: Nannizzia gypsea (Nannizzi) Stockdale, Sabouraudia 3:
119, 1963. N. gypsea (Nannizzi) Stockdale var. gypsea, Mycol. Pa-
per 96:32, 1964
A. Conidial state
Microsporum gypseum (Bodin) Guiart & Grigorakis, sensu lato
Lyon Med. 141:369–378, 1928.
B. Description
Heterothallic. Ascocarp globose, pale buff, 500–1250 µm in
diameter, excluding appendages. Peridial hyphae hyaline, pale
buff, septate, branched with thin, densely verruculose walls.
Up to four branches arising in succession at the apex of the
same cells. Appendages are of three kinds: (1) straight, slender,
smooth-walled, septate hyphae, up to 250 µm long, 2.5–4.0 µm
at the base tapering to 1.5–2.0 µm; (2) slender, smooth-walled,
septate, spiral hyphae, rarely branched, 2.5–3.5 µm at the base
tapering to 1.5–2 µm, loosely to tightly coiled with a variable
number of turns; (3) moderately thick-walled, verrucose, ellip-
soid or fusiform macroconidia, 35–55 ⫻ 10–13.5 µm, with up
to five septa. Asci are subglobose, thin-walled, evanescent, 5–
7 µm in diameter, with eight ascospores. Ascospores smooth,
lenticular, 1.5–2 ⫻ 2.5–4 µm, yellow in mass.
Colonies on glucose peptone agar are coarsely granular,
Figure 4 Macroconidia of Microsporum gypseum, ⫻875.
radiating, with arachnoid edge, surface rosy-buff, while the re-

verse is buff to cinnamon. Macroconidia ellipsoid to fusiform,
25–58 ⫻ 8.5–15 µ, with moderately thick, verrucose walls
(Fig. 4). Slender whiplike appendages, up to 30 µm long, borne
on the apical cells of the conidia, are seen in some isolates but
not in others.
Microconidia are clavate, 1.7–3.5 ⫻ 3.3–8.3 µm, unicellular,
smooth-walled, or slightly roughened, sessile or on pedicels,
borne laterally on the hyphae. Spiral hyphae similar to those
borne on the ascocarp are also formed on the vegetative myce-
lium.
C. Discussion
This species is worldwide in distribution. It is found in the soil
and causes an inflammatory tinea corporis and tinea capitis in
humans (66). Small-spore ectothrix involvement of hairs is
seen but generally no fluorescence. The fungus also infects a
variety of animals (1, 2, 41). A genetic study of a phenomenon
of pleomorphism has been made by means of induced mutants
160 Howard et al.
of this fungus and of A. incurvatum (67–69). The results of

such studies have been comprehensively reviewed (39, 57).
VI. Arthroderma incurvatum (Stockdale) Weitzman, McGinnis, Padhye
et Ajello, comb. nov., Mycotaxon 25:505, 1986; basionym: Nanniz-
zia incurvata Stockdale, Sabouraudia 1:46, 1961
A. Conidial state
Microsporum gypseum (Bodin) Guiart & Grigoraksi, sensu lato
Lyon Med. 141:369–378, 1928.
B. Description
Heterothallic. Ascocarp globose, pale buff to yellow, 350–650
µm in diameter, excluding appendages. Peridial hyphae pale
buff, hyaline, septate, verticillately branched with up to five
branches, which usually curve in toward their main axis. Cells
moderately thick-walled, densely asperulate, with one to three
more or less symmetrical constrictions, usually up to 7–8 µm
in diameter but occasionally reaching 11 µm. Free ends numer-
ous and appendages of three kinds: (1) elongate, slender,
smooth-walled, septate, occasionally branched hyphae, up to
300 µm long, 3.0–4.5 µm in diameter at the base tapering to
1.5–2.0 µm in diameter, straight or loosely coiled; (2) elongate,
slender, smooth-walled, septate, occasionally branched hyphae,
2.5–3.5 µm in diameter at the base tapering to 1.5–2.0 µm in
diameter, tightly coiled with up to 30 spirals; (3) macroconidia
thick-walled, asperulate spindle-shaped, 40–57 ⫻ 10–12.5 µm,
with 1–5 septa. Asci globose to ovate, 5–7 µm in diameter,
eight-spored. Ascospores yellow, smooth-walled, lenticular,
2.8–3.5 ⫻ 1.5–2.0 µ.
Colonies grow rapidly on most laboratory media, becom-
ing powdery and buff to cinnamon-brown in color. Reverse
pale yellow, tan or reddish-brown, occasionally red in some
isolates. Microconidia clavate, thin-walled, and sessile on the
hyphae. Macroconidia numerous, large, echinulate, thick-
walled, ellipsoid, with 3–9, commonly 4–6 septa, 8–12 ⫻ 30–
50 µm.
C. Discussion
This species produces tinea corporis or tinea capitis. Hair
involvement is of the small-spored ectothrix type and the hairs
usually do not fluoresce. The fungus, which also infects ani-
mals, is found in soil throughout the world.
A description of the morphogenesis of the sexual phase
of growth was included with the original description (34) and
has been re-examined in detail by Kwon-Chung (71).
VII. Arthroderma obtusum (Dawson et Gentles) Weitzman, McGinnis,

Padhye et Ajello, comb. nov., Mycotaxon 25:505, 1986; basionym:
Nannizzia obtusa Dawson et Gentles, Sabouraudia 1:56, 1961
A. Conidial state
Microsporum nanum (Fuentes, Aboulafia & Vidal) Fuentes,
Mycologia 48:613–614, 1956.
B. Description
Heterothallic. Ascocarp globose, pale buff, 250–450 µm in di-
ameter, excluding appendages. Peridium about 50 µm thick.
Peridial hyphae pale yellow, hyaline, septate, branching mostly
dichotomous, occasionally verticillate, angle between branch
and main hypha usually obtuse. Cells thick-walled, echinulate,
cylindrical, 8–20 µm. Average 13 ⫻ 4–7 µm, may have one or
two slight constrictions. Appendages of two sorts: (1) septate,
smooth-walled, tightly coiled, lateral or terminal; (2) elongate,
slender, septate, hyphae up to 450 µm long, terminal. Asci sub-
globose, thin-walled, evanescent, 5.5–6.5 ⫻ 5–6 µm, eight-
spored. Ascospores hyaline, smooth or finely roughened, lentic-
ular, 2.7–3.2 ⫻ 1.5–2 µm, yellow in mass.
Colonies white, cottony, and spreading, becoming granu-
lar and buff-colored. Reverse red to brown. Macroconidia (Fig.
5) small, clavate, thick-walled, echinulate, generally with one
septum but a few spores may have two or even three septa,
12–18 ⫻ 4–7.5 µm.
C. Discussion
This species is worldwide in distribution. The fungus causes
tinea capitis, in which the hairs display the small-spored ec-
tothrix type of involvement but do not fluoresce. Arthroderma
obtusum is primarily a pathogen of pigs (1, 2).
VIII. Arthroderma otae (Hasegawa et Usui) McGinnis, Weitzman, Pad-
hye et Ajello, comb. nov., Mycotaxon 25:505, 1986; basionym:
Nannizzia otae Hasegawa et Usui, Jpn J Med Mycol 16:151, 1975
A. Conidial state
Microsporum canis Bodin, Arch Parasitol 5:5–30, 1902. Dis-
tinctive variety: Microsporum distortum di Menna & Marples,
Trans Br Mycol Soc 37:372–374, 1954.
B. Description
Heterothallic. Ascocarps are globose, white becoming buff
with age, 280–700 µm in diameter, exclusive of appendages.
Peridial hyphae hyaline, septate, echinulate, constricted at the
cell junction, usually dichotomously but occasionally verticil-
lately branched. The tips of peridial hyphae are blunt and
162 Howard et al.
Figure 5 Macroconidia of Microsporum nanum, ⫻675.
curved toward the ascocarp. Appendages of three kinds: (1)

long (up to 150 µm), slender, straight, smooth-walled, and sep-
tate hyphae; (2) spiral or long, coiled hyphae with 10–15 turns;
and (3) hyphal appendages with thick-walled, echinulate mac-
roconidia (Fig. 6a). Asci are subglobose, thin-walled, evanes-
cent, 5–7 µm in diameter, with eight ascospores. Ascospores
are smooth, lenticular, 2–2.5 ⫻ 2.5–4.8 µm.
Colonies on glucose peptone agar grow fairly rapidly,
forming a cottony or woolly mycelium, white to buff in color.
The reverse is yellow to orange-brown. (The distortum variety
may be colorless.) Macroconidia are numerous, large, thick-
walled, spindle-shaped, echinulate, 8–20 ⫻ 40–150 µm, with
6–15 septa. The macroconidia of the distortum (Fig. 6b) variety
are basically spindle-shaped, but all bend and are grossly
distorted. Their size is roughly similar to the canis variety
but difficult to measure because of the distortions. Microconi-
dia are usually scarce, clavate to elongate, sessile, or on short
pedicels borne laterally on the hyphae. Racquet hyphae, pec-
Figure 6 Macroconidia of Microsporum canis var. canis (a, ⫻875) and M. canis var.
distortum (b, ⫻450).
tinate hyphae, nodular bodies, and chlamydospores may be

seen.
C. Discussion
The fact that A. otae (M. canis var. canis) grows on polished
rice grains whereas M. audouinii does not was one of the earli-
est uses of nutritional tests in identification of dermatophytes
(49). Arthroderma otae (M. canis var. canis) causes tinea capi-
tis, tinea corporis, including primary invasive infections (70),
and rarely, tinea unguium (72). A mixed infection with T. men-
tagrophytes was reported in an AIDS patient (73). The source
of infection is almost invariably an animal. The infected hairs
fluoresce under a Wood’s lamp and the small-spores ectothrix
type of hair shaft involvement is seen. The fungus is zoophilic,
being frequently isolated from dogs and cats and occasionally
from monkeys, horses, rabbits, and pigs. The mating behavior
of A. otae has been studied (74).
A. otae (M. canis var. distortum) species produces tinea
capitis. As with the canis variety, the hairs show ectothrix
involvement and fluoresce under a Wood’s lamp. Cases have
been reported in monkeys, dogs, horses, and pigs (41). The fun-
gus occurs in the United States, Australia, and New Zealand (41).
164 Howard et al.
IX. Arthroderma persicolor (Stockdale) Weitzman, McGinnis, Padhye et

Ajello, comb. nov., Mycotaxon 25:505, 1986; basionym: Nannizzia
persicolor Stockdale, Sabouraudia 5:357, 1967; syn: Nannizzia quin-
ckeani Balabanov et Schick, Dermatol Venerol 9:35–36, 1970
A. Conidial state
Microsporum persicolor (Sabouraud) Guiart & Grigorakis, Lyon
Med. 141:369–378, 1928.
B. Description
Heterothallic. Ascocarps are globose, 350–900 µm in diameter,
pale buff to buff, and composed of a central mass of asci sur-
rounded by a peridium of loosely interwoven hyphae. Peridial
hyphae are hyaline, pale buff, septate, and branching, with up
to four branches arising from the apex of the same cell. The
distal branches curve back over the ascocarp, and the ultimate
branches are composed of up to five cells. Cells of the peridial
hyphae have rather thin, verruculose walls and are 5.5–27.5 µm
long ⫻ 2.5–7.5 µm in diameter at their widest part. The cells
of the distal branches are dumbbell-shaped, with one slight con-
striction in the middle; they are symmetrical in both face and
side views. Appendages are numerous, branched, spirally coiled
hyphae. Asci subglobose, thin-walled, evanescent, 4.5–6.0 ⫻
5.0–7.0 µm in diameter, and contain eight ascospores. Asco-
spores are hyaline, smooth-walled, lenticular, 2.5–3.3 ⫻ 1.6–
2.1 µm, pale yellow.
Colonies on glucose peptone agar are fast-growing, flat at
first, becoming fluffy and folded, yellowish-buff to pink. The
reverse is variable from peach, rose, to deep ochre. Macroconidia
are not abundantly produced, rather thin-walled, clavate to fusi-
form, echinulate at the tip, usually with six septa. Microconidia
produced in abundance, clusters, clavate, fusiform, or globose.
Stalked, elongate, clavate conidia are distinctive.
C. Discussion
This species is worldwide in distribution, producing tinea infec-
tions in wolves, bats, shrews, mice, horses, and more rarely, in-
fection of humans (2, 75). It has also been isolated from soil (79).
At one time the fungus was considered to be a Trichophyton, T.
persicolor, closely related to—or in some individuals’ views,
synonymous with—T. mentagrophytes. (See Ref. 45.) Stockdale
established that A. persicolor was a distinct taxon and expressed
surprise that the conidial state should be Trichophyton (80). She
recorded the slightly verruculose walls of the macroconidia, a
feature used subsequently to support inclusion of the species in
Microsporum as M. persicolor.
X. Arthroderma racemosum (Rush-Munro, Smith et Borelli) Weitz-

man, McGinnis, Padhye et Ajello, comb. nov., Mycotaxon 25:505,
1986; basionym: Nannizzia racemosa Rush-Munro, Smith et Bore-
lli, Mycologia 62:858, 1970
A. Conidial state
Microsporum racemosum, Borelli, Acta Med. Venez. 12:148–
151, 1965.
B. Description
ameter, exclusive of appendages. Peridial hyphae hyaline, sep-
tate, verticillately and dichotomously branched. Up to four
branches arising in succession at the apex of the same cell,
many branches curve back over the ascocarp. Cells are thin-
walled, echinulate, often more or less symmetrically con-
stricted with one to three constrictions, up to 8 µm in diameter.
Appendages of three kinds: (1) slender, smooth-walled, septate
hyphae, 2.3–3.0 µm in diameter at the base tapering to 1.5–
2.0 µm at the tip, coiled with up to 15 turns; (2) straight, slen-
der, smooth-walled hyphae, 3.0–4.0 µm in diameter at the base,
tapering to 1.7–2.0 µm at the tip, up to 380 µm long; (3) deli-
cate, smooth-walled hyphae, 0.7–0.9 µm in diameter, which
are up to 200 µm long. Asci globose or oval, 4.4–5.5 µm in
diameter, containing eight spores. Ascospores hyaline, smooth-
walled, oval, 2.5–3.0 ⫻ 1.2–1.5 µm, yellow in mass. The dis-
tinctive feature of the ascocarps of this fungus is the flagel-
lumlike appendages (45).
Colonies are very fast-growing on glucose peptone agar.
Said to be the fastest-growing dermatophyte (45). The surface
of the colony is powdery cream-white. The reverse is grape-
red. Macroconidia spindle-shaped, echinulate, rather thick-
walled, up to 60 µm long, 5–10 septa. The macroconidia
frequently have a terminal filament. Microconidia are quite
distinctive. They are club-shaped, mostly stalked, and are borne
in large, wandlike clusters (racemes) (81).
C. Discussion
This fungus has been reported from soils of South America and
Romania (82) but is probably worldwide. Infections in humans
have been reported in Illinois (83, 84).
XI. Arthroderma simii Stockdale, Mackenzie & Austwick, Sabouraudia
4:113, 1965
A. Conidial state
Trichophyton simii (Pinoy) Stockdale, Mackenzie & Austwick,
Sabouraudia 4:114, 1965; synonyms: Epidermophyton simii Pi-
166 Howard et al.
noy, C R Soc Biol (Paris) 72:59, 1912; Pinoyella simii (Pinoy)

Castellani & Chalmers, Manual of Tropical Medicine. 3rd ed.
London: Bailliere, Tindall & Cox, 1919, p. 1023.
B. Description
Heterothallic. Ascocarp globose, pale buff to buff, 200–700
µm, thin, excluding appendages. Peridial hyphae pale buff, sep-
tate, with somewhat thin, verruculose walls. Up to three sec-
ondary branches may arise from the apex of the same cell of
a peridial hypha. Distal branches curve over the ascocarp, and
the ultimate branches are rarely composed of more than two
or three cells. Cells of the inner parts of the peridial hyphae
are 12.5–21 ⫻ 3–4.5 µm, not constricted but swelling toward
the apex from which the branches arise, 5–7 µm in diameter.
Cells of the outer branches are asymmetrically constricted, 6.5–
12.5 ⫻ 4.2–6.7 µm at their widest part. Appendages slender,
smooth-walled, septate, spiral hyphae. Smooth-walled, cylin-
drical to fusiform macroconidia are produced by the peridial
hyphae. Asci subglobose, thin-walled, evanescent, 5–6.7 µm
in diameter, eight-spored. Ascospores hyaline, smooth, oblate,
2.5–3.5 ⫻ 1.5–2.5 µm, yellow in mass.
Colonies velvety with a finely granular uniform surface
and a fluffy asteroid margin. The surface is pale buff and the
reverse straw to salmon. Macroconidia numerous, borne termi-
nally on complex branched hyphae, hyaline, smooth-walled,
fusiform, occasionally cylindrofusiform, 30–80 ⫻ 6–11 µm,
with 4–7 (up to 10) septa. In older cultures the macroconidia
become constricted at the septa as a result of an increase in the
diameter of individual cells forming intercalary, thick-walled
chlamydospores. Microconidia rare in young cultures, more
abundant in old cultures, clavate to pyriform, 2–6.5 ⫻ 1.5–4
µm, sessile or on short pedicels, and borne singly along the
sides of the hyphae. Spiral hyphae are moderately abundant on
the vegetative mycelium of old cultures.
C. Discussion
Arthroderma simii is of restricted geographic distribution, as
most of the cases have been reported in or from India (41, 45);
also reported from Brazil and Guinea (45). The fungus pro-
duces tinea in monkeys, poultry, and dogs (85). Several human
cases have been reported, and the type of hair involvement is
endothrix (1, 2). The organism also occurs in the soil (1). Some
workers have considered A. simlii and A. persicolor to be geo-
philic (1, 87, 88) while others consider them to be zoophilic
(2, 86). Both species have been isolated from soil and from the
hair of animals that did not display lesions (79, 87).
A fascinating attribute of A. simii is that it induces a form
of stimulated growth of the mycelium of several different spe-
cies of dermatophytes (89). This stimulation occurs in strains
of mating type opposite that of the tester A. simii type. The
interaction, of course, never goes on to formation of fertile as-
cospores within asci except in opposite mating types of A. simii
itself, but the observation of stimulated growth has been used
to presume the juxtaposition of opposite mating types. In some
instances isolates of species not known to engage in a sexual
form of growth have been identified as being of a single mating
type. For example, all isolates of T. rubrum were shown in
this manner to be the (⫺) mating type (90). Such observa-
tions strengthen the argument that the reason certain species
cannot be induced to reveal a sexual phase of growth is that
they are all of one mating type. Thus, if virulence were associ-
ated with mating type, one could imagine the evolutionary dis-
appearance of a mating type. This would be especially likely
to happen if the environment were highly restricted. It is inter-
esting to reflect on the fact that only one of the anthropophilic
species (A. vanbreuseghemii) has been shown to have a perfect
state.
The chromosome number of A. simii is 4 (59), and the in-
compatibility system has been subjected to a comprehensive
study (91). At one time it was suggested that A. simii was one of
the ‘‘three’’ teleomorphs of the T. mentagrophytes complex (92).
Although this concept has been reiterated on one recent occasion
(93), it would appear that the results of careful studies affirm the
distinctness of T. simii as the single anamorph of A. simii (57, 94,
95). The relationships are, however, close (57, 95).
XII. Arthroderma vanbreuseghemii Takashio, Ann Soc Belge Med Trop
53:427, 1973
A. Conidial state
Trichophyton mentagrophytes (Robin) Blanchard, Traite de pa-
thologie generale, vol. 2. Paris: Masson et Cie, 1896, p. 811.
B. Description
Heterothallic. Ascocarps are globose, 300–650 µm in diameter,
straw- or buff-colored. Peridial hyphae are hyaline or slightly
buff, septate, interwoven, dichotomously branched, and thin-
walled. Cells dumbbell-shaped, echinulate, asymmetrically con-
stricted, 8–12 ⫻ 4.5–5.2 µm. Appendages of two sorts: (1) elon-
168 Howard et al.
gate, smooth-walled hyphae tapering at the apex, 60–200 µm

long; (2) smooth-walled spiral hyphae with up to 15 turns. Asci
globose to ovate, 4.2–7.2 ⫻ 3.6–6 µm, thin-walled,
evanescent, with eight ascospores. Ascospores are hyaline,
smooth, oblate, 1.5–1.8 ⫻ 2.5–2.8 µm, yellow in mass.
Colonies described under A. benhamiae.
C. Discussion
This species is the second perfect state of the T. mentagrophytes
complex. (See A. benhamiae.) The differences are the larger
ascospores of A. vanbreuseghemii and no interspecific crosses.
Mating studies in the complex have been comprehensively re-
corded by Takashio (94). Variations in the colonial morphology
have been presented (94). The disease pattern and distribution
have been described under A. benhamiae.
D. Pathogenic Species: Teleomorphs Unknown

Epidermophyton Sabouraud, Arch Med Exp Anat Pathol 19:754–762,
1907; This genus is characterized on the basis of the macroconidia, which are
6–10 ⫻ 8–15 µm in size, clavate, smooth-walled, with 0–4 (usually 2–3) septa.
No microconidia are found. The type species is Epidermophyton floccosum (Harz)
Langeron & Milochevitch, 1930.
XIII. Epidermophyton floccosum (Harz) Langeron & Milochevitch, Ann
Parasitol Hum Comp 8:495–497, 1930
A. Description
Colonies. The fungus grows slowly on glucose peptone agar.
The colonies have a velvety to powdery surface that is gently
folded in a number of radiating furrows and is khaki-yellow in
color. The reverse is yellow to tan. Usually, isolates mutate
early and almost invariably, with the production tufts of white,
sterile hyphae that soon overgrow the entire colony. Macroco-
nidia (Fig. 7) clavate, smooth, fairly thick-walled, with 0–4
(usually 2–3) septa; the macroconidia are borne in characteris-
tic clusters of twos and threes. Microconidia are not produced.
Chlamydospores, racquet hyphae, and nodular bodies are often
abundantly produced.
B. Discussion
This species is worldwide in distribution. The fungus produces
tinea pedis and tinea cruris. There are rare reports of tinea ungu-
ium (41) and one report of tinea capitis (96). A dog has been
reported to have yielded E. floccosum, but animals are generally
not infected (41).
Figure 7 Macroconidia of Epidermophyton floccosum, ⫻475.
Microsporum Gruby, C. R. Acad. Sci. (Paris) 17:301–303, 1843. The genus

is characterized on the basis of the macroconidia, which are large, thick-walled,
echinulate, fusiform to obovate conidia with 1–15 septa, generally borne singly.
Smaller one-celled microconidia are produced on short pedicels or are sessile on
the hyphae. Pectinate hyphae, racquet hyphae, nodular bodies, coils, and chla-
mydospores may be present. Type species is Microsporum audouinii Gruby,
1843.
XIV. Microsporum audouinii Gruby, C R Acad Sci (Paris) 17:301–303,
1843; synonyms: Sabouraudites (Microsporum) langeronii Van-
breuseghem, Ann Parasitol Hum Comp 25:409–417, 1950; Mi-
crosporum rivalieri Vanbreuseghm, Sabouraudia 2:215–224, 1965
A. Description
Colonies on glucose peptone agar grow rather slowly, form a
matted, velvety surface with straggly edges, and are light tan
to grayish-white in color. The reverse is buff-salmon to orange-
brown. Macroconidia are rarely found in cultures. When ob-
served they are distorted and very irregular with thick, echinu-
late, or smooth walls. Some may be nearly spindle-shaped. The
number of septa is from 2 to 8. Microconidia, when present,
are sessile or borne on short pedicels along the hyphae, clavate,
and single-celled. Racquet hyphae, pectinate hyphae, nodular
bodies, and chlamydospores may be present and are not infre-
170 Howard et al.
quently the only microscopic structures visible. As pointed out

earlier, the poor growth of M. audouinii on rice grains serves
to distinguish the species from M. canis (A. otae) and M. gyp-
seum (A. gypsea and A. incurvata). Also, M. audouinii does
not perforate hair whereas M. canis does.
B. Discussion
This species produces classic, epidemic tinea capitis in chil-
dren. The hairs show ectothrix involvement and fluoresce under
a Wood’s lamp. Rare in the United States and Europe, mainly
present in Africa, Rumania, and Haiti (2). Microsporum audou-
inii is one of the forms of ringworm most susceptible to griseo-
fulvin therapy, and the incidence of disease has been remark-
ably reduced wherever the drug is used. A few animal
infections have been reported, but M. audouinii is clearly an-
thropophilic (41).
XV. Microsporum equinum (Delacroix & Bodin) Gueguen, Les champig-
nons parasites de l’homme et des animaux domestiques, Josephy
Van In & Cie, Lierre, 1904, p. 144
A. Description
Colonies on glucose peptone agar are downy to powdery, pale
buff to pinkish-buff. The reverse is yellow to buff-amber. Mac-
roconidia elliptical to fusiform, thick-walled, echinulate, 18–
62 ⫻ 5–13.5 µm, with 4–8 septa and borne terminally in clus-
ters (97, 98). Microconidia sessile or on short pedicels,
pyriform to clavate, 3–8 ⫻ 2–3.5 µm. Spiral hyphae and chla-
mydospores have been seen (97). The description of this spe-
cies in the early literature (97) varies in detail from that more
recently published (98).
B. Discussion
This species is now included in most standard lists of Mi-
crosporum spp. (1, 2, 40). It was originally reported in Europe,
Java, and Uruguay (99), and is said to cause tinea of horses.
Small-spored ectothrix involvement is described (97). The fun-
gus is said to infect humans (98). The results of a recent study
support the view that M. equinum is a distinct species and not
a synonym of M. canis (98).
XVI. Microsporum ferrugineum (Ota), Bull Soc Pathol Exot 15:588–596,
1922; synonym: Trichophyton ferrugineum (Ota) Langeron & Milo-
chevitch, Ann Parasitol Hum Comp 8:422–436, 1930
A. Description
Colonies on glucose peptone agar are slow-growing, heaped
with many deep furrows, glabrous and waxy, deep yellow to
orange in color. Reverse is buff to brownish. A white velvety

cover may form over the colony. Sectoring occurs with great
variations in color intensities. Macroconidia are rarely ob-
served. Such structures have been reported when isolates are
cultured on a potato dextrose charcoal agar or diluted Sabour-
aud agar (41, 91, 100), however. The macroconidia so gener-
ated were typical for the genus Microsporum (i.e., spindle-
shaped, echinulate, thick-walled, 40.5–47 ⫻ 8.5–10.5 µm, with
2–8 septa). The mycelium comprises hyphae, some of which
have prominent cross walls (‘‘bamboo’’ hyphae). Chlamydo-
spores may be seen.
B. Discussion
The fungus causes tinea capitis in children. Hair involvement
is of the small-spored ectothrix sort. The hairs fluoresce under
a Wood’s lamp. The organism is strictly anthropophilic (41)
and is found in middle Europe, Asia, the former USSR, and
Africa (41).
XVII. Microsporum gallinae (Megnin) Grigorakis, Ann Dermatol Syphilol
10:18–53, 1929; synonym: Trichophyton gallinae Silva & Benham,
J Invest Dermatol 18:432–472, 1952
A. Description
Colonies on glucose peptone agar grow moderately rapidly, are
flat at first with radial folds. Edges of the colonies may be irreg-
ular. The reverse of the colony is deep strawberry-red. The pig-
ment diffuses into the medium. Macroconidia relatively thin-
walled, echinulate at the tips, 6–8 ⫻ 15–50 µm, frequently
curved, with 2–10 septa. Microconidia pyriform to clavate,
smooth-walled, borne laterally on the hyphae.
B. Discussion
This species is a very rare incitant of tinea capitis, tinea corpo-
ris, and tinea cruris in humans. The ectothrix type of hair
involvement is seen. More typically seen as a cause of tinea in
chickens and other fowl (1, 41). The distribution is worldwide.
XVIII. Microsporum praecox Rivalier, Ann Inst Pasteur 86:276–284, 1953
A. Description
Colonies on glucose peptone agar are moderately fast-growing,
cream to yellowish-tan; powdery, with a yellowish-orange re-
verse. There is a report of an isolate from a case of tinea capitis
whose macroscopic appearance differed from that of the origi-
nal description by Rivalier (77). Macroconidia long, narrow,
lanceolate, with fairly thick, roughened walls, 60–65 ⫻ 8–10
µm, with 6–9 septa. Microconidia are absent.
172 Howard et al.
B. Discussion.
This species was isolated from a pustular, vesicular tinea lesion
on the wrist from an adult (45), from tinea capitis (77), from
saprophytic sites associated with horses (76), and from a tinea
capitis in a patient with sickle cell anemia (78).
Trichophyton Malmsten, Trichophyton tonsurans harskararde Mogel, 1845
(transl.), Arch Anat Physiol Wiss Med 1–19, 1848.
The genus is characterized on the basis of the macroconidia, which are elongate,
clavate, to fusiform, generally thin-walled, smooth, and have 0–10 septa. The size
ranges from 8–50 ⫻ 4–8 µm. Smaller one-celled microconidia are usually pro-
duced. These cells are globose, 2.5–4 µm, or clavate, to pyriform, and 2–3 ⫻ 2–
4 µ. Spiral hyphae and chlamydospores may be produced. The type species is Tri-
chophyton tonsurans Malmsten, 1845.
XIX. Trichophyton concentricum Blanchard, Traite de pathologie generale.
vol. 2. Paris; Masson et Cie, 1895, pp. 811–926
A. Description
Colonies on glucose peptone agar are very slow-growing and are
raised deeply folded, smooth and white, becoming cream to
amber or brown, and covered with short, gray hyphae. The re-
verse is cream to brown. Macroconidia not observed. Microscopi-
cally similar to T. schoenleinii. The hyphae are swollen, bearing
chlamydospores and aborted branches. Microconidia have been
reported (45).
B. Discussion
The fungus causes tinea imbricata [i.e., a form of tinea corporis
in which concentric rings of scales occur on the skin (1, 2)]. The
disease has been reported in the South Pacific islands, Guatemala,
southern Mexico, and central Brazil (1).
XX. Trichophyton equinum (Matruchot & Dassonville) Gedoelst, Les
champignons parasites de l’homme et des animaux domestiques. Jo-
seph Van In & Cie, Lierre, 1902, p. 88
A. Description
Colonies grow rapidly on glucose peptone agar and are flat, de-
veloping folds with age, surface white, cottony with yellow color
in the edge around the new growth. The reverse is bright yellow,
becoming dark pink to brown with age. Macroconidia are rarely
found in cultures grown on glucose peptone agar but may be
produced on wort agar (101). They are slightly clavate, thin-
walled, smooth, with 3–4 septa. Microconidia are nearly spheri-
cal to slightly pyriform, borne laterally along the hyphae, sessile
or on short pedicels and in clusters.
B. Discussion
This species—considered by some as being synonymous with or
a variety of T. mentagrophytes—was re-evaluated as a separate

taxon (101). The fungus is rare in humans and commonly causes
ringworm in horses (102). The large-spored ectothrix type of hair
involvement is seen. There is a variety recorded from New
Zealand and Australia with less exacting nutritional requirements
than is customary. This form has been referred to as T. equinum
var. autotrophicum (103).
XXI. Trichophyton gourvilii Cantanei, Bull Soc Pathol Exp 26:377–381,
1933
A. Description
Colonies on glucose peptone agar are waxy with a heaped and
folded surface, becoming velvety with age. The color is la-
vender to deep garnet-red. The reverse is reddish-purple.
The colonies are said to resemble those of T. violaceum
and T. soudanense (41). Macroconidia smooth-walled, cylin-
drical. Microconidia pyriform, smooth-walled, borne laterally
on the hyphae. Not all isolates produce micro- and macro-
conidia.
B. Discussion
This fungus is found in Africa (1), where it produces tinea capi-
tis and tinea corporis. Endothrix type of hair involvement is
observed.
XXII. Trichophyton megninii Blanchard, Traie de pathologic generale. vol.
2. Paris: Masson et Cie, 1896, pp. 811–926; synonym: Trichophyton
kuryangei Vanbreuseghem & Rosenthal, Ann Parasitol Hum Comp
36:797–802, 1961 (see Ref. 46)
A. Description
Colonies grow slowly on glucose peptone agar and are cottony
to velvet, white at first, becoming pink with age. A nondiffus-
ible rose to red pigment develops on the reverse side. Macroco-
nidia are scarce, clavate, thin and smooth-walled, with 2–10
septa, said to resemble those of T. rubrum (45). Microconidia
are numerous, small, pyriform to clavate, borne singly or in
clusters along the hyphae.
B. Discussion
Produces tinea barbae predominantly and to a lesser extent
tinea corporis and tinea capitis. Hair involvement of the large-
spored ectothrix type. Predominantly anthropophilic, it has
been reported on dogs (41). The fungus is found in Europe and
Africa.
XXIII. Trichophyton rubrum (Castellani) Sabouraud, Br J Dermatol 23:389–
390, 1911; synonym: Trichophyton fluviomuniense Pereiro Miguens,
Sabouraudia 6:312–317, 1968 (see Ref. 71)
174 Howard et al.
A. Description
Colonies on glucose peptone agar are cottony and white, later
becoming velvety. The reverse side is reddish to rose-purple.
Macroconidia elongate, cylindrical, thin and smooth-walled,
with 3–8 septa. Generally, these are scarce on glucose peptone
agar but are formed in cultures on heart infusion tryptose agar
(15, 57). Microconidia (Fig. 8) numerous, clavate, 2–3 ⫻ 3–5
µm, borne singly along the hyphae. Chlamydospores, racquet
hyphae, and nodular bodies may be seen in primary cultures. A
wide variety of colonial types has been described (1).
B. Discussion
This species is worldwide in distribution and clinically is among
the most important dermatophyte pathogens. It produces tinea
corporis (see Ref. 104 for a recent report on invasive infections),
tinea pedis, tinea cruris, and tinea unguium. Nail lesions have
been refractory to treatment, but newer drugs have succeeded in
producing a clinical and mycological cure. The fungus rarely
causes disease in animals, but instances are recorded (41). Tri-
chophyton rubrum does not perforate autoclaved hair, a test use-
ful in distinguishing isolates of it from T. mentagrophytes (105).
Mating studies with A. simii (90) have revealed that all
Figure 8 Microconidia of Trichophyton rubrum, ⫻560.

tested isolates of T. rubrum were the same mating type (⫺). It

may be that virulence or some other selective factor has elimi-
nated the (⫹) mating type of T. rubrum.
XXIV. Trichophyton schoenleinii (Lebert) Langeron & Milochevitch, Ann
Parasitol Hum Comp 8:465–508, 1930
A. Description
Colonies are very slow-growing on glucose peptone agar,
heaped with many irregular folds, waxy smooth, later becoming
velvety white. Macroconidia have never been reported. Micro-
conidia are virtually never seen in primary isolation; old cul-
tures become velvety and develop microconidia. Antlerlike ter-
minal branching (‘‘favic chandeliers’’) is the single most
obvious morphological feature. Chlamydospores may be seen.
B. Discussion
The fungus is widespread in Eurasia and North Africa (1),
where it produces favus or tinea favosa, which is a chronic,
cicatricial form of tinea capitis distinguished by the formation
of scutula, crusts, scarring, atrophy, and permanent hair loss.
Hair involvement is quite distinctive. Within the hair, hyphae
and air spaces accompanied by degenerate hyphae are seen. A
dull bluish-white fluorescence of the hairs under a Wood’s
lamp has been described. Tinea corporis has been observed, as
have infections in animals (41).
XXV. Trichophyton soudanense Joyeux, C R Soc Biol (Paris) 73:15–16,
1912; synonym: Langeronia soudanensis Vanbreuseghem, Ann Par-
asitol Hum Comp 25:493–508, 1950
A. Description
Colonies are very slow-growing, flat to folded, suede-like, yel-
low, dried-apricot colored, often with a fringed border (106).
Sterile rapidly growing hyphae develop readily in culture. Mac-
roconidia not seen. Microconidia are seen in cultures grown on
potato dextrose agar and are pyriform, smooth-walled, borne
laterally on hyphae, sessile or on short pedicels (106). The my-
celium is characterized by hyphae that break up into segments
or arthroconidia and by reflexive branching. Produces dark
brown colonies on Lowenstein Jensen medium.
B. Discussion
This species produces tinea capitis and tinea corporis. It is
found principally in Africa, but cases have been reported in
England, Brazil, and the United States (41). The differentiation
between M. ferrugineum and T. soudanense has been studied
(107).
176 Howard et al.
XXVI. Trichophyton tonsurans (Malmsten, 1845). Trichophyton tonsurans

harskarande Mogel, Stockholm (transl.) Arch Anat Physiol Wiss
Med 1–99, 1848
A. Description
Colonies grow fairly slowly on glucose peptone agar, flat, some-
what powdery or velvety, white, yellowish to pinkish surface.
The reverse is mahogany-red. The sulfureum variety produces
pale to deep yellow colonies. Macroconidia are not frequently
seen, but when found are thin-walled, club-shaped, or sinuous,
with 3–5 septa. Some more abundantly sporulating strains have
been described (26). Microconidia are numerous, clavate, 2–5
⫻ 3–7 µm, and are borne laterally along the hyphae on pedicels
of various lengths (Fig. 9). Chlamydospores and racquet hyphae
may be seen. Growth stimulated by thiamine.
B. Discussion
This species produces black-dot tinea capitis and tinea corporis
[caused institutional and family outbreaks—role of families
also—tinea corporis gladiatorum (contact spreads outbreaks)]
Figure 9 Microconidia of Trichophyton tonsurans, ⫻875.

and tinea pedis. Hair shaft involvement is of the endothrix sort.

Animal infections have been rarely reported. Two varieties have
been described: T. tonsurans var. tonsurans and T. tonsurans var.
sulfureum (108, 109).
XXVII. Trichophyton verrucosum Bodin, Les champignons parasites de
l’homme. Paris: Masson et Cie, 1902, p. 121
A. Description
Colonies are very slow growing on glucose peptone agar,
heaped, deeply folded, glabrous and waxy, sometimes with a
fine white velvety surface. Colonial variants have been de-
scribed and named [var. ochraceum, var. album, and var. dis-
coides (45)]. These variants were originally described as spe-
cies (1, 105, 106), but are not currently so considered (40).
On glucose peptone agar generally only chlamydospores and
hyphae are observed microscopically. On enriched media mi-
croconidia are produced. They are clavate to pyriform, sessile,
borne laterally along the hyphae. Macroconidia are rare and are
‘‘rat-tailed’’ or ‘‘string bean’’-shaped with 3–5 septa, smooth,
thin-walled. All strains require thiamine; most require thiamine
and inositol. Growth stimulated at 35–37°C.
B. Discussion
This species produces highly inflammatory tinea capitis, tinea
barbae, and tinea corporis. The hairs show ectothrix type of
involvement. The fungus is worldwide in distribution. Charac-
teristically, it is a zoophilic species found primarily in cattle,
and has also been recorded in donkeys, dogs, goats, sheep, and
horses (41).
XXVIII. Trichophyton violaceum Bodin, Les champignons parasites de
l’homme. Paris: Masson et Cie, 1902, p. 113
A. Description
Colonies are slow-growing on glucose peptone agar, waxy,
wrinkled, heaped up or verrucous, with a deep purplish-red pig-
mentation. Colonies tend to produce sector variants. Generally,
only torturous, tangled hyphae are observed. Pyriform micro-
conidia borne laterally may be seen, but only rarely. Clavate
macroconidia (45) with 3–5 septa, and smooth, thin walls have
been reported. Growth stimulated by thiamine.
B. Discussion
This species produces tinea corporis and tinea capitis of the
black-dot variety. Hair invasion is of the endothrix sort. World-
wide in distribution, although unusual in the United States (1).
Reported to infect animals other than humans (41).
178 Howard et al.
XXIX. Trichophyton yaoundei, Cochet & Doby-Dubois, Sem Hop Paris 33:
26–30, 1957.
A. Description
Colonies are very slow growing on glucose peptone agar, gla-
brous, raised, folded, white to cream in color at first, becoming
tan to brown with age. The pigment diffuses into the medium.
Sectoring variants devoid of diffusible pigment on the reverse
occur regularly. Macroconidia are not formed. Microconidia
rare, but when seen are pyriform and borne laterally on the
hyphae. Chlamydospores are present.
B. Discussion
This species produces endothrix-type tinea capitis and is found
in equatorial Africa (1).
E. Saprophytic Species: Teleomorphs

XXX. Arthroderma borellii (Moraes, Padhye et Ajello) Padhye, Weitzman,
McGinnis et Ajello, comb. nov., Mycotaxon 25:505, 1986; basi-
onym: Nannizzia borellii Moraes, Padhye et Ajello, Mycologia 67:
1112, 1975
A. Conidial state
Microsporum amazonicum Moraes, Borelli & Feo, Med. Cuta-
nea 2:201–205, 1967.
B. Description
Heterothallic. Ascocarp globose, dull white or pale yellow,
300–500 µm in diameter, excluding appendages. Peridial hy-
phae are hyaline, septate, and dichotomously branched. Cells
are thin-walled, finely echinulate, slightly constricted in the
middle, and swollen near the septa. Appendages are of two
kinds: (1) elongate, septate, smooth-walled, tapering hyphae up
to 150 µm in length, 2–3 µm in diameter at the base and 1.2–
1.5 µm at the tip; (2) elongate, smooth-walled, slender hyphae
coiled into spirals; macroconidia may be at the end of some
hyphal appendages. Asci are hyaline, subglobose, 4–5 µm in
diameter with eight ascospores. Ascospores hyaline, oblate, 2–
3 ⫻ 2.5 µm in diameter, yellow in mass.
Colonies on glucose peptone agar are fluffy to powdery
with a distinctive gray-olive-buff color (45). Macroconidia are
symmetrical, spindle-shaped, echinulate, thick-walled, with 1–
4 septa. Microconidia not observed.
C. Discussion
The fungus was isolated from hair of spiny rats (Proechimys
guyannensis) and from hair of a rat belonging to the genus Ory-

zymys. Although the organism is associated with animals, it is
a saprophyte since naturally occurring lesions have not been
described.
XXXI. Arthroderma ciferrii Varsavsky & Ajello, Riv Patol Veg 4:358–359,
1964
A. Conidial state
Trichophyton georgia Varsavsky & Ajello, Riv Patol Veg 4:
357–358, 1964.
B. Description
Homothallic. Ascocarp globose, pale buff to pale brownish vi-
naceous, 450–700 µm in diameter, excluding appendages. Pe-
ridial hyphae hyaline, septate, uncinately branched with curled
end. Cells of the peridial hyphae dumbbell-shaped, symmetri-
cally or asymmetrically constricted, densely asperulate, 8–12
µm long, 7–8.5 µm at enlarged ends in width. Appendages of
two kinds: (1) slender, smooth-walled spiral hyphae, borne ter-
minally, and (2) slender, smooth-walled, septate, tapering hy-
phae extending outward, 60–96 µm long. Asci subglobose,
thin-walled, evanescent, 4.8 ⫻ 6 µm, 8-spored. Ascospores hy-
aline, smooth, oblate, 2.5–3 ⫻ 1.4–2.8 µm, yellow in mass.
Colonies on glucose peptone agar are flat and granular
with an umbonate center. Center pale vinaceous and downy.
Flat area surrounding umbonate center powdery to granular,
vinaceous brown. Reverse irregularly spotted with dark red or
dark vinaceous brown. Macroconidia thought not to occur, but
microconidia have septa and might be regarded as macroco-
nidia (45). Microconida variable in size and shape, elongate,
clavate, occasionally pyriform to subglobose, borne laterally or
terminally, singly on short pedicels, or sessile on the hyphae.
Microconidia smooth-walled, usually nonseptate, occasionally
with two or three septa, 4–6.5 ⫻ 2–2.5 µm.
C. Discussion
This fungus is found in soil, on hair and feathers, or in birds’
nests or excreta (111). The organism has been reported from
the United States, Venezuela, Australia, Czechoslovakia, and
Yugoslavia (111).
XXXII. Arthroderma cookiellum (de Clercq) Weitzman, McGinnis, Padhye
et Ajello, comb. nov., Mycotaxon 25:505, 1986; basionym: Nanniz-
zia cookiella de Clercq, Mycotaxon 18:23, 1983
A. Conidial state Microsporum de Clercq, Mycotaxon 18:23–28,
1983.
180 Howard et al.
Heterothallic. Ascocarp globose, pale buff to yellow, 400–800 µm

in diameter. Peridial hyphae pale buff, verrucose, septate, dividing
in two or more branches. Distal branches straight or curved in
‘‘running legs.’’ Peridial cells 10–20 ⫻ 2–5 µm, asperulate, with
appendages that are either slender, smooth-walled, 64–400 ⫻ 2
µm, or as 10–14 µm wide spirals with 8–22 turns. Asci subglo-
bose, 4–6 ⫻ 4 µm, 8-spored, evanescent. Ascospores golden-yel-
low in mass, smooth-walled, 2–3 µm. Macroconidia numerous,
oval-shaped, dumpy, 18–34 ⫻ 17 µm, predominantly four-celled,
thick-walled, 2–4 µm, very densely verruculose, the ‘‘warts’’ re-
sembling sometimes ‘‘pseudopodes.’’ Microconidia numerous,
pyriform, sometimes very elongate, 1–2 ⫻ 2–8 µm.
On glucose peptone agar, colonies rapidly growing. Ae-
rial mycelium, ochraceous powdery, at the margin white and
fluffy; the down becomes violet after 7 days. Reverse brown-
ish-purple with a nondiffusing pigment and a violet border by
transparency. There is no growth at 37°C. Inoculation on the
scarified skin of guina pigs is negative. Habitat in soil of Ivory
Coast. Keratinophilic.
XXXIII. Arthroderma corniculatum (Takashio et de Vroey) Weitzman,
McGinnis, Padhye et Ajello, comb. nov., Mycotaxon 25:505, 1986;
Basionym: Nannizzia corniculata Takashio et de Vroey, Mycotaxon
14:384, 1982
A. Conidial state
Microsporum boullardii Dominik & Majchrowicz, Ecol Pol Ser A
13:415–447, 1965.
B. Description
Heterothallic. Ascocarp globose, pale-buff to yellow-buff, 370–870
µm diameter. Peridial hyphae pale-buff, hyaline, septate, verticil-
lately branched at the distal ends of hyphal cells, with up to four
branches, straight or curved. Distal branches composed of one to
five cells curve moderately or coil to form semicircle, or a com-
plete circle or even more. Cells 10–28 (⫺34) µm long, with maxi-
mal breadth of 3–7 µm and with minimal breadth of 3–5 µm, moder-
ately thick-walled, densely asperulate, slightly thickened toward
their ends, except the terminal cells, which are slightly tapering
and have blunt ends. These terminal cells may have an appendage:
elongate, slender, smooth-walled hyphae, 2–2.5 µm in diameter,
more or less tightly coiled with up to 11 spirals of (10-) 12–20 µm
in diameter. Asci globose to ovate 4–5 ⫻ 5–5.5 µm diameter, eight-
spored. Ascospores yellow, smooth-walled, lenticular, 1.7 ⫻ 2.7
µm.
Colonies on glucose peptone agar are rapid growing, flat with

a dense downy to floccose chamois-like surface, tawny-buff to pink-
buff in color. Reverse red to purplish, resembles some isolates of
M. fulvum. (See A. fulva.) Macroconidia are fairly thick-walled,
echinulate, clavate to bullet-shaped, 24–58 ⫻ 7.5–12 µm, 4–6 septa,
borne singly. Microconidia single-celled, clavate, and abundant.
C. Discussion
This fungus has been isolated from soil of Guinea, Africa (4, 45).
Seemingly, this is the only report of its isolation (99). Its ability to
produce disease is unproven.
XXXIV. Arthroderma flavescens Rees, Sabouraudia 5:206–207, 1967
A. Conidial state
Trichophyton flavescens Padhye & Carmichael, Can J Bot 9:
1535, 1971.
B. Description
ameter, excluding appendages. Peridial hyphae hyaline, sep-
tate, with two inwardly curving branches developed from apex
of some peridial cells. Cells dumbbell-shaped, thick-walled,
asymmetrically constricted, echinulate, 11–16.5 ⫻ 3–4.8 µm.
Appendages smooth-walled, septate; spiral hyphae usually ter-
minal. Smooth-walled, septate macroconidia are rarely attached
to peridial hyphae. Asci subglobose, thin-walled, evanescent,
5.9–8.3 ⫻ 4.8–7.5 µm, eight-spored. Ascospores hyaline,
smooth, oblate, with equatorial rim, 3.8–4 ⫻ 1.8–2 µm, yellow
in mass.
Colonies on glucose peptone agar white to pale yellow
with buff center. Reverse bright yellow, darkening to yellow-
brown. Microconidia borne laterally or terminally smooth, thin-
walled, sessile or on short pedicels, unicellular, occasionally
two-celled, pyriform, ovate, 5–16 ⫻ 4–8 µm. Macroconidia
numerous, hyaline, usually 2–6 septa, smooth, thin-walled, cy-
lindrical with rounded apices, 26–86 ⫻ 8–14 µ.
C. Discussion
This species is found in Australia, where it was isolated from
feathers (111).
XXXV. Arthroderma gertleri Bohme, Mykosen 10:251, 1967
A. Conidial state
Trichophyton vanbreuseghemii Rioux, Jarry & Jiminer, Nat
Monspel Ser Bot 16:153–162, 1964.
B. Description
182 Howard et al.
diameter, excluding appendages. Peridial hyphae hyaline, sep-

tate, uncinately branched, curving over the ascocarp. Cells
dumbbell-shaped, echinulate, usually asymmetrically, but
occasionally symmetrically constricted. Appendages septate
spirals varying in length and number of turns, borne termin-
ally. Multiseptate, thin-walled, smooth, cylindrical macro-
conidia are sometimes attached to the pyridial hyphae. Asci
subglobose, thin-walled, evanescent, 4 ⫻ 5 µm, eight-spored.
Ascospores hyaline smooth, oblate, 1.5–2.8 ⫻ 2–2.5 µm;
yellow in mass.
Colonies flat or with radial grooves, floccose at first, be-
coming velvety [described as the texture of ‘‘fine glove
leather’’ (45)] with a finely granular center, white to buff. Re-
verse creamy white to pale buff. Microconidia pyriform, sub-
globose, ovoid, thin-walled, unicellular, sessile or on short ped-
icels, borne laterally or terminally, 2–7 ⫻ 1.5–2.5 µm.
Macroconidia numerous, thin-walled, smooth, cylindrical,
multiseptate, borne singly, 30–55 ⫻ 6–8 µm.
C. Discussion
This dermatophyte is probably worldwide in distribution and
has been isolated from a tinea of the hand (45). Large-spored
ectothrix involvement of guinea pig hair is reported (41). The
fungus has been isolated from soil (45, 111).
XXXVI. Arthroderma gloriae Ajello, Mycologia 59:257, 1967.
A. Conidial state
Trichophyton gloriae Ajello, Mycologia 59:257, 1967.
B. Description
diameter, excluding appendages. Peridial hyphae thin-walled,
septate, hyaline, uncinately branched. Cells dumbbell-shaped,
echinulate, symmetrically constricted, 7.5 ⫻ 5 µm. Append-
ages moderately slender, thin-walled hyphae pointed at the end,
150 µm long. Asci subglobose, thin-walled, evanescent, 3.5 ⫻
4.6 µm, eight-spored. Ascospores hyaline, thin-walled, smooth,
oblate, 2–2.5 ⫻1.5–2 µm, yellow in mass.
Colonies downy, slightly folded, pale cream with a
brownish center. Reverse yellow with yellow-brown center.
Microconidia pear-shaped, borne singly on short pedicels or
sessile, 1.5–6 ⫻ 1.5–2.5 µm. Macroconidia numerous, borne
singly, more often in clusters of 4–30, cylindrical, multiseptate,
with 1–10 septa, smooth walls up to 10 µm thick, 9–60 ⫻ 3–
7 µm.
C.Discussion
This fungus has been reported from the soil and on animal hair
from several states of the United States (45).
XXXVII. Arthroderma insingulare Padhye & Carmichael, Sabouraudia 10:47,
1972
A. Conidial state
Trichophyton terrestre, sensu lato Durie and Frey, Mycologia
49:401, 1957.
B. Description
Heterothallic. Ascocarp globose, white to pale yellow, 250–500
µm in diameter, excluding appendages. Peridial hyphae pale yel-
low, hyaline, septate, uncinately branched, usually on the outside
of the main hypha. Cells thick-walled, echinulate, asymmetri-
cally dumbbell-shaped, 8–12 µm long, 5–6 µm wide at enlarged
ends and 3–4 µm wide at the internode. Appendages spiral, sep-
tate, smooth-walled, terminal hyphae, which vary considerably
in length and number of turns. Asci subglobose, thin-walled, eva-
nescent, 4–6 ⫻ 3.5–5 µm, eight-spored. Ascospores hyaline,
smooth, oblate, 2.5–3 ⫻ 2.2–2.5 µm, yellow in mass.
Colonies on glucose peptone agar are white and fluffy, be-
coming pale yellow, and downy to granular. Reverse yellowish-
brown. Some strains develop a red pigment on the reverse. Micro-
conidia elongate, pyriform, borne singly or in groups, 3–6.5 ⫻
1.5–3.5 µm. Macroconidia cylindrical or rarely clavate, smooth,
thin-walled, sessile, 8–52 ⫻ 4–5 µm, with 2–6 septa. Intermedi-
ate forms present. Terminal or intercalary chlamydospores, spi-
rals, racquet hyphae, antler-like hyphae, and nodular bodies oc-
casionally seen. The colonial description of T. terrestre given at
this point for convenience should more properly have been intro-
duced under Arthroderma quadrifidum, where it was first used.
C. Discussion
This species has been isolated from soil, hair, and feathers. The
fungus is known to occur in Canada, the United States, Hun-
gary, and Czechoslovakia.
XXXVIII. Arthroderma lenticularum Pore, Tsao & Plunkett, Mycologia 57:
970–971, 1965
A. Conidial state
Trichophyton terrestre, sensu lato Durie & Frey, Mycologia
49:401, 1957.
B. Description
ameter, excluding appendages. Peridial hyphae pale yellow, hy-
184 Howard et al.
aline septate, uncinately branched usually to one side, the out-

side of the main hyphae. Cells thick-walled, echinulate,
dumbbell-shaped, symmetrically constricted, 5.5–8.5 ⫻ 7–10
µm. Appendages septate, spiral hyphae borne terminally. Asci
subglobose, thin-walled, evanescent, 4–4.8 ⫻ 5–5.6 µm, eight-
spored. Ascospores hyaline, smooth, oblate, 2.2–3 ⫻ 1.5–7.5
µm, yellow in mass.
For colonies see description of A. insingulare. This fun-
gus is found in soil in the United States (41).
XXXIX. Arthroderma quadrifidum Dawson & Gentles, Sabouraudia 1:56–
57, 1961
A. Conidial state
Trichophyton terrestre, sensu lato Durie & Frey, Mycologia
49:401, 1957. Synonym: Trichophyton thuringiense Koch, My-
kosen 12:287–290, 1969. (See Ref. 63.)
B. Description
aline, septate, uncinately branched usually to one side, the out-
side of the main hypha. Cells thick-walled, strongly echinulate,
dumbbell-shaped when young but when mature resembling a
short humerus bone with condyles much accentuated and
formed on one face only, 8–13 ⫻ 5–9 µm. Appendages septate
spirals varying considerably in length and number of turns,
borne terminally. Asci subglobose thin-walled, evanescent, 4–
6 ⫻ 3.5–5 µm, eight-spored. Ascospores hyaline, smooth, ob-
late, 2.5–3 ⫻ 2.2–3 µm, yellow in mass.
Colony (see description of A. singulare).
C. Discussion
This fungus is of worldwide occurrence and is found in soil,
on feathers, and on animal hair (41).
Pore and Plunkett (112) reported three more teleomorphs
in the T. terrestre complex, but these new species were not
named or described by the authors and so have not been included
in compilations of the T. terrestre complex (38, 41, 45, 106).
XL. Arthroderma uncinatum Dawson & Gentles, Sabouraudia 1:55, 1961
A. Conidial state
Trichophyton ajelloi (Vanbreuseghem) Ajello, Sabouraudia 6:
148, 1968. Synonym: Keratinomyces ajelloi Vanbreuseghem,
Bull Acad R Med Belg. 38:1076, 1952.
B. Description

aline, septate, uncinately branched, usually to the outside of the
main hyphae. Cells thick-walled, strongly echinulate, symmet-
rically dumbbell-shaped, 7–11 ⫻ 4–7 µm. Appendages septate,
smooth-walled, spirals that vary considerably in length and
number of turns, borne terminally. Sometimes smooth-walled,
multiseptate, fusiform macroconidia are produced by peridial
hyphae. Asci subglobose, thin-walled, evanescent, 5.4–7.2 ⫻
5–6.5 µm, eight-spored. Ascospores hyaline, smooth, oblate,
2.5–3.2 ⫻ 1.8–3 µm, yellow in mass.
Colonies flat to folded, with a downy to powdery surface,
cream to orange-tan. Reverse with or without diffusable, vina-
ceous red pigment that becomes bluish-black with age. Macro-
conidia produced in abundance, cylindrical to fusiform, multi-
septate (8–12 septa), thick-walled, smooth, 18–60 ⫻ 8–12 µm.
Microconidia present in some strains, pyriform to obovate, 3–
9 ⫻ 2–5 µm (56).
C. Discussion
It is to be noted that there are two colonial forms of T. ajelloi:
one producing a diffusible bluish-black pigment, the other not.
In the original description of A. uncinatum it was recorded that
nonpigmented varieties did not mate with pigmented ones (38).
Moreover, the size of the macroconidia of the two forms has
been reported to differ (113). Thus, the suggestion was made
that the nonpigmented isolates might represent a distinct taxon
(113). Weiztman et al. (114), however, and Padhye and Carmi-
chael (111, 115) examined a larger number of strains, observed
fertile crosses between pigmented and nonpigmented isolates,
and concluded that the lack of pigment did not denote a separate
taxon.
Weitzman and Silva-Hutner (116) studied the genetics of
pigmentation in A. uncinatum and concluded from the segrega-
tion pattern that there was a single gene difference between
pigmented and nonpigmented parents. Arthroderma uncinatum
has a haploid complement of four chromosomes (59). A variety
of T. ajelloi with very small macroconidia has been described
at T. ajelloi var. nanum (117, 118)
F. Saprophytic Species: Teleomorphs Unknown

XLI. Epidermophyton stockdaleae Prochacki & Englehardt-Zasada, My-
copathol. Mycol. Appl. 54:341–345, 1974
186 Howard et al.
A. Description
Colonies grow fairly rapidly on glucose peptone agar, flat, with
a slightly umbonate center, powdery to granular, and buff
cream-colored. The reverse is yellow, becoming ferruginous
to blush ferruginous. Macroconidia numerous, smooth-walled,
clavate, or cylindrical, borne laterally or terminally, 20–50 ⫻
3.8–12.9 µ, with 2–9 septa. Microconidia not formed. Degen-
erate macroconidia such as those seen in A. simii are observed.
B. Discussion
This species was isolated from soil in Szezecin, Poland.
XLII. Microsporum magellanicum Coretta & Piontelli, Sabouraudia 15:
1–10, 1977
A. Description
Colonies on glucose peptone agar are finely granular, powdery,
cream to yellowish in color; reverse yellow to ochre. Macroco-
nidia are hyaline, ovate or clavate, rarely cylindrical, measuring
14.4–21.6 ⫻ 4.8–7.2 µm, verrucous and pedicillate-walled also
verruculose, with 1–2 or rarely 4–6 septa, borne singly. Micro-
conidia not seen, but young macroconidia are pyriform or cla-
vate with a truncate base, lightly verruculose, measuring 2.4–
3 ⫻4.8–5.8 µm, sessile or pedunculate often with a septum.
Chlamydospores observed rarely.
B. Discussion
This keratinophilic fungus was isolated from soil collected in
the extremity of Chile, in southern Shetland, and the Antarctic
continent. It has only been described in this saprophytic situation.
XLIII. Microsporum ripariae Hubalek & Rush-Munro, Sabouraudia 11:
287–289, 1973
A. Description
Colonies on glucose peptone agar are densely powdery to vel-
vety and floccose with some zonate growth. Reverse is light
yellow or lemon yellow and brownish in the center. Macroco-
nidia are produced in limited numbers, borne singly, hyaline,
relatively thin-walled, verrucose to verruculose, fusiform to
cigar-shaped or almost cylindrical, 27–47 ⫻ 6.3–11 µm, with
4–6 septa. Microconidia are numerous, unicellular, hyaline,
smooth, thin-walled, and sessile or borne on short, lateral
branches, often in bunches. They are pyriform and on the aver-
age 3.2 ⫻ 2.1 µm. Arthroconidia and terminal chlamydospores
are occasionally observed.
B. Discussion
This fungus has only been reported one time. It was isolated
from feathers and nests of the sand martin (Riparia riparia) in

central Europe. Although isolated from a seemingly sapro-
phytic source, it is capable of causing experimental infections
in guinea pigs and humans.
XLIV. Trichophyton fischeri Kane, Sabouraudia 15:231–241, 1977 (50)
A. Description
Colonies develop moderately slowly, are velvety to cottony,
and white. The reverse is blood red. Macroconidia are sinuous,
few in number, and approximately 50 µm long by 3–5 µm
wide. Filaments bear club-shaped lateral projections that are
9–12 µm long by 6–8 µm wide. These projections have a broad
attachment to the hyphae and have 0–3 septa. Two types of
microconidia are seen: slender, club-shaped conidia 4–5 µm
long by 2 µm wide, and subglobose microconidia 3–4 µm long
by 2–3 µm wide. Both types are borne singly on the hyphae
(46, 50).
B. Discussion
T. fischeri appears to be a saprophyte (46). A recent report of
the first isolation of the fungus in the United States again af-
firms its saprophytic occurrence (119). T. fischeri resembles
closely T. rubrum and must be distinguished from the pathogen
(114).
XLV. Trichophyton longifusum (Florian & Galgoczy) Ajello, Sabouraudia
6:148, 1968
A. Description
Colonies on glucose peptone agar are fluffy to powdery, and
white to pale yellow in color. The reverse is yellowish-brown.
Macroconidia are long and cylindrical, 7–9 µm in diameter,
and up to 300 µm long, occurring in clusters, and characteristi-
cally may arise from one another and merge into terminal hy-
phae. Microconidia not reported in original description (117).
B. Discussion
Isolated from soil in Hungary (120). The species was trans-
ferred to the genus Trichophyton by Ajello (117) and otherwise
has seemingly received scant attention.
XLVI. Trichophyton mariatii Tapia de Fossaeri, Mizrachi, Padhye et
Ajello, Proc. 5th Int’l. Conf. on the Mycoses, Sci. Publ. no. 396,
154–158, 1980
A. Description
Colonies grow fairly rapidly on glucose peptone agar and are
flat and velvety, pale yellow; reverse dark yellow to apricot.
Macroconidia are scanty, smooth, thin-walled, carrot-shaped,
188 Howard et al.
3 to 8 single cells on short stalks or sessile, 35–56 ⫻ 6–18

µm. Microconidia sessile or with short stalks; varying sizes and
shapes: elongated, clove- or pear-shaped; mostly one-celled,
rarely two- or three-celled, 3–6.5 ⫻ 1.3–2.0 µm.
B. Discussion
A saprophyte isolated from soil in Caracas, Venezuela. The
basis for differentiating T. mariatii from other geophilic species
is the shape of the macroconidium: broad at the base (8–10
µm) and narrower at the tip (4–5 µm), resembling a carrot.
Attempts to cross T. mariatii with strains of A. benhamiae, A.
lenticularum, and A. quadrifidum failed.
XLVII. Trichophyton phaseoliforme Borelli & Feo, Acta Med Venez 13:
176–177, 1966
A. Description
Colonies on glucose peptone agar are powdery, white to bright
cinnamon colored. Microconidia are cylindrical and produced
in terminal clusters. Microconidia are curved and ‘‘cashew-
nut’’ shaped, borne laterally on the hyphae and in large num-
bers on thickened and enlarged hyphae in the ascocarp-like
(pycnidia?) structures.
B. Discussion
This species deserves further work to establish various morpho-
logical features that might alter its current taxonomic treatment.
It was isolated from soil in Romania, from an apparently nor-
mal rodent in Venezuela, and is perhaps worldwide in distribu-
tion (45).
NOTE ADDED IN PROOF
During the last revision of the manuscript for this chapter, a review article on
tinea capitis appeared in the literature. Since the review contained 354 references
and dealt predominantly with therapy, it is included here as an addendum which
will interest some readers of this chapter (121).
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6
Ascomycetes
The Onygenaceae and Other Fungi
from the Order Onygenales
Lynne Sigler
University of Alberta, Edmonton, Alberta, Canada
I. INTRODUCTION
The ascomycete order Onygenales is important from the medical perspective be-
cause it includes the sexual stages of the true fungal pathogens of humans and
animals (i.e., the dermatophytes and the dimorphic fungi capable of causing dis-
ease in an otherwise healthy host). The Onygenales includes three families: the
Arthrodermataceae, including dermatophytes (treated in this volume, Chap. 5);
the Onygenaceae, including the dimorphic fungi; and the Gymnoascaceae. Al-
though a prior version of this volume (1) treated the Onygenaceae as part of the
Eurotiales, the most recent treatment of the ascomycetes (2) maintains it within
the order Onygenales separately from the Eurotiales. This chapter describes the
pathogenic members of the Onygenaceae as well as some nonpathogenic species
that may resemble them and that are rare to common contaminants in clinical
specimens. A few members of the Gymnoascaceae are also treated. A fourth
family, the Myxotrichaceae, formerly included within the Onygenales (3, 4), ap-
pears instead to have affinities to the inoperculate discomycetes (5). Notwith-
standing its probable disparate relationship to onygenalean fungi, some members
of the Myxotrichaceae are included here for convenience.
Although phylogenetic relationships of many of the true pathogenic fungi
are known, most medical mycologists continue to use the name of the anamorph
195
196 Sigler
(asexual or mitotic stage) rather than the name of the teleomorph (sexual or mei-
otic stage). The reasons are because a vast body of literature has been published
in which these names are used and because the majority of these fungi are hetero-
thallic in compatibility. The anamorph is the stage commonly encountered in
primary isolation from the specimen, and the teleomorph is seldom seen.
The species described in this chapter are ones with known or inferred place-
ment within the order Onygenales. A few nonpathogenic ‘‘look-alikes’’ of uncer-
tain affinity are included for convenience. Species are described under the name
in common use (i.e., usually the name for the anamorph). Table 1 lists the species
in the order in which they are covered and provides comments on their known
or inferred teleomorphs and occurrence as pathogens.
II. TAXONOMIC CONCEPTS
Heterothallic compatibility is common among onygenalean fungi and the teleo-

morph can be obtained only through mating trials. These tests are of great value
in confirming identity and in establishing biological relationships, but are rarely
employed in diagnostic laboratories because of the need for appropriate test iso-
lates, the length of time required for development and maturity of the sexual
structures, and—at least for the dimorphic fungi—the risks of handling the fungi.
For this reason, medical mycologists have paid greater attention to the character-
istics of the anamorph (asexual or mitotic stage) because it is the stage encoun-
tered in primary isolation from the specimen. Even though connections between
anamorphs and teleomorphs have been firmly established for many fungi, the
physical separation between the anamorph and teleomorph means that relation-
ships often have been difficult to discern. The correct phylogenetic position of
Cooccidioides immitis, for example, a fungus known for 100 years and for which
there is a vast body of literature, remains uncertain, but modern approaches place
it clearly within the Onygenaceae. A suggestion of a close relationship among
Histoplasma capsulatum, Blastomyces dermatitidis, and Emmonsia species,
made first in the late 1940s, has been borne out by evidence that their teleom-
orphs occur in the same genus of the Onygenaceae and by molecular phyloge-
netic studies.
III. CHARACTERISTICS OF THE ORDER AND FAMILIES
Members of the Onygenales are united by their formation of prototunicate asci

with eight ascospores and by ascospores that are small (usually less than 8 µm
in length), single-celled, and light colored (never dark brown or black). Prototuni-
cate asci are usually globose or subglobose; they are irregularly arranged within
Table 1 Overview of the Species in Order of Their Coverage and Comments on Their Known or Inferred Teleomorphs
and Pathogenic Potential
Figure
Anamorph Teleomorph Family affinity Occurrence as a pathogen number
Histoplasma capsulatum Ajellomyces capsulatus Onygenaceae Dimorphic pathogen; common 1, 2

but limited in endemic areas;
may be associated with out-
breaks
Histoplasma capsulatum var. Ajellomyces capsulatus Onygenaceae Dimorphic pathogen; sporadic
duboisii even in endemic areas
Histoplasma capsulatum var. Not known; relationship to Ajel- Onygenaceae Dimorphic pathogen; sporadic,
farciminosum lomyces inferred mainly causing infection in
horses in endemic areas
Blastomyces dermatitidis Ajellomyces dermatitidis Onygenaceae Dimorphic pathogen; sporadic 1, 3
even in endemic areas; may
be associated with outbreaks
Ascomycetes: Onygenaceae and Other Fungi
Emmonsia crescens Ajellomyces crescens Onygenaceae Dimorphic pathogen; very rarely 1, 4

pathogenic to humans; com-
mon in rodents and small
mammals as shown by sur-
veys
Emmonsia parva Not known; relationship to Onygenaceae Dimorphic pathogen; very rarely
Ajellomyces inferred pathogenic to humans; spo-
radic in rodents and small
mammals as shown by sur-
veys
197
Emmonsia pasteuriensis Not known; relationship to Onygenaceae Dimorphic pathogen; known

Ajellomyces inferred from a single case of infection
Table 1 Continued
Figure
198
Anamorph Teleomorph Family affinity Occurrence as a pathogen number
Paracoccidioides brasiliensis Not known; relationship to Onygenaceae Dimorphic pathogen; common 5

Ajellomyces inferred but limited in endemic areas
Coccidioides immitis Not known; relationship to Onygenaceae Dimorphic pathogen; common in 6
Uncinocarpus inferred endemic areas; may be associ-
ated with outbreaks
Chrysosporium Aphanoascus fulvescens Onygenaceae Rarely pathogenic; common cuta- 7
neous contaminant and rare
agent of cutaneous infection.
Chrysosporium zonatum Uncinocarpus orissi Onygenaceae Pathogenic; soil fungus known 8
from a single case of deep in-
fection and two cases of pul-
monary colonization
Chrysosporium Nannizziopsis vriesii Onygenaceae Pathogenic; uncommon agent of
cutaneous infection in reptiles
Chrysosporium Renispora flavissima Onygenaceae Nonpathogenic; soil fungus 9
shown for comparison with
H. capsulatum
Chrysosporium keratinophilum Aphanoascus keratinophilus Onygenaceae Nonpathogenic; uncommon con- 10
taminant of cutaneous speci-
mens; shown for comparison
Chrysosporium articulatum Not known Onygenaceae Nonpathogenic; uncommon but 11
regular contaminant of cutane-
ous specimens; compare C.
Sigler
anamorph of Aphanoscus
fulvescens
Chrysosporium carmichaelii Not known Not known Nonpathogenic; uncommon but 12
ous specimens; compare C.
undulatum
Chrysosporium lobatum Not known Not known Nonpathogenic; uncommon but 13
ous specimens
Malbranchea Uncinocarpus reesii Onygenaceae Nonpathogenic; uncommon con- 14
taminant; shown for compari-
son with C. immitis
Malbranchea gypsea Not known Not known ?Pathogenic; status as agent of 15
onychomycosis not confirmed
Onychocola canadensis Arachnomyces nodosetosus Gymnoascaceae Pathogenic; uncommon agent of 16
onychomycosis
anamorph absent Gymnascella dankaliensis Gymnoascaceae Pathogenic; uncommon agent of 17
onychomycosis
anamorph absent Gymnascella hyalinospora Gymnoascaceae Pathogenic; uncommon contami- 18

nant; known from single case
of pulmonary infection
Geomyces pannorum Not known Myxotrichaceae Nonpathogenic; common con- 19
taminant of cutaneous speci-
mens
anamorph absent or arthro- Myxotrichum deflexum Myxotrichaceae Nonpathogenic; uncommon con- 20, 21
conidia taminant of cutaneous speci-
mens
Ovadendron sulphureo- Not known ?Myxotrichaceae Pathogenic; rare agent of eye in- 22
199
ochraceum fection
200 Sigler
the ascomata (ascocarps, sexual fruiting bodies) and their cell walls lyse at or
near maturity, allowing for passive discharge of the ascospores. Ascomata have
varied morphologies ranging from nonostiolate, usually globose, pseudoparen-
chymatous structures (i.e., cleistothecia) to a loose network of differentiated hy-
phal cells or of branched hyphae (i.e., often called gymnothecia) that sometimes
extend into elaborate hooked, spiralled, or branched appendages; to clusters of
more or less naked asci and ascospores. Under cultural conditions, the ascomatal
appendages are sometimes found associated with the anamorph. Anamorphs are
solitary, single-, or multicelled aleurioconidia or alternate arthroconidia in which
part or all of the subtending or intervening cell lyses to release the conidium.
Characters that have been used to separate families include features of the as-
cospore wall ornamentation, correlated to some extent with ascospore shape,
habitat, capacity to degrade keratinous or cellulosic substrates or lacking these
enzymatic capacities, and features of the anamorph. Members of the Arthroder-
mataceae and Onygenaceae (Table 2) are often keratinolytic, and these families
are distinguished by their ascospore wall morphologies (smooth in the former
and punctate or punctate-reticulate in the latter) and conidial types. The key char-
acters for the Onygenaceae are described below.
A. Family Onygenaceae
Members of the Onygenaceae have an ability to degrade keratinaceous substrates,
and are often associated with mammals. Ascospore walls are ornamented with
small pits or depressions (also called puncta), with netlike ridges (reticulate) or
a combination of both, or with minute spiny projections (muriculate). Anamorphs
are aleurioconidia that are predominantly single-celled and placed in the genera
Histoplasma, Blastomyces, Emmonsia, Paracoccidioides, and Chrysosporium, or
are alternate arthroconidia and placed in the genus Malbranchea (Table 2). It
should be noted, however, that the latter two anamorph genera are not monophy-
letic; some species are of uncertain affinity.
Species of Medical Relevance

1. Ajellomyces McDonough & Lewis (Fig. 1).
Synonym: Emmonsiella Kwon-Chung.
Ascomata (gymnothecia) solitary, discrete, globose, becoming irregularly
stellate by formation of coiled appendages, buff, small, 80 to 350 µm in
diameter. Peridial hyphae pale brown, smooth, branched, individual cells
obtusely diamond-shaped (swollen near the center and constricted at the
septa) or unswollen. Appendages helically coiled, thick-walled, yellowish-
brown. Asci subglobose, clavate or pyriform, evanescent, eight-spored.
Ascospores small, globose, hyaline, measuring 1 to 1.5 µm in diameter,
Ascomycetes: Onygenaceae and Other Fungi 201
muriculate (having short, sharp outgrowths) by scanning electron micros-

copy, but appearing almost smooth by light microscopy.
Comments. Similarities in growth habit and conidial type have long been
noted among the dimorphic fungi Histoplasma, Blastomyces, Emmonsia,
and Paracoccidioides, but the species have been retained in separate gen-
era largely based on differences in their parasitic forms, in their virulence,
and in the clinical syndromes that they elicit. Recent studies have con-
firmed a close relationship among them. Cultural mating experiments
proved that Blastomyces dermatitidis, Histoplasma capsulatum, and Em-
monsia crescens are heterothallic ascomycetes and that their ascomata and
ascospores are characteristic of the genus Ajellomyces (6–8). Phylogenetic
trees derived from comparison of large subunit ribosomal and ITS region
DNA sequences place sexual and asexual species of all four genera as
members of the Ajellomyces clade (9, 10) and show that Emmonsia and
Blastomyces species are more closely related to each other than to His-
toplasma species. These findings lend some support to arguments that have
been made in favor of maintaining Emmonsiella as a separate genus, but
molecular phylogenetic studies all show the Ajellomyces clade to be well
supported (9–11), even though the relationships within it are not clearly
resolved. Ambiguities are found in ubiquinone data that show that the
principal ubiquinones are Q-10(H 2) for H. capsulatum and Emmonsia spe-
cies but Q-10 for B. dermatitidis (12, 13). Paracoccidioides has been
placed within the Ajellomyces clade, suggesting that its teleomorph (if
found) will belong there, but its closest relative is not yet clearly elucidated
(9, 10, 14). This evidence of close relationship opens the possibility of
combining the anamorphic genera. This could be useful from the taxo-
nomic point of view because relationships become more obvious, but is
disadvantageous because the anamorphic names are clinically relevant and
in widespread use.
Molecular phylogenetic studies show the Ajellomyces clade to be
separated from other members of the family Onygenaceae. It may belong
in a separate family of the Onygenales. The minute spiny projections on
the ascospore wall are unusual within the family, and similar features are
found only in one other monotypic genus (8).
a. Ajellomyces capsulatus (Kwon-Chung) McGinnis & Katz, Mycotaxon

8:158, 1979
Synonym: Emmonsiella capsulata Kwon-Chung, Science 177:368,
1972.
Description as for genus. Ascomata 80 to 250 µm in diameter. Peridial
hyphae uniform in diameter (unswollen) and not constricted at the
septa. Appendages helically coiled, smooth, originating near the center
Table 2 Comparison of Arthrodermataceae and Onygenaceae (Onygenales)
202
General features of Arthrodermataceae and Onygenaceae

• Often keratinolytic
• Conidia having lytic dehiscence (solitary, single-, or multicelled aleurioconidia
and/or alternate arthroconidia)
• Prototunicate asci
• Ascomata with varied morphologies (naked asci, gymnothecia, cleistothecia)
• Ascospores light or brightly colored, single-celled
• Often heterothallic
Anamorphs
Conidia multicelled (macroconidia) Conidia predominantly single-celled

and/or single-celled
Epidermophyton Microsporum Trichophyton Chrysosporium Histoplasma Malbranchea

Blastomyces Coccidioides
Emmonsia
Paracoccidioides
Macroconidia only Macroconidia Macroconidia Multicelled absent Multicelled absent Multicelled absent
• Smooth/warty • Rough-walled • Smooth-walled
• Thin-walled • Thick/thin walled • Thin/rarely thick
• No microconidia • ⫹Microconidia • ⫹Microconidia ↔ single-celled Single-celled Alternate
• Rarely 1 septate arthroconidia
• ⫹/⫺Alternate
arthroconidia
Sigler
Teleomorph(s)
Arthroderma Arthroderma
Unknown or unknown or unknown Arthroderma
哸
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -哸
-
Arthrodermataceae
Aphanoascus Ajellomyces Uncinocarpus
Uncinocarpus or unknown Auxarthron
Nannizziopsis other genera or
other genera unknown
or unknown
Onygenaceae
Onygenales
Conidial and ascomatal types in the Onygenales
Source: illustrations of teleomorphs courtesy of  R. S. Currah.

203
204 Sigler
Figure 1 Ajellomyces species ascocarp and ascospores (Note that these structures are
drawn to different scales; ascospore scale bar approx 1 µm.) Source: ascocarp  R. S.
Currah (with permission).
of the ascoma, 2 to 10 (commonly 3–5) per ascoma, 1.7 to 3 µm in

diameter, 30 to 100 µm long. Asci pyriform or clavate, to 10 µm wide.
Ascospores globose, hyaline, muriculate, 1.2 to 1.5 µm in diameter.
Heterothallic. Anamorphs Histoplasma capsulatum var. capsulatum,
H. capsulatum var. duboisii.
i. Histoplasma capsulatum var. capsulatum Darling, JAMA 46:1285,
1906 (Fig. 2).
Occurrence as a pathogen. Etiologic agent of histoplasmosis cap-
sulati, an infection having several clinical manifestations, includ-
ing asymptomatic infection (most common), acute or chronic
pulmonary infection, and disseminated disease involving the re-
ticuloendothelial system (15–18).
Figure 2 Histoplasma capsulatum in vitro and in vivo. Bar approximately 10 µm.

Description (See also Table 3.) Detailed descriptions of the tissue

and colonial and microscopic morphologies have been published
in medical mycology texts (e.g., 15, 16, 18, 19). In culture, sin-
gle-celled conidia of two sizes are produced and are commonly
referred to as macro- and microconidia. Conidia are sessile
(borne directly on the sides of the hypha) or borne at the ends
of short or long stalks that have parallel sides (i.e., are unswollen)
or are slightly swollen. Macroconidia are globose or broadly pyr-
iform, sometimes smooth, but typically tuberculate; that is, hav-
ing tuberous or fingerlike projections that vary in length (2–6
µm) and width (0.5–2 µm). Microconidia are globose, smooth
to slightly roughened, and predominantly sessile. As shown by
Berliner (20) in a detailed study of phenotypic variation, primary
subculture may yield albino (also called A type) or brown (B
type) colonies or both. Albino colonies are white and cottony
and have slightly broader aerial hyphae. They are more likely to
have smooth to slightly warty macroconidia and abundant micro-
conidia, but sporulation may be lost in serial subculture. Addi-
tionally, they are difficult to convert to the yeast form. Brown
colonies are buff to pale brown with a diffusible tan pigment,
velvety to almost granular in age, and have narrow hyphae (ⱕ2
µm wide). Sporulation is more stable and consists predominantly
of the typical tuberculate macroconidia.
Comments. Use of a commercially available DNA probe (Accu-
Probe; Gen-Probe Inc., San Diego, CA) (18, 21) and/or exoanti-
gen test are the methods now most commonly used to confirm
identification of an isolate suspected of being H. capsulatum.
The DNA probe proved useful in delineating an unusual case of
dual infection in which culture yielded a mixed growth of H.
capsulatum and B. dermatitidis (22). Conversion to the yeast
phase may be achieved by subculture onto enriched media con-
taining cysteine and incubation at 35–37°C, but conversion can
be difficult to achieve and may require multiple subcultures. His-
topathologic staining of biopsy specimens or smears of fluids
demonstrating presence of intracellular small oval yeast cells and
history of residence or travel in endemic areas are useful con-
firmatory findings.
Only a few soil fungi produce large, echinulate to tubercu-
late conidia with morphologies resembling the macroconidia of
H. capsulatum, and these can be distinguished by the absence of
the microconidial state and differences in colonial morphology.
These species are rarely isolated as contaminants in clinical
206
Table 3 Characteristics of the Dimorphic Fungi 1

Ecology & Parasitic form Confirmatory Selected
Distribution Mycelial form Teleomorph in vivo tests refs
Histoplasma capsula- Soil associated with Narrow hyphae form- Ajellomyces capsulatus Intracellular, rarely ex- Convert to yeast form 15, 16, 17, 18, 19, 20,
tum var. capsulatum dung of birds & ing solitary sessile tracellular, small using enriched me- 21, 23, 26
bats. microconidia and tu- yeast cells, 3 to 5 dia (brain heart infu-
North America in areas berculate macroco- µm in length, with sion or blood agar
along Mississippi, nidia at the ends of single narrow based with added cysteine)
Missouri & Ohio unswollen stalks. bud at 37°C; multiple
River valleys, rarely Microconidia sub- subculture may be
in Central & South globose or broadly required for com-
America, Asia, Eu- pyriform, 4–5 µm plete conversion. Ex-
rope long & 3–4 µm oantigen shows spe-
wide. Macroconidia cific m, or h and m,
broadly pyriform, precipitin bands
8–17 µm long & 8– DNA probe test.
13 µm wide, or glo-
bose, 8–13 µm in
diameter, bearing tu-
berous projections
2–6 µm long and
0.5–2 µm wide.
Histoplasma capsula- Rarely isolated from Similar to var. capsula- Ajellomyces capsulatus Extracellular, thick- Convert to yeast form 15, 16, 17, 18, 19, 26,
tum var. duboisii soil, intestinal con- tum walled yeast cells, using enriched me- 27
tents of bats, aard- 12–15 µm in length, dia) at 37°C. Differ-
varks, baboons; habi- with single narrow entiate from var. cap-
tat poorly known. based bud, occasion- sulatum by larger
Central Africa, Mada- ally in short chains size of yeast cells.
gascar
Sigler
Histoplasma capsula- Isolated from equines. Similar to var. capsula- Not known. Relation- Intracellular, rarely ex- Convert to yeast phase 15, 16, 17, 26, 28, 29
tum var. farcimino- Africa, Asia, Eastern tum but very slow ship to Ajellomyces tracellular, small on brain heart infu-
sum Europe growing and not spor- inferred. yeast cells, 3 to 5 sion agar (with or
ulating on rich me- µm in length, with without blood) at
dia. single narrow based 37°C; several subcul-
bud tures may be re-
quired.
No specific exoantigen
test available.
Blastomyces dermati- River & forest soil, bea- Narrow hyphae form- Ajellomyces dermati- Large yeast cells, 10 to Convert to yeast form 6, 8, 9, 15, 16, 18, 19,
tidis ver dams, habitat ing solitary sessile tidis 12 µm in diameter, on enriched (brain 21, 23, 30, 32, 33,
poorly known. conidia and 1, rarely with thick, double heart infusion agar) 34, 36
North America in areas 2, conidia at the ends contoured wall and at 37°C or on conver-
along Mississippi & of narrow, usually un- single broad-based sion media (Kane) at
Ohio River valleys, swollen stalks. Co- bud. 37°C or lower; serial
eastern & central nidia mostly pyri- subculture may be re-
Canada, rarely Af- form, 4–5 µm quired.
rica & Asia long & 3–4 µm wide Exoantigen shows spe-
cific A precipitin
band.
DNA probe test.
Emmonsia crescens Many animal hosts Narrow hyphae form- Ajellomyces crescens Oval or globose adi- Maximum growth 8, 9, 10, 15, 23, 38, 42,
(about 118 species of ing solitary sessile aspores, 50 to 500 temperature 37°C 43
rodents, lago- conidia and 1–3 co- µm, commonly 300 (lower for some
morphs, carnivores, nidia at the ends of µm in diameter, strains); forming adi-
insectivores, marsu- narrow, often slightly walls 10–70 µm aspores (enlarged
pials), rarely recov- swollen stalks. Co- thick chlamydospore-like
ered from soil or nidia subglobose, of- structures) measur-
other habitats. ten slightly broader ing 20–140 µm
Worldwide except Af- than long, 2.5–4 µm Exoantigen tests may
rica, Australia long & 3–5 µm show non-specific
wide, smooth to precipitin lines
finely roughened. against B. dermati-
tidis and H. capsula-
tum antisera
207
208
Table 3 Continued
Ecology & Parasitic form Confirmatory Selected
Distribution Mycelial form Teleomorph in vivo tests refs
Emmonsia parva Few animal hosts Narrow hyphae form- Not known Oval or globose adi- Maximum growth tem- 8, 9, 10, 15, 23, 38, 42,
(about 21 species of ing solitary sessile aspores, 10 to 40 perature 40°C, form- 43a
rodents, lago- conidia and 1–3 co- Relationship to Ajello- µm in diameter, ing adiaspores mea-
morphs, carni- nidia at the ends of myces inferred walls thinner suring 8–20 µm
vores & marsupi- narrow, often dis- diameter.
als), rarely tally swollen stalks. Exoantigen tests may
recovered from soil Conidia subglo- show non-specific
or other habitats. bose, often slightly precipitin lines
Geographically re- broader than long, against B. dermati-
stricted (parts of 2.5–4 µm long & tidis and H. capsula-
US, Kenya, Europe, 3–5 µm wide; tum antisera
Australia) smooth to finely
roughened.
Emmonsia pasteuriana Known only from Italy. Narrow hyphae form- Not known Small and large yeast- Conversion to yeast 8, 39, 44
ing solitary sessile like cells; oval to glo- phase at 37°C on en-
conidia and 1–8 co- Relationship to Ajello- bose with narrow riched media; no adi-
nidia at the ends of myces inferred based buds. aspores formed.
narrow, distally swol-
len stalks. Conidia
subglobose to oval,
often slightly broader
than long, 2 µm
long & 3–4 µm
wide; smooth to
Sigler
finely roughened.
Paracoccidioides bra- Rarely isolated from Narrow hyphae, often Not known. Thick walled central On enriched media 9, 10, 14, 15, 16, 18,
siliensis soils (coffee planta- remaining sterile; oc- Relationship to Ajello- cell up to 30 µm in (brain heart infusion 19, 45, 46, 47, 48
tion, rain forest), ar- casionally forming myces inferred. diameter, bearing 1 agar or blood agar),
madillos, habitat con- lateral or terminal to many daughter converts to yeast
sidered to be forest pyriform conidia and cells of varying size stage at 37°C.
areas of moderate cli- solitary intercalary and joined to mother Exoantigen test shows
mate, medium to arthroconidia that are cell by narrow pedi- specific 1, 2, 3 pre-
High annual rainfall. often slightly swol- cel, occasionally oc- cipitin bands.
Central and South len (2–5.5 ⫻ 2–3.5 curring in short
America. µm). chains.
Coccidioides immitis Soil of arid regions, ani- Hyphae forming alter- Not known Spherules 30 to 60 µm In vitro conversion not 15, 16, 18, 19, 33, 49,
mal burrows, has nate arthroconidia. with walls 2 µm recommended and 50, 51, 52, 53, 54,
been found in mixed Arthroconidia cylin- Relationship to Uncino- thick and containing rarely done. 55, 56, 57
infections with E. drical 3–8 µm long carpus (Onygena- endospores 2–5 µm Exoantigen test shows
parva in lungs of ro- and 3–5 µm wide. ceae) inferred in diameter. specific F precipitin
dents & other bur- band. DNA probe
rowing animals. test.
Geographically re-
stricted to arid re-
gions of southwest
U.S. & parts of Cen-
tral & South
America
1
Modified from Sigler (38).
209
210 Sigler
specimens, but laboratorians may encounter them in proficiency

testing programs. They include the Chrysosporium anamorph
of Arthroderma tuberculatum (Arthrodermataceae) (23), the
Chrysosporium anamorph of Renispora flavissima (Onygena-
ceae) (23), the Botryotrichum anamorph of Chaetomium his-
toplasmoides (Chaetomiaceae) (24), and some Sepedonium spe-
cies (anamorphs of Hypocreaceae) (23). None is closely related
to H. capsulatum. Indeed, as mentioned above, the closest rela-
tives to species of Histoplasma are Blastomyces dermatitidis and
species of Emmonsia.
Studies of compatibility among ⬎1000 soil and human iso-
lates of H. capsulatum of North American origin showed that
mating type was significantly associated with pathogenicity, with
a ratio of 7(⫺) : 1(⫹) among human isolates compared with 1:1
for soil isolates, and with conversion to the yeast form, with a
higher percentage of conversion among ⫺ than ⫹ mating type
strains (25). Recent studies have revealed genetic differences
among populations of H. capsulatum. Based on DNA thermal
denaturation, Guého et al. (9) could distinguish three geographic
populations of H. capsulatum, but the LSU rDNA sequences
were identical or showed only one base difference. Peterson and
Sigler (10) observed significant genetic difference between two
North American isolates, including one from a human source
and another derived from germination of a single ascospore of
A. capsulatus. Kasuga et al. (26) have suggested that the differ-
ences are sufficient to recognize six groups that may be consid-
ered as phylogenetic species.
ii. Histoplasma capsulatum var. duboisii (Vanbreuseghem) Ciferri,
Manual di Micologia Medica, Tomo II:342, 1960.
Synonym: Histoplasma duboisii Vanbreuseghem, Ann. Soc.
Belge Med. Trop. 32: 578, 1952.
Occurrence as a pathogen. Etiologic agent of histoplasmosis du-
boisii, a systemic infection endemic in parts of central Africa,
in which lesions occur most commonly in the bone, skin, and
subcutaneous tissues (15–18).
Description (See also Table 3.) The colonial and microscopic fea-
tures correspond with those described for the variety capsulatum
(15–19).
Comments. Initially recognized as a distinct species because of the
larger size of yeast cells in tissue and different clinical manifesta-
tions, H. duboisii was later given variety status. Evidence sup-
porting this decision came from mating studies (27) that demon-
strated compatibility between H. duboisii ⫻ H. capsulatum.

Ascospores produced in these crosses failed to germinate, how-
ever, leaving open a possibility that these may be separate spe-
cies, except that failure to germinate occurred also among some
H. capsulatum ⫻ H. capsulatum crosses. As with the latter, ⫺
mating types predominated. Comparison of partial rRNA gene
sequences showed a level of variation within the genus not ex-
ceeding 1% and supported the varietal status of duboisii (9, 14),
but comparison of DNA sequences of protein-coding genes sup-
ported recognition of H. duboisii as a distinct species (26).
iii. H. capsulatum var. farciminosum (Rivolta) Weeks, Padhye &
Ajello, Mycologia 77:969, 1985.
Synonym: H. farciminosum (Rivolta) Redaelli & Ciferri, Boll. Sez.
Ital. Soc. Int. Microbiol. 6:377, 1934.
Occurrence as a pathogen. Etiologic agent of histoplasmosis far-
ciminosi or epizootic lymphangitis, a systemic infection of
equines that affects mainly the subcutaneous lymph nodes and
lymphatics of the neck (15–17, 28, 29).
Description (See also Table 3.) Weeks et al. (29) obtained sporu-
lation only when isolates were grown on a weak medium, such
as soil extract agar. Colonies were very slow growing, attaining
diameters of only 5 to 6 mm after 12 weeks incubation at 30°C.
Sporulating areas of the colony were buff in color and demon-
strated abundant micro- and macroconidia having similar mor-
phologies to those of var. capsulatum.
Comments. Varietal status has been proposed based on antigenic
and morphologic similarities in the yeast cells and macroconidia
(29). No sexual stage is known. Groupings based on comparison
of partial LSU rRNA sequences support the distinction of farci-
minosum at the variety level except that genetic differences are
slightly greater between it and the other varieties (9, 14). Analy-
sis of different genes suggested a close relationship between
equine isolates and some var. capsulatum isolates from South
America (26).
b. Ajellomyces dermatitidis McDonough & Lewis, Mycologia 60:77, 1968

Description as for genus. Ascomata 200 to 350 µm in diameter. Perid-
ium is composed of hyphae in which individual cells are obtusely dia-
mond-shaped (swollen near the center and constricted at the septa). Ap-
pendages helically coiled, generally 3 to 5 per ascoma, breadth of helix
12 to 40 µm. Asci globose or subglobose, 3.5 to 7.5 µm. Heterothallic.
Anamorph Blastomyces dermatitidis.
212 Sigler
Figure 3 Blastomyces dermatitidis in vitro and in vivo.
i. Blastomyces dermatitidis Gilchrist & Stokes, J Exp Med 3:76, 1898

(Fig. 3).
Nomenclature. This name remains in common use for the agent of
blastomycosis even though it has long been recognized as invalid
under the International Code of Botanical Nomenclature. See
Carmichael (30), van Oorschot (31), or de Vries (1), for detailed
lists of synonyms and explanation of the problem. Recent
changes to the code suggest that a proposal for conservation
might be successful and ensure retention of this important name.
Occurrence as a pathogen. Etiologic agent of blastomycosis
(Gilchrist’s disease or North American blastomycosis), a sys-
temic infection involving the lung but frequently progressing to
other sites, including the skin and bone (15, 16, 18, 32).
Description (See also Table 3.) The tissue, colonial, and micro-
scopic morphologies have been described extensively (e.g., 15,
16, 18, 19, 23, 30). Aleurioconidia are sessile (borne directly on
the sides of the hypha) or borne at the ends of short stalks that
are unswollen (having parallel sides) or slightly swollen at the
apical end (nearest to the conidium). Conidia are smooth to echi-
nulate (spiny walled), broadly pyriform or subglobose, and often
flattened apically, so that they are slightly broader than long, and
have a narrow truncate base. Occasional isolates show conidia of
larger dimensions that are smooth or ornamented with irregular
protrusions (8), or helical coils and peridial hyphae characteristic
of the sexual stage. Colonies are initially white, becoming buff
to pale brown, sometimes with a diffusible tan pigment, velvety
to almost granular in older cultures. Sporulation is heavy in buff-
pigmented colonies, but may be absent or sparse in isolates that
are glabrous (having little aerial hyphae).
Comments. The presence of large, thick-walled yeast cells with

a single broad-based bud in cutaneous material or biopsy speci-
mens provides presumptive evidence of infection. The commer-
cial probe (AccuProbe) has been evaluated for specificity in con-
firming B. dermatitidis in culture (18, 21, 33). The specificity
was 100% when the nontarget isolates were morphologically
similar but not biologically related (21), but dropped to 59%
when isolates of the biologically related Paracoccidioides bra-
siliensis (i.e., a member of the Ajellomyces clade) were included
(33). Conversely, the exoantigen test was found to be 100% spe-
cific for isolates of both species (33). These species occur in
different endemic areas and express different morphologies in
their yeast stages. Isolates may be converted to the yeast phase
by subculture onto enriched or conversion media (e.g., Blasto
‘‘D’’ medium; see Refs. 34, 35) and incubating at 35–37°C. Al-
though temperature is considered the primary determinant for
dimorphism in this species (36), medium constituents also play
a role in transition (34). Glabrous and poorly sporulating strains
are more difficult to convert and may require multiple subcul-
tures.
Fungi that form conidia resembling those of B. dermati-
tidis have been reviewed recently in detail (23). The most similar
conidial morphologies are found among species of Emmonsia.
(See Table 3.) As has long been hypothesized (30, 37), Emmon-
sia species are the closest biological relatives to B. dermatitidis
(see also discussion under H. capsulatum and Emmonsia) (8, 9,
10, 38), but they express different in vivo morphologies, forming
nonreplicating thick-walled adiaspores rather than budding cells.
Isolates demonstrating transitional features have recently been
reported from human cases, however (9, 10, 39, 40); thus the
features defining the genera are not as clear as previously sup-
posed. Arguments in favor of combining the genera have been
made, but to retain use of the name Blastomyces rather than Em-
monsia, it must first be conserved. (See Ref. 10.)
Kwon-Chung (41) studied compatibility among ⬎40 hu-
man isolates of B. dermatitidis from North America and Africa.
She demonstrated that incompatibility was of the bipolar type;
that mating competence was greater in buff-pigmented colony
types; and that ascospores showed a 1:1 ratio of ⫹ to ⫺ mating
type. She also confirmed previous findings of incompatibility be-
tween North American and African isolates. As reviewed by di
214 Sigler
Salvo (32), antigenic and morphologic differences have been re-

ported for African strains. In comparing sequences of a 592 nu-
cleotide region covering the two most divergent regions of the
LSU rRNA, Guého et al. (9) found nine base pair differences
between African and North American isolates, almost the same
level of difference as found between the latter and E. parva (eight
bases).
c. Ajellomyces crescens Sigler, J Med Vet Mycol 34:305, 1996

Description as for genus. Ascomata 80 to 250 µm in diameter. Peridium
is composed of hyphae in which individual cells are obtusely dia-
mond-shaped (swollen near the center and constricted at the septa).
Heterothallic. Anamorph Emmonsia crescens.
i. Emmonsia crescens Emmons & Jellison, Ann NY Acad Sci 89:96,

1960 (Fig. 4).
Synonym: Chrysosporium parvum var. crescens (Emmons & Jelli-
son) Can J Bot 40:1164, 1962.
Occurrence as a pathogen. Etiologic agent of adiaspiromycosis, a
fairly common pulmonary infection of rodents and small bur-
rowing animals. Infection in humans is rare, usually confined to
the lung, with rare progression to other sites (15, 38, 42).
and cultural and microscopic morphologies have been published
recently (8, 15, 23, 38). The colonial and microscopic morpholo-
gies resemble those of Blastomyces dermatitidis, but Emmonsia
species differ in their propensity to form one to three conidia at
the ends of narrow, often slightly swollen stalks. Growth of E.
crescens isolates is moderately to strongly inhibited by addition
of cycloheximide (400 µg/ml) to culture media and almost com-
pletely inhibited at 35–37°C. At this temperature, most isolates
will develop thick-walled adiaspores (globose or subglobose
Figure 4 Emmonsia species in vitro and in vivo.

chlamydosporelike structures) that measure 20 to 140 µm in diam-

eter.
Comment. Adiaspores in vivo may be much larger and reach di-
mensions of 50 to 500 µm with walls from 10 to 70 µm thick.
Adiaspores are unusual parasitic forms since they arise from
swelling or enlargement of inhaled conidia rather than by multi-
plication of the conidia within the tissues. Available evidence
suggests that Emmonsia species are weak pathogens and that the
functional disability and elicitation of symptoms is dependent
upon the number of conidia inhaled (43). The presence of these
large and thick-walled structures in tissue provides presumptive
evidence of infection by E. crescens. Discovery of their presence
has often been made accidentally during histopathological exam-
ination of tissues when other syndromes were suspected. Al-
though E. crescens has been obtained in culture from several
animal species by dissection of lung tissue, so far there has been
no documented recovery of an authentic isolate from human tis-
sue. Several isolates from human sources that were examined by
Peterson and Sigler (10) demonstrated genetic differences that
placed them outside the E. crescens clade.
d. Species Within the Ajellomyces Clade Lacking Known Teleomorphs
i. Emmonsia parva (Emmons & Ashburn) Ciferri & Montemartini,
Mycopath Mycol Appl 10:314, 1959.
Synonym: Chrysosporium parvum var. parvum (Emmons & Ash-
burn) Carmichael, Can J Bot 40:1164, 1962.
Occurrence as a pathogen. Etiologic agent of adiaspiromycosis in
animals, but less widely distributed and in fewer animal hosts
than E. crescens. There is a single report of disseminated infec-
tion in a human with AIDS (15, 38, 42, 43a).
and cultural and microscopic morphologies have been published
recently (8, 15, 23, 38). E. parva isolates are slightly inhibited by
cycloheximide (at 400 µg/ml) in culture media and moderately
inhibited at 37°C. At 40°C, growth is almost completely inhib-
ited, and at this temperature most isolates develop thin to slightly
thick-walled adiaspores (globose or subglobose chlamydospore-
like structures) measuring 8 to 20 µm in diameter.
Comment. Molecular phylogenetic analysis demonstrated a close
relationship between E. parva and B. dermatitidis and also re-
vealed considerable genetic variation among isolates originally
thought to represent E. parva (10). Isolates from human skin
216 Sigler
lesions appeared to be distinct (10, 40). Similarly an isolate caus-

ing cutaneous disseminated mycosis in a patient with AIDS was
found to be molecularly and morphologically distinct and was
described as a new species, E. pasteuriana (9, 39, 44).
ii. Emmonsia pasteuriana Drouhet, Guého & Gori, J Med Mycol 8:
70, 1998.
Occurrence as a pathogen. There is a single report of disseminated
cutaneous infection in a human with AIDS.
Description (See also Table 3.) The following is based on the pub-
lished description of the ex-type culture and only known isolate
(39, 44). Colonies are moderately slow growing, yellowish-
white, densely woolly, furrowed or zonate with powdery or gla-
brous sectors. The fungus is tolerant of cycloheximide. Hyphae
are narrow. Conidia are solitary and formed on the sides of the
hyphae (sessile), or one to eight conidia are borne at the tips
of narrow stalks that are distally swollen to form a vesiclelike
structure. Conidia appear smooth to finely roughened by light
microscopy, tuberculate by scanning electron microscopy, and
are slightly broader than long. Conversion to the yeast phase may
be obtained by subculture onto enriched media and incubation
at 37°C. Colonies on brain heart infusion agar appear smooth,
light greyish-brown, and consist of oval to lemon-shaped, small
(2–4 µm) yeastlike cells having narrow-based buds. In vivo,
yeast cells are more variable in size and shape.
Comments. Variation in colony texture appears similar to that re-
ported for other species of Emmonsia (8). E. pasteuriana differs
in forming clusters of up to eight conidia on a swollen stalk, by
lacking expression of adiaspores, and by conversion to a yeast-
like stage in vitro. Notwithstanding the unusual expression of a
yeast phase, the microscopic morphology and comparison of par-
tial LSU rDNA sequences suggested that the Italian isolate from
a patient with AIDS fit within the concept of the genus Emmonsia
and was a close relative of E. crescens.
iii. Paracoccidioides brasiliensis (Splendore) Almeida, C R Soc Biol
Paris 106:316, 1930 (Fig. 5).
Occurrence as a pathogen. Etiologic agent of paracoccidioido-
mycosis, a systemic infection endemic in parts of Central and
South America that manifests as benign or progressive lung in-
fection but often progresses to form chronic granulomatous le-
sions of the skin or mucous membranes, especially of the mouth,
nose, and gastrointestinal tract (15, 16, 18, 45).
Figure 5 Paracoccidioides brasillensis in vitro and in vivo.
and colonial and microscopic morphologies have been published

in medical mycology texts (15, 16, 18, 19, 45). Colonies are
very slow growing and vary in texture from glabrous to densely
velvety. They may be furrowed and are initially white, but
darken to buff. Sporulation is often absent on richer media, such
as Sabouraud dextrose agar, but may be induced by growing an
isolate on nutritionally poor media such as tap water, glucose
salts, or yeast extract agars (46).
Comments. Diagnosis and identification may be confirmed by ob-
servation of the distinctive yeast stage, either in vivo or in vitro
by subculture onto enriched media and incubation at 37°C. Di-
morphism in P. brasiliensis has been reviewed (47) and stated to
be strictly thermal. In a case of imported paracoccidioidomycosis
known to the author, however, the primary isolate was recovered
in the yeast form on phytone yeast extract agar at 28–30°C, and
could be maintained for several subcultures under these condi-
tions without becoming hyphal. As discussed earlier, the DNA
probe test may fail to differentiate isolates of P. brasiliensis from
B. dermatitidis, but these species occur in different endemic re-
gions and express different yeast morphologies. The ecology and
substrate preferences of P. brasiliensis are still rather poorly un-
derstood, as the fungus has rarely been isolated from soil or other
habitats (48).
Recent molecular phylogenetic studies (9, 10, 14) have
confirmed that the closest biological relatives of P. brasiliensis
are members of the Ajellomyces clade; that is, Blastomyces, Em-
monsia, and Histoplasma, as suggested by Dowding (37), but
the relationships among them require further study.
2. Coccidioides immitis Stiles in Rixford & Gilchrist, Johns Hopkins Hosp Rep
1:243, 1896 (Fig. 6).
218 Sigler
Figure 6 Coccidioides immitis in vitro and in vivo.
Teleomorph: unknown but relationship to Uncinocarpus inferred.

Occurrence as a pathogen. Etiologic agent of coccidioidomycosis, a sys-
temic infecton with several clinical manifestations, including asymptom-
atic benign infection (most common), acute or chronic progressive lung
infection, and disseminated disease, especially involving the skin, subcuta-
neous tissues, and bone (15, 16, 18, 49).
Description (See also Table 3.) Detailed descriptions of the tissue and colo-
nial and microscopic morphologies have been published in medical mycol-
ogy texts (15, 16, 18, 19, 49). Conidia are formed in an intercalary position
by concentration of cytoplasm and organelles in some cells while other
cells become devoid of contents and the walls autolyse. Conidia formed
in this way have been called alternate arthroconidia, arthroaleuries, or en-
teroarthric conidia. In C. immitis, arthroconidia are formed in branched
fertile hyphae and are closely spaced, with intervening cells often (but not
always) shorter than the arthroconidia (50). They are cylindrical to barrel-
shaped or cuneiform if terminal or lateral, and measure from 3 to 5 µm
in width. Vegetative hyphae are septate and often show swellings at one
end of the cell (i.e., racquet hyphae). Colonies are moderately fast grow-
ing, white to yellowish-white or pale buff, velvety to powdery, occasion-
ally glabrous. Variation in colonial texture and pigmentation has been re-
ported in the literature (e.g., see Ref. 15), but some isolates having unusual
features have been reidentified as species of Malbranchea (50).
Comments. The demonstrated presence of characteristic spherules containing
endospores in fluid or tissue specimens or in animal tissue following inoc-
ulation is confirmatory evidence for diagnosis of coccidioidomycosis;
however, spherules are sometimes lacking or structures are not definitive
(51). The exoantigen test and DNA probe are commercially available tests
commonly used to confirm identification of an isolate (33). In vitro conver-
sion is not recommended because of the potential for misidentification
when arthroconidia of similar Malbranchea species swell and round up

but lack endospores.
The endosporulating spherules and lack of a sexual cycle has made
the taxonomic position of C. immitis the subject of considerable specula-
tion. The alternate arthroconidia of its saprobic phase are typical of some
Malbranchea species (50), and it has been hypothesized that its closest
relatives would be found among some keratinophilic species of Malbran-
chea having meiotic stages in the Onygenaceae (52). Depending upon the
fungi included in the analysis, molecular phylogenetic studies have placed
C. immitis within the order Onygenales (9, 53) or within the family Ony-
genaceae with Uncinocarpus reesii, an ascomycete with a Malbranchea
anamorph, as a close relative (11, 54). The sexual stage of C. immitis
remains undiscovered, but Burt et al. (55) report molecular evidence of
recombination. Some isolates produce helically coiled hyphae (56, 57).
Helical, spiral, or uncinate hyphae often occur as ascomatal appendages
in onygenalean fungi, and their presence in cultures of anamorphs suggests
a potential for sexual reproduction.
3. Chrysosporium Corda
Type species: C. merdarium (Link) Carmichael.
Teleomorphs are placed in Aphanoascus, Nannizziopsis, Renispora, Uncino-
carpus, Arthroderma, and some other genera (as on page 303).
Comments. Many species of Chrysosporium are anamorphs of onygenalean
fungi. The genus as circumscribed by Carmichael (30) was heterogeneous,
but has been useful for the placement of anamorphic fungi that produce
solitary, usually nonseptate aleurioconidia (i.e., conidia with lytic dehis-
cence), and occasional arthroconidia. He broadened the concept of the
genus to include Blastomyces and Emmonsia, but their exclusion is now
supported by genetic data. Some Chrysosporium species are encountered
as rare to common contaminants from cutaneous specimens, and a few
species have the potential to cause infection (23). Some reports concerning
Chrysosporium species as etiologic agents must be viewed with caution,
however, since the isolated organism has neither been identified to species
nor documented well enough to confirm the etiology, and a representative
culture has not been maintained (58, 59).
Species of Medical Relevance.
a. Aphanoascus fulvescens (Cooke) Apinis, Mycopathol Mycol Appl 35:
101, 1968 (Fig. 7).
Occurrence as a pathogen. Rare cause of nail, skin, and scalp infection
(35).
Description. Detailed descriptions of the colonial and microscopic mor-
phologies have been published recently (35). Colonies grow moder-
220 Sigler
Figure 7 Aphanoascus fulvescens (note conidia and ascospores are not drawn to the
same scale). Bar approx 5 µm. Source: ascomata R. S. Currah (used with permission).
ately rapidly, are tolerant of cycloheximide, and are somewhat inhib-

ited at 37°C. They are yellowish-white, becoming buff to greyish as
ascomata develop, velvety to granular. The Chrysosporium anamorph
is distinguished by the development of sessile or terminal pyriform
or clavate aleurioconidia on short or long branches and numerous in-
tercalary cylindrical or barrel-shaped arthroconidia (i.e., alternate
arthroconidia; see Fig. 7). Conidia are fairly large, and measure 6 to
20 by 3 to 5(8)µm. Ascomata usually develop within 3 weeks and are
globose yellowish-brown cleistothecia containing yellowish-brown,
ovoid, irregularly reticulate ascospores measuring 4 to 5 by 2.5 to 3.5
µm.
Comments. Species of Aphanoascus occur in soil. They are strong kera-
tinophiles and commonly isolated by hair bait techniques. Isolates are
often obtained in the anamorphic stage, and with the exception of A.
fulvescens, it can be difficult to obtain or maintain the teleomorph in
culture. The anamorph of A. fulvescens has been suggested to be C.
keratinophilum, but the teleomorph of the latter is a different species.
The data of Leclerc et al. (14) place the two species on one branch
of the phylogenetic tree of the Onygenales.
b. Chrysosporium zonatum Al Musallam & Tan, Persoonia 14:69, 1989
(Fig. 8).
Synonym: C. gourii P.C. Jain, Deshmukh & S.C. Agrawal, Mycoses 36:
77, 1993.
Teleomorph: Uncinocarpus orissi (B. Sur & G.R. Ghosh) Sigler & Flis,
Can J Bot 76:1627, 1998.
Synonyms: Pseudoarachniotus orissi B. Sur & G.R. Ghosh, Kavaka 12:
67, 1985; Gymnoascus arxii Cano & Guarro, Stud Mycol 31:61, 1989.
Figure 8 Chrysosporium zonatum (Uncinocarpus orissi).
Occurrence as a pathogen. Known from two cases of pulmonary coloni-

zation and a single case of disseminated infection involving the lung
and bone in a patient with chronic granulomatous disease (59, 60).
Description. Detailed descriptions of the colonial and microscopic mor-
phologies have been published recently (57, 59). C. zonatum is
thermotolerant and cycloheximide tolerant. Colonies grow faster at
37°C than at 25°C, reaching diameters of 7.5 to 8 cm in 14 days. They
are initially yellowish-white but darken to buff, especially on potato
dextrose agar at the higher temperature. Aleurioconidia are borne at
the ends of short or long stalks that are characteristically curved, or
are sessile. They are smooth or warty, clavate or broadly obovoid,
rounded at the tip, and measure (3.5) 4 to 8 (13) by (2.5) 3 to 5 µm.
Intercalary arthroconidia are rare. C. zonatum is heterothallic, and the
teleomorph is obtained by mating compatible isolates. Ascomata are
discrete, more or less globose gymnothecia composed of reddish-
brown ascospores surrounded by loose wefts of undifferentiated rac-
quet hyphae that may form conidia on side branches. Ascospores are
oblate, appearing round in face view and flattened with truncate ends
in side view, and measure 4.5 to 7 by 3 to 4.5 µm. They appear smooth
to minutely roughened under light microscopy and pitted under SEM
(57).
Comments. This strongly keratinolytic and thermotolerant species ap-
pears to have a broad geographic distribution in subtropical and
warmer temperate regions. The anamorph and teleomorph have been
described independently and under more than one name; the connec-
tions among them have been established through matings (57).
c. Chrysosporium anamorph of Nannizziopsis vriesii (Apinis) Currah, My-
cotaxon 24:164, 1985.
Occurrence as a pathogen. Reported as the cause of cutaneous mycoses
in lizards and snakes with rare invasion into deep tissues (61, 62).
222 Sigler
Description. Detailed descriptions of the tissue and colonial and micro-

scopic morphologies have been published recently (61, 62). Asexual
isolates of this fungus have come from several infected reptiles. Colo-
nies are moderately fast growing, flat or umbonate, and sometimes
zonate (showing concentric zones of denser and thinner mycelium),
yellowish-white, and powdery. Growth is similar on media containing
cycloheximide but is highly restricted at 37°C. Aleurioconidia are pyr-
iform or clavate, single-celled or rarely one-septate, and commonly
sessile or borne at the ends or sides of short stalks that arise at 90°
angles. The conidia are commonly 3 to 6 by 1.5 to 2.5 µm, but may
be up to 12.5 µm in length. Arthroconidia measuring 1.5 to 3.5 by 4
to 9 µm long form either in an alternate position (i.e., separated by
cells that ultimately lyse) or in chains, and these are separated by
fission at the septum (i.e., schizolytic dehiscence). A characteristic
feature is the development of solitary lateral branches that are undulate
(wavy) and sparsely septate. Rarely these fragment to form arthro-
conidia. The teleomorph has been found in cultural conditions only in
two soil isolates. Ascomata are discrete, yellowish-white, and com-
posed of branched asperulate hyphae that are slightly constricted at the
septa. Ascospores are globose, punctate-reticulate, and measure 2.5 to
3 µm in diameter. Development of ascomata occurs optimally at 30°C.
Comments. Histopathologic sections of the skin lesions on affected rep-
tiles show presence of hyaline hyphae that in some instances erupt
through the surface of the epidermis to form aerial terminal arthrocon-
idia (61, 62). The sessile or short stalked aleurioconidia borne at right
angles to the hyphae are reminiscent of some Trichophyton species,
including T. mentagrophytes and T. terrestre, but this dermato-
phytelike fungus differs in its propensity to form schizolytic arthro-
conidia and undulate hyphae, in its failure to form macroconidia, and
in comparison with T. mentagrophytes, in its restricted growth at
37°C. Preliminary results from molecular genetic testing suggest that
the pathogenic anamorphic isolates are closely related to teleomorphic
soil isolates (63), but further testing is being done to assess the degree
of genetic diversity among them. Currah (3) placed Nannizziopsis in
the Onygenaceae, but Guarro et al. (64) treated it within the Eurotiales
and transferred some other species to the genus.
d. Notes on Saprophytic Species
i. Chrysosporium anamorph of Renispora flavissima Sigler, Gaur,
Lichtwardt & Carmichael, Mycotaxon 10:134, 1979 (Fig. 9). Colo-
nies are moderately slow growing, lemon yellow, and powdery.
The conidia are large, reaching a size similar to those of the macro-
Figure 9 Chrysosporium an. of Renispora flavissima.
conidia of Histoplasma capsulatum (Table 3) and demonstrating

tubercles on the surface. This is a soil fungus that is known only
from its original habitat in Kansas. Growth is strongly inhibited at
37°C and there is no conversion to a yeast phase. It is heterothallic,
forming gymnothecial ascomata in mated isolates.
ii. Chrysosporium keratinophilum (Frey) Carmichael, Can J Bot 40:
1157, 1962 (Fig. 10). Colonies are moderately rapid growing, flat,
dense, velvety to granular, yellowish-white. Large (mostly 10 to
12 by 6 to 8 µm), smooth, broadly pyriform conidia are formed in
clusters at the ends of acutely branched, unswollen stalks or ses-
sile (23). The teleomorph, Aphanoascus keratinophilus Punsola &
Cano, is rarely recovered, and the conditions for its inducement in
strictly anamorph strains are not yet known. As shown by Leclerc
et al. (14) and expected by their known teleomorphs, C. keratino-
philum and Aphanoascus fulvescens, cluster together in the phylo-
genetic tree of the Onygenales.
iii. Chrysosporium articulatum Scharapov, Nov. Syst. niz. Rast. 15:
146, 1978 (Fig. 11). The microscopic morphology is very similar
to the C. anamorph of Aphanoascus fulvescens (Fig. 7), but C. arti-
Figure 10 Chrysosporium keratinophilum.

224 Sigler
Figure 11 Chrysosporium articulatum.
culatum is not known to form a teleomorph, and colonies are more

rapid growing and usually cottony to woolly (23).
iv. Chrysosporium carmichaelii van Oorschot, Stud Mycol 20:15,
1980 (Fig. 12). Colonies are moderately slow growing, yellowish-
white, umbonate, velvety to woolly. Growth is enhanced in the
presence of the vitamin thiamine but there is no growth at 37°C.
Fertile hyphae bear small aleurioconidia (mostly 3.5 to 4 by 2.5 to
3 µm) at the ends or on the sides of short, often slightly curved
branches that often occur in dense clusters associated with sterile
undulate or curved hyphae (23).
v. Chrysosporium lobatum Scharapov Nov. Syst. niz. Rast. 15:144,
1978 (Fig. 13). Colonies are slow growing, velvety or felty, with
an irregular lobate margin, initially pale grayish green and often
developing pinkish-grey to violet tints in the center and diffusible
brown pigment. Colonies are stimulated by the vitamin thiamine.
Small (mostly 2.5 to 3.5 by 2 to 2.5 µm), smooth to rough conidia
form at the ends of short, very narrow, peglike stalks that arise at
a 90° angle from the vegetative hyphae and occur in clusters (23).
Figure 12 Chrysosporium carmichaelii.

Figure 13 Chrysosporium lobatum.
4. Malbranchea Saccardo
Type species: M. pulchella Saccardo & Penzig.
Teleomorphs. Teleomorphs of keratinolytic species of Malbranchea are
placed in the genera Aphanoascus, Auxarthron, and Uncinocarpus and
some other genera of the Onygenaceae. Some cellulolytic species have
known or inferred affinities within the genus Myxotrichum.
Comments. There is no evidence to confirm a pathogenic role for any species
of Malbranchea. A few species are seen occasionally as clinical contami-
nants. It has been shown that the arthroconidia of some species survive
in and may be recovered from animal tissue after inoculation (50). The
genus encompasses species producing alternate arthroconidia, (i.e., arthro-
conidia that are separated from each other by one or more cells that un-
dergo lytic dehiscence). Sigler and Carmichael (50) divided the species
into two groups: one containing species in which the fertile hyphae are
strongly curved or arcuate and the other in which they are straight. Species
among the latter group show closest morphologic similarity to Coccidi-
oides immitis, and two of the species described were based on isolates that
had previously been thought to represent atypical isolates of C. immitis. In
a key to the species, the Malbranchea anamorph of Uncinocarpus reesii
was placed in a couplet with C. immitis as the most similar species, and
a close phylogenetic relationship between these species has subsequently
been demonstrated by molecular methods (11, 54).
Notes on Saprophytic Species.
a. Malbranchea anamorph of Uncinocarpus reesii Sigler & Orr, Myco-
taxon 4:462, 1976 (Fig. 14). Colonies are moderately fast growing,
flat, dense, velvety to powdery, yellowish-white to buff. Growth is
strongly to moderately inhibited at 37°C. Arthroconidia are separated
by one or more cells of irregular length, and the intervening cells often
show signs of collapse (23, 50, 57). Arthroconidia are cylindrical to
slightly barrel-shaped, club or slightly wedge-shaped if terminal, and
measure 3.5 to 6 (8) by 2.5 to 3.5 (4) µm. Thick-walled, brown uncinate
(curved at the tip) appendages develop from the vegetative hyphae in
many isolates. U. reesii is heterothallic and when compatible strains are
226 Sigler
Figure 14 Uncinocarpus reesii showing ascocarp and Malbranchea anamorph (not

drawn to same scale). Source: ascomata  R.S. Currah (used with permission).
mated, the resultant gymnothecia are composed of a loose net-

work of these uncinate appendages (23, 50, 57). Ascospores are oblate
with truncate or slightly rounded apices and are ornamented on the
surface with small pits that are hardly discernible by light micro-
scopy. They are pale yellowish-brown individually and reddish-brown
in mass.
Comments. The closely spaced, fairly broad arthroconidia of Coccoidi-
oides immitis are unlikely to be confused with those of Malbranchea
species, and isolates suspected to be C. immitis may be confirmed by
the use of probe or exoantigen tests.
b. Malbranchea gypsea Sigler & Carmichael, Mycotaxon 4:455, 1976 (Fig.
15). Colonies are slow growing, white, often furrowed and velvety. Cy-
lindrical to slightly barrel-shaped, narrow arthroconidia measuring (2.5)
3 to 6(9) by 2 to 2.5 µm are formed in straight, branched fertile hyphae
and separated by one or more cells (23, 50).
Figure 15 Malbranchea gypsea. Bar approximately 5 µm.

Comments. This slow-growing fungus is unlikely to be confused with

C. immitis. Its recovery from a dystrophic nail on three occasions is
suggestive of a possible role in onychomycosis, but its role has not
been substantiated (23).
B. Family Gymnoascaceae
Members of the Gymnoascaceae are a diverse group (3), and Currah (4) has
suggested that some taxa may belong outside the family. They are not keratino-
lytic and not or only weakly cellulolytic as determined by in vitro methods. Asco-
spores are smooth or ornamented with thickened knobs and are oblate or discoid,
sometimes with one or two longitudinal ridges. Anamorphs are lacking for many
species or are arthroconidia or aleurioconidia. (See also Table 1.)

1. Onychocola canadensis Sigler & Congly, J. Med. Vet. Mycol. 28:409, 1990
(Fig. 16). Teleomorph: Arachnomyces nodosetosus Sigler & Abbott, J Med
Vet Mycol 32:280, 1994.
Colonies are very slow growing, often digging into the agar and
eventally cracking it. They are initially glabrous, yellowish-white to pale
grey, gradually developing floccose tufts of white to grey aerial mycelium
and often developing diffusible tan pigments. Isolates are cycloheximide-
tolerant. Arthroconidia are formed in the aerial mycelium and are one- or
two-celled, initially cylindrical but commonly rounding up, and remaining
connected in adherent chains (35, 65). Dehiscence occurs by splitting
(schizolysis) of adjacent conidia or by lysis of thin-walled segments of the
hyphae. Detached conidia measure 4 to 8 by 2 to 5 µm if 0-septate and 8
to 17 by 2.5 to 5.5 µm if 1-septate. Brown, knobbed, uncinate, or spiralled
appendages form in older cultures and especially on media such as phytone
Figure 16 Onychocola canadensis arthroconidia and seta.

228 Sigler
yeast extract or blood agar. O. canadensis is heterothallic, but the teleomorph

has been obtained among matings of only a few isolates after prolonged
incubation. Ascomata are globose cleistothecia, and bear knobbed append-
ages; ascospores are oblate, pale brown, and smooth (65, 66).
Comments. As mycologists have gained familiarity with this slowgrowing
and often poorly sporulating fungus, they have recorded its isolation from
nails, and less commonly from the skin of feet or hands in geographically
separated areas (Canada, Australia, New Zealand, the United Kingdom,
and Europe). Gupta et al. (67) have suggested that O. canadensis may be
isolated occasionally from dystrophic nails without evidence of pathology.
In one unusual finding, it was incidentally isolated from a bronchial wash-
ing (Sigler, unpublished data).
2. Gymnascella dankaliensis (Castellani) Currah, Mycotaxon 24:77, 1985 (Fig.

17). Colonies are moderately slow growing and appear yellowish-white and
sterile on routine isolation media. Ascosporulation occurs on media such as
oatmeal salts and Takashio or Leonian’s agars (35), and as ascospores ma-
ture, colonies darken to yellowish- or brownish-orange. Asci develop as na-
ked clusters sometimes associated with slightly differentiated hyphae. Ma-
ture ascospores are oblate and pale orange-brown, and measure 5.5 to 7 by
3 to 4.5 µm (3, 68). They appear round with irregular thickenings in face
view and rhomboidal (broader in the center than on the ends) with a longitu-
dinal rim and polar thickenings in side view. No anamorph is formed.
Comments. This weakly cellulolytic species is mainly recorded from the soil
of warmer climates. In the one reported case of nail infection, hyphal
elements in tissue were irregularly swollen (68).
Figure 17 Gymnascella dankaliensis ascospores. (Note that ascospores shown here are
drawn to a different scale than conidia shown in other figures.) Bar approximately 3 µm.
Source:  R. S. Currah (used with permission).
Figure 18 Gymnascella hyalinospora ascospores (Note that ascospores shown here are
drawn to a different scale than conidia shown in other figures.) Bar approximately 3 µm.
Source:  R.S. Currah (used with permission).
3. Gymnascella hyalinospora (Kuehn, Orr & Ghosh) Currah, Mycotaxon 24:

84, 1985 (Fig. 18). Colonies are moderately slow growing, initially white
and glabrous or with fine nap of aerial mycelium, and gradually form sectors,
patches, or tufts of yellowish-orange to yellowish-green aerial hyphae on
potato dextrose or oatmeal salts agar. G. hyalinospora is thermotolerant and
slightly cellulolytic. Asci develop in the colored areas of the colonies and
are naked or loosely associated with yellow, slightly thick walled hyphae
(3). Ascospores are oblate, yellow, smooth-walled under light microscopy,
and measure 2 to 3.5 by 2 to 2.5 µm. No anamorph is formed.
Comments. G. hyalinospora is mainly recorded from soil, and has been re-
covered rarely from lesions of humans and animals without confirmation
of a pathogenic role. A recent report documenting invasive pulmonary
infection in a immunosuppressed patient confirms its pathogenic potential
(69).
C. Family Myxotrichaceae
Members of the Myxotrichaceae have fusiform or ellipsoidal ascospores that are
striate or smooth; they degrade cellulosic substrates, and some have Geomyces
or Malbranchea anamorphs (3, 50). (See also Table 1.)

1. Geomyces pannorum (Link) Sigler & Carmichael, Mycotaxon 4:377, 1976
(Fig. 19). Colonies are slow growing, smooth or furrowed, white, tan, pale
yellow or pale grey, and glabrous, fasciculate, powdery or cottony in texture.
G. pannorum is tolerant of cycloheximide and is psychrophilic, growing bet-
230 Sigler
Figure 19 Geomyces pannorum.
ter at 18°C than at 25°C and failing to grow at 37°C. The species is distin-
guished by short, slender conidiophores that branch acutely at the tip to form
three to four verticillate branches (23, 30, 50). The septate fertile branches
develop basipetally into short chains of slightly swollen conidia that are sepa-
rated from each other by a short cell. Conidia may also develop on the sides
of the branch (sessile). Conidia are cuneiform (wedge-shaped) or barrel-
shaped, smooth or roughened, and measure 2 to 5 by 2 to 4 µm. G. pannorum
is a common fungus found in temperate soils worldwide. It is a regular con-
taminant, especially of nails, but it has not been substantiated as a cause of
onychomycosis (23). No teleomorph is known for isolates recognized as G.
pannorum. Geomyces vinaceus encompasses isolates with similar micro-
scopic features, but having colonies that are purplish-red or vinaceous; the
teleomorph is Pseudogymnoascus roseus (23, 50).
2. Myxotrichum deflexum Berkeley, Ann. Nat. Hist. 1:260, 1838 (Figs. 20, 21).
Colonies are slow growing, yellowish-white to light grey, often developing
patches of pink or wine red, and expressing a pink diffusible pigment on
some media. With development of ascomata on media such as oatmeal salts
and Takashio or Leonian’s agars (35), colonies darken to grey or black. Asco-
mata are discrete or confluent and are composed of a meshlike network of
branched dark brown to black hyphae (3). Commonly the lateral branches
of the ascomatal hyphae are bent downwards (deflexed) and terminate in
hyaline filaments that eventually disintegrate. Ascospores are ovoid to fusi-
form, slightly striate, and measure 4 to 5.5 by 2.5 to 3.5 µm. Alternate arthro-
conidia are formed by some isolates. M. deflexum is uncommonly isolated
from nails, but has not been substantiated as a cause of onychomycosis.
3. Ovadendron sulphureo-ochraceum (van Beyma) Sigler & Carmichael, My-
cotaxon 4:392, 1976 (Fig. 22). Colonies are moderately slow growing, yel-
lowish-white to yellowish-green, and velvety. Narrow curved or loosely heli-
Figure 20 Myxotrichum deflexum ascocarp. Source:  R. S. Currah (used with permis-

sion).
Figure 21 Myxotrichum deflexum ascospores. Bar approximately 4 µm. Source:  R. S.

Currah (used with permission).
Figure 22 Ovadendron sulphureo-ochraceum.

232 Sigler
cally coiled lateral branches arise from the narrow vegetative hyphae. These
fertile branches become basipetally septate and develop into a chain of
slightly swollen arthroconidia separated by short cells. Mature arthroconidia,
released by lytic dehiscence, are barrel-shaped and measure 2.5 to 4 by 1.5
to 2.5 µm (50). O. sulphureo-ochraceum is a rare fungus that has been re-
ported as an agent of eye infection following lens implantation (70). It has
been isolated several times from a patient with suspected mycotic keratitis
and incidentally recovered from sputum. It is moderately cellulolytic and
has been recovered from decayed wood. Although it has been reported to
be slightly keratinolytic (50), this capability requires retesting with additional
available isolates.
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7
Ascomycetes
Aspergillus, Fusarium, Sporothrix, Piedraia, and
Their Relatives
Richard Summerbell
Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands
This chapter also includes pathogenic and opportunistic members of the Eu-
rotiales, Hypocreales, Ophiostomatales, and Pseudeurotiaceae ss. str., plus the
families Didymosphaeriaceae and Piedraiaceae of the order Dothideales and
mycetoma-causing members of the genus Leptosphaeria (Pleosporales).
I. INTRODUCTION
The hierarchical classification of organisms into taxa begins with the species,
and then advances to the genus, family, order, class, phylum or division, and
kingdom. Each of these major levels may also contain some recognized subtaxa
(e.g., species may contain varieties). In the years immediately prior to the advent
of molecular phylogeny, the families, orders, and classes of fungi in the phylum
Ascomycota were in disrepute. It was realized that they were partially based on
relatively uncomplicated form characteristics that could easily have evolved con-
vergently, so that to some extent they represented artificial groups rather than
naturally biologically related groups. Fortunately, studies based on sequencing
genes such as the 18S and 26S ribosomal subunits have allowed more natural
groupings of ascomycetous fungi to be clarified. To a large extent, the traditional
taxonomy of these organisms is retained, while the correct assignment of species
in groups long suspected of heterogeneity, such as the Pseudeurotiaceae (1), is
237
238 Summerbell
facilitated. Currently, the orders Eurotiales and Hypocreales appear to be sup-

ported by molecular phylogenetic studies, although the latter is paraphyletic if
its offshoot, the Clavicipitales or Clavicipitaceae, is considered a separate order.
In the past, formally delineated biological groups based on sexual species
were kept separate from anamorphic species described based on asexual states.
The reason was that the asexual states usually did not display sufficient informa-
tion to allow them to be meaningfully biologically grouped. Recently, however,
ongoing biological studies, particularly those involving sequencing, have dis-
closed the biological affinities of the majority of anamorphs of medical and eco-
nomic interest, if these were not already known. In the presentation below, there-
fore, anamorphic species are represented along with teleomorphic species in the
orders and families named. It should be noted, however, that anamorphs are still
accorded separate generic names; therefore, even though Sporothrix schenckii is
very closely related to the teleomorph genus Ophiostoma, it remains in the sep-
arate anamorph genus Sporothrix under our current nomenclatural rules and
does not become ‘‘Ophiostoma schenckii’’ (as one recent publication erroneously
named it). The close teleomorphic affinities of anamorphs, where they are known,
will be emphasized in this chapter through the device of inserting the name of
a sufficiently closely related teleomorph genus in braces between anamorph genus
name and epithet in the primary entry for the species (e.g., Sporothrix {aff. Ophi-
ostoma} schenckii). This is an extension of the traditional device of inserting
additional information about superseded relationships into the middle of binomi-
als [e.g., Candida (Torulopsis) glabrata]. As with its prototype, the device used
here should not be taken to imply a new nomenclatural combination. Again, as
with the prototype, it is not a formal nomenclatural usage, but rather a literary
one. The standard taxonomic abbreviation ‘‘aff.’’ (affine) is used both to obstruct
the appearance of a new coinage and also to indicate that in current taxonomy,
where Hennigian phylogenetic principles are not always strictly adhered to, a
highly phenotypically divergent teleomorph might still be placed in a separate
genus, even if phylogenetic indications seemed to contradict this. The prototype
for this decision is the genus Homo, which is generally held apart from the
chimpanzee/bonobo genus Pan, even though cladistic analysis tends to amalgam-
ate the genera into a revised genus Homo (2).
In this chapter, emphasis is given to the form in which the fungus is most
likely to be seen in the clinical laboratory. In cases in which one morph is very
unlikely to be seen in the clinical lab, it is generally mentioned but not described.
It should be noted that some phylogenetic studies [e.g., that of Suh and
Blackwell (1)] have found the affinity between the Eurotiales (classically consid-
ered to contain the holomorphs of Aspergillus, Penicillium, and relatives) and
the Onygenales (containing the holomorphs of dermatophytes, Blastomyces, Coc-
cidioides, and relatives) to be so great that the two orders are combined into an
Ascomycetes 239
expanded concept of the Eurotiales. In fact, this concept coincides with some
older concepts that unified these two groups, such as the concept used by de
Vries (3). This expanded concept is not followed here, but the close relationship
between these two medically important groups is noted with interest. Possibly
in the future additional phylogenetic studies will generate sufficient supporting
evidence that the two groups can comfortably be combined.
A. Notes on the Descriptions

Unless mentioned otherwise, descriptions are based on growth on modified Leo-
nian’s agar (4) at 7 days at 25°C in the dark. For most fungi such descriptions
differ minimally from those obtained on other common fungal sporulation media
with moderate nutrient levels, such as malt extract agar and potato glucose agar.
Colonial appearances and growth rates may differ substantially on media low in
nutrients, such as soil extract agar, or high in nutrients, such as any formulation
of Sabouraud’s peptone-glucose agar.
B. A Note on the List of Included Species

It is axiomatic that in a book entitled Pathogenic Fungi of Humans and Animals
the described species should be pathogenic. On the other hand, medical literature
concerning less common opportunistic fungi contains a nearly overwhelming
number of spurious case reports based on false attributions of pathogenicity to
contaminating organisms, as well as similarly misleading reports based on mis-
identified organisms. Also, many likely credible reports have been rendered ‘‘ir-
reproducible results’’ from the technical point of view because the authors did
not describe unusual organisms well enough that identifications could be verified,
and/or did not deposit voucher cultures in professional culture collections. Medi-
cal mycology is an unusual mycological subfield to review, in that a high propor-
tion of the published work is the product of professionals in intersecting fields
who are nonetheless mycological amateurs, whether physicians or laboratory
technologists, and the result, as with all work done by amateurs, covers the whole
range from thoroughly professional to tragicomic. Considerable effort, however,
has been made to ensure that the present chapter is minimally influenced by
erroneous literature, including that extensively cited in other books and reviews.
In order to inform readers why some apparent records of pathogenicity have been
rejected and to correct the record in general, some notes are included on rejected,
reinterpreted, or questionable case reports. These appear in the general paragraph
discussing the pathogenicity of described organisms and occasionally in notes
on species previously reported as pathogenic for which no cases can be verified.
In general, every effort has been made to avoid making or recapitulating ambigu-
240 Summerbell
ous statements such as ‘‘this species has been isolated from infected skin’’ or
‘‘this fungus has been reported from peritonitis,’’ which, despite their apparent
promise of etiologic relevance, may well contain no medical information whatso-
ever and in some cases may further the accumulation of a pseudo-literature sup-
porting and promoting diagnostic error.
Mycology is entering an era in which molecular identification will greatly
increase the accuracy of case reports involving unusual organisms. It is therefore
useful to know which elements of the past 100 years of medical literature are
suitably reliable, based on standard criteria of taxonomic documentation and re-
producibility, in order to be meaningfully compared with the results that will be
generated in upcoming decades. Also, since more accurate identification methods
can in some cases simply generate better quality, spurious reports imputing etiologic
status to incidental organisms, the basic gold standards of medical fungal ecology
that are used in the reliable confirmation of cases are rather rigorously adhered
to in the text below. For the sake of accuracy, no charity is given to ambiguity.
II. EUROTIALES
The order Eurotiales, as roughly defined here, contains three families: Eremomy-
cetaceae, Thermoascaceae, and Trichocomaceae. By far the greatest number of
fungal pathogens is in the last-named family. Eremomycetaceae are represented
in medical mycology only by Arthrographis kalrae, the anamorph of Eremo-
myces langeronii. It is dealt with in Chap. 11. The family Thermoascaceae con-
tains Thermoascus and Byssochlamys species, as well as some related Paecilo-
myces anamorphs, by far the most biomedically prominent of which is P. variotii.
Another medically important Paecilomyces species, P. lilacinus, is completely
unrelated, with affinities in the order Hypocreales, family Clavicipitaceae, rather
than Eurotiales. It is therefore included in the section of this chapter devoted to
Hypocrealean fungi rather than being retained with P. variotii. The major human
and animal pathogens in the Eurotiales consist of medically important aspergilli,
as well as Penicillium marneffei and P. variotii.
A. Aspergillus Fr: Fr.

The medically important members of the genus Aspergillus comprehend ana-
morphs related to seven teleomorph genera: Emericella, Eurotium, Fennellia,
Hemicarpenteles, Neopetromyces, Neosartorya, and Petromyces. Teleomorphs
are described where species forming them have been recorded in connection with
human and animal disease. In addition, the recently described Neopetromyces is
added in order to distinguish it from the similar Petromyces. Species are described
in biological groups rather than in pure alphabetical order. The reason for this
Ascomycetes 241
organization is that it highlights the biological patterns that emerge when Asper-
gillus species are considered in the context of their natural relationships. These
are as outlined by Samson (5), with some modifications to reflect recent molecular
studies (6). Within each natural subgroup, teleomorphic species are considered
before anamorphic species, and within formal taxonomic sections of the genus,
the namesake and type species of the section is treated first, then related species
are listed in alphabetical order beneath it (e.g., A. versicolor is listed first in the
section Versicolores). In the section Aspergillus, where there is no such name-
sake, species are just listed alphabetically.
To make species easy to find, Table 1 displays the species in the order in
which they appear in this chapter. At the same time, it summarizes the teleo-
morphs most closely corresponding to common anamorphs seen in the clinical
lab. See also the rapid ‘‘Aspergillus finder’’ (Table 2).
An overview of the clinically important aspergilli and their characteristic
features is presented in Table 3.
In nature, aspergilli are diversely competent, mostly generalist saprobes,
usually possessing most (but not all) of the following capabilities: thermotoler-
ance, osmotolerance, cellulose degradation, opportunistic human/animal patho-
genesis, plant seed pathogenesis/degradation, insect pathogenesis/degradation,
and hydrocarbon degradation. Some species are particularly strongly specialized
for osmotolerance; that is, growth on materials of low water activity. Although
aspergilli are commonly called ‘‘soil fungi,’’ in biomedical literature they are
common in soil only in tropical or subtropical areas, deserts, some grasslands,
and indoor plantings. In any case, soil is only one of their many characteristic
niches.
In general the anamorph genus Aspergillus consists of fungi that have no
melanized elements, excepting black conidia in some species. They generally
form erect, aseptate, thick-walled conidiophores with an expanded vesicle at the
apex and a thick-walled ‘‘foot cell’’ integrating more or less at a 90° angle into
the subtending hypha in the growth medium at the base. On the apical vesicle,
fertile elements are formed synchronously (not sequentially, as in members of
the genus Penicillium); that is, primordia of fertile elements in the initial stages
of formation can be seen to be all the same size on each individual vesicle. Fertile
elements may consist of phialides alone or of short branches (metulae) bearing
clusters of phialides apically. Conidia are formed in interconnected chains and
are strongly hydrophobic. Conidia en masse may be black, white, or ‘‘brightly
colored’’ (e.g., green, blue, yellow, sandy-brown, ochraceous).
1. Eurotium Link: Fr.

Ascomata are globose cleistothecia, 100 to 150 µm in diameter, and usually bright
yellow to orange-red. Asci are eight-spored, globose, and evanescent. Ascospores
242
Table 1 Natural (Teleomorph-Based) Groups of Aspergillus Species as Arranged in This Chapter
Aspergillus subtaxon
Raper/Fennell Species:
Teleomorph Subgenus Section group teleomorph, (anamorph)
Eurotium Aspergillus Aspergillus A. glaucus grp. Eurotium amstelodami/(Aspergillus vitis)
E. chevalieri/(A. chevalieri)
E. herbariorum/(A. glaucus)
E. repens/(A. reptans)
E. rubrum/(A. rubrobrunneus)
Restricti A. restrictus grp. (A. restrictus)
(A. caesiellus)
(A. conicus)
(A. penicillioides)
Emericella Nidulantes Nidulantes A. nidulans grp. Emericella nidulans/(A. nidulans)
E. dentata/(A. nidulans var. dentatus)
E. echinulata/(A. nidulans var. echinulatus)
E. quadrilineata/(A. tetrazonus)
E. rugulosa/(A. rugulovalvus)
E. unguis/(A. unguis)
Versicolores A. versicolor grp. (A. versicolor)
(A. granulosus)
(A. janus)
(A. sydowii)
(A. varians)
Usti A. ustus grp. (A. ustus)
(A. deflectus)
Summerbell
Fennellia Nidulantes Flavipedes A. flavipes grp. Fennellia flavipes/(A. flavipes)
F. nivea/(A. niveus)
(A. carneus)
Terrei A. terreus grp. (A. terreus)
‘‘Circumdati’’ a Candidi A. candidus grp. (A. candidus)
Ascomycetes
Neopetromyces Circumdati Circumdati A. ochraceus grp. (A. ochraceus)

(A. sclerotiorum)
Petromyces Flavi A. flavus grp. Petromyces alliaceus/(A. alliaceus)
(A. flavus)
(A. oryzae)
(A. avenaceus)
(A. tamarii)
Nigri A. niger grp. (A. niger)
(A. japonicus)
Neosartorya Fumigati Fumigati A. fumigatus grp. Neosartorya pseudofischeri/(A. thermomu-
tatus)
N. fischeri/(A. fischerianus)
(A. fumigatus)
Hemicarpenteles Clavati A. clavatus grp. (A. clavatus)
(A. clavato-nanica)
a
Recent molecular studies have shown that this group was particularly strongly displaced from its natural affinities by phenotypic taxonomy, and it is
arranged with its related groups. The related subgeneric nomenclature, however, has not yet been changed.
243
244 Summerbell
Table 2 Aspergillus Finder [Look up the Aspergillus or

Teleomorph Name (or Common Synonym) Alphabetically
and Locate It in the Biological Arrangement]
Related Page
Name teleomorph group number
Aspergillus alliaceus Petromyces 298

A. amstelodami Eurotium 252
A. avenaceus Petromyces 303
A. caesiellus Eurotium 264
A. candidus Fennellia 290
A. carneus Fennellia 288
A. chevalieri Eurotium 256
A. clavatonanica Hemicarpenteles 315
A. clavatus Hemicarpenteles 313
A. conicus Eurotium 266
A. deflectus Emericella 283
A. fischeri Neosartorya 308
A. flavipes Fennellia 285
A. flavus Petromyces 298
A. fumigatus Neosartorya 310
A. glaucus Eurotium 257
A. granulosus Emericella 277
A. janus Fennellia 278
A. japonicus ?Petromyces 306
A. nidulans Emericella 267
A. niger ?Petromyces 304
A. niveus Fennellia 286
A. ochraceus Neopetromyces 294
A. oryzae Petromyces 301
A. penicillioides Eurotium 266
A. quadrilineatus Emericella 271
A. reptans Eurotium 262
A. restrictus Eurotium 263
A. rubrobrunneus Eurotium 263
A. rugulosus Emericella 272
A. sclerotiorum Neopetromyces 295
A. sydowii Emericella 279
A. tamarii Petromyces 303
A. terreus Fennellia 290
A. tetrazonus Emericella 271
A. unguis Emericella 274
A. ustus Emericella 281
A. varians Emericella 280
Ascomycetes 245
Table 2 Continued
Related Page
Name teleomorph group number
A. versicolor Emericella 275

A. vitis Eurotium 252
Emericella dentata (T) 270
E. echinulata (T) 270
E. nidulans (T) 267
E. quadrilineata (T) 271
E. rugulosum (T) 272
E. unguis (T) 274
Eurotium amstelodami (T) 252
E. chevalieri (T) 256
E. herbariorum (T) 257
E. repens (T) 262
E. rubrum (T) 263
Fennellia flavipes (T) 285
F. nivea (T) 286
Neosartorya fischeri (T) 308
N. pseudofischeri (T) 308
N. spinosa (T) 308
Petromyces alliaceus (T) 298
Note: (T) indicates teleomorph names.
are rough or smooth, oblate, usually with at least one of the following: equatorial
crests, an equatorial furrow, or a distinct flattened equatorial band. The anamorphs
are members of Aspergillus subgenus Aspergillus section Aspergillus, formerly
considered the A. glaucus series (7). They have uniseriate conidiophores.
Species with a Known Teleomorph and with Anamorphs in A. Subgenus
Aspergillus Section Aspergillus (Formerly Aspergillus glaucus Group). This
is a highly unified and distinct group of species, in which most isolates readily
form the teleomorph homothallically in culture on common media, and do so
even more reliably on concentrated media used for osmotolerant fungi. In ecol-
ogy, members of this group are osmotolerant colonizers of dry substrates or other
substrates with low water activity due to high solute concentrations (e.g., salted
materials, high-sugar substrates). They are thus often encountered in food my-
cology, and are also major colonizers of household dust, where they may
grow on relatively dry shed-skin scales. Growth on leather stored in slightly hu-
mid conditions, as well as other semidry surfaces, is common. Their isolation
Table 3 Laboratory Characters of the Medically Important Aspergilli and Species Frequently Confused with Them
Aspergillus
246
Species head structure Typical colony surface color Stipe Special features
Uniseriate group
Eurotium amstelodami Uni a Dull green with yellow tufts Smooth Ascospores with central
(Aspergillus vitis) (ascomata) groove and rough valves
E. chevalieri (A. Uni Dull green with yellow tufts Smooth Ascospores with central
chevalieri) (ascomata) groove and two broad sur-
rounding ridges; valves
smooth
E. herbariorum Uni Dull green with yellow, red, Smooth Ascospores with shallow
(A. glaucus) and orange tufts (ascomata groove and sometimes
and sterile hyphae) small ridges; valves smooth
E. rubrum Uni Dull green with yellow, red, Smooth Ascospores with shallow
and orange tufts (ascomata groove, valves smooth;
and sterile hyphae) growth rate on special me-
dia differs from E. herbari-
orum
E. repens Uni Dull green with yellow to Smooth Ascospores with flat equato-
orange tufts (ascomata rial band; valves smooth
and sterile hyphae)
A. restrictus Uni Dark green Smooth Small colonies; small vesicles
with persistent chains of
long-ellipsoidal conidia; no
growth at 42°C
A. fumigatus Uni Blue-green to smoky blue- Smooth, greenish Conidial masses in upright col-
green umns; good growth at 42°C
A. fumigatus Uni or Dirty white to pale green Poorly formed No conidiation, or aberrant,
(dysgonic) irregular small heads; conidia also
Summerbell
sometimes aberrant; good

growth at 42°C
Neosartorya pseudo- Uni Creamy (white ascomata) Smooth Ascospores with erect triangu-
fischeri lar flaps on valves, a central
groove, and broad sur-
rounding ridges
N. fischeri Uni Creamy (white ascomata) Smooth Ascospores with netlike reticu-
lum on valves, plus central
Ascomycetes
groove and surrounding

ridges
N. spinosa Uni Creamy (white ascomata) Smooth Ascospores with small conical
spines on valves, plus cen-
tral groove and surrounding
ridges
A. clavatus Uni Blue-green Smooth Elongate club-shaped vesicles
A. japonicus Uni Purple-black Smooth Large, round vesicle; conidia
spiny
Uni- or biseriate group (all with rough, apically expanding stipes)
A. flavus Uni or bi a Yellow-green Roughened, expanded Metulae extend around whole
toward apex vesicle surface; conidia
smooth to finely roughened
A. parasiticus Mostly uni, Dark olive green Roughened, expanded Metulae if present extend
few bi toward apex around whole vesicle sur-
face; conidia strongly rough-
ened
A. tamarii Uni or bi Olive brown Roughened, expanded Metulae extend around whole
toward apex vesicle surface; conidia
strongly roughened
Biseriate group with metulae surrounding vesicle
A. avenaceus Bi Dull olive green Almost smooth (very Metulae extend around whole
fine roughening); vesicle surface; black sclero-
247
parallel sides tia present

Aspergillus
A. ochraceus Bi Pale yellow-brown Roughened, parallel Metulae extend around whole

sides vesicle surface; conidia
smooth or finely rough; scle-
rotia absent or pink/purple
A. sclerotiorum Bi Pale yellow Roughened, with parallel Metulae extend around whole
sides vesicle surface; conidia
smooth or finely rough; scle-
rotia abundant, cream-
colored
A. niger Bi Dirty black Smooth Metulae extend around whole
vesicle surface; conidia
roughening irregular ridges
and bars
A. japonicus b Uni Purple-black Smooth No metulae; conidia rough-
ening small conical spines
A. candidus Bi White Smooth or finely rough- Metulae extend around whole
ened, hyaline vesicle surface; no aleurio-
conidia in substrate myce-
lium
Biseriate group with metulae on upper portion of vesicle
A. flavipes Bi Pinkish-buff Smooth or finely rough; Metulae from upper 3/4 vesi-
brownish cle; aleurioconidia present
in substrate mycelium
A. niveus Bi White Smooth, hyaline Metulae from upper 2/3 of of
vesicle; aleurioconidia may
Summerbell
be detected
A. carneus Bi Pinkish to pinkish brown Smooth, hyaline or faint Metulae from upper 2/3 of
brown vesicle; irregularly disposed
phialides may be seen; aleu-
rioconidia in substrate myce-
lium
A. terreus Bi Sandy brown Smooth Metulae from upper 2/3 of
vesicle in nearly parallel ar-
Ascomycetes
rangement; conidial masses

columnar; aleurioconidia in
substrate mycelium
Emericella nidulans Bi Forest green, later with yellow Smooth, brown Metulae from upper 3/4 of
(A. nidulans) tufts (ascomata) vesicle; ascospores purple-
red with two crests, valves
smooth; round hülle cells
present
E. echinulata Bi Forest green, later with yellow Smooth, brown Metulae from upper 3/4 of
tufts (ascomata) vesicle; ascospores purple-
red with two crests; valves
with small spines; round
hülle cells present
E. quadrilineata Bi Dull green, later with yellow Smooth, brown Metulae from upper 3/4 of
tufts (ascomata) vesicle; ascospores purple-
red with four crests, two
large ones flanked by two
smaller ones; valves
smooth; round hülle cells
present
E. rugulosa Bi Dull green, soon yellow to Smooth, pale brown Metulae from upper 3/4 of
purple brown with heavy vesicle; ascospores purple-
ascoma formation red with two crests, valves
rugose (irregular folded
249
roughening); round hülle

cells present
Aspergillus
A. unguis Bi Dull green Smooth, brown Metulae from upper 2/3 of

vesicle; ascomata usually
not formed; colony surface
bears erect spinelike hyphae
(‘‘setae’’)
A. versicolor Bi Grey-green, emerald green, Smooth, hyaline Metulae from upper 3/4 of
pinkish or tawny vesicle; round hülle cells
sometimes present; small
penicillate conidiophores
may be present
A. sydowii Bi Turquoise grey to dull blue- Smooth, hyaline Metulae from upper 3/4 of
green vesicle; small penicillate co-
nidiophores may be present
A. granulosus Bi Dull green to purple-brown Smooth, hyaline Metulae from upper 3/4 of
vesicle; colony surface with
clumps of hülle cells
A. ustus Bi Grey-brown Smooth, brown Metulae from upper 3/4 of
vesicle; colony reverse yel-
low; long, sinuous hülle
cells may be present
A. deflectus Bi Grey-brown Smooth, red-brown, vesi- Metulae from upper 3/4 of
cle deflected abruptly vesicle; colony reverse yel-
downward low to reddish, sinuous
hülle cells may be present
a
uni ⫽ uniseriate; bi ⫽ biseriate.
Summerbell
b
A. japonicus is given a second entry to facilitate comparison with A. niger.
Ascomycetes 251
and growth are favored by high-solute media not used in medical mycology, such
as Czapek’s ⫹ 20% sucrose agar and dichloran 18% glycerol agar, but most
isolates will grow and sporulate, at least to some degree, on common medical
mycology sporulation media such as potato dextrose agar. In secondary metabo-
lism, the members of the group investigated so far form a similar spectrum of
compounds, including physcion and echinulin, which are not considered myco-
toxins in the strict sense of Frisvad and Thrane (8).
The species in this group have often not been distinguished from one an-
other, and the group as a whole—or A. glaucus used as a general term—has a
pathogenic record that cannot be attributed to individual species. In some cases
authors have attributed responsibility to A. glaucus, but lack of detail—and in
larger surveys, failure of more common Eurotium species to appear—leads one
to suspect that all Eurotium anamorphs were lumped under this name. Nonethe-
less, such attributions will be discussed under E. herbariorum (anamorph A. glau-
cus) below. All species in Aspergillus subgenus Aspergillus produce small asper-
gilli with uniseriate heads, leading to possible confusion with dysgonic (poorly
and aberrantly sporulating), host-adapted isolates of A. fumigatus, as is illustrated
in several definite examples of mistaken identity mentioned under A. subgenus
Aspergillus, section Restricti below. Only a small number of records contain suf-
ficient detail to allow verification that the identification was correct.
Young et al. (9) ascribed three cases of pathologically verified systemic
aspergillosis in immunocompromised patients to members of the A. glaucus
group, not further identified. These cases made up 5% of total aspergilloses seen
in a 39-patient survey (7.7% of the patients seen were attributed A. glaucus infec-
tion; the difference between the two percentages arises from the occurrence of
mixed aspergilloses in the patient population). One attributed A. glaucus group
infection was pulmonary, one disseminated, and one affected the spleen concomi-
tant with an A. fumigatus pulmonary infection. No identification details were
given. This report came from a prestigious institution (the National Institutes of
Health) and may well be legitimate, despite its absence of taxonomic documenta-
tion and its indefinite identifications. It raises, however, the question of why A.
glaucus group members did not continue to cause a similar proportion of invasive
aspergilloses through the swell of well-documented opportunistic mycoses of the
later 1970s and onward. In fact, as can be seen in the present writing, a high
proportion of the reports of invasive opportunistic mycoses caused by Aspergillus
species with small, green aspergilli, potentially confused with host-adapted A.
fumigatus (i.e., Aspergillus subgenus Aspergillus, and for workers experiencing
the initial difficulties of distinguishing biseriate and uniseriate structures, the sec-
tion Versicolores) are indeed from more than 25 years ago. This finding is difficult
to explain except by postulating at least a degree of influence arising from a shift
from less accurate to more accurate identification. The inclusion of the ‘‘A. glau-
cus group’’ in the bibliographically unsupported list of invasive aspergilloses
252 Summerbell
originated by Rinaldi (10) and reproduced and extended by Rippon (11) appears
to derive from the report of Young et al. (9), based on a comparison of the body
sites listed. Well-verified examples of comparable cases must be documented
before these records can be accepted.
Table 4 gives statistics on the number of reports obtained in the online
Medline database with keywords designed to roughly determine the proportion
of aspergilloses caused by different species in immunocompromised patients as
of August 8, 2000. (See the footnote in Table 4 for the keywords used.) The
records obtained were scanned to eliminate inappropriate entries, but no attempt
was made to enumerate multiple cases recorded in individual reports or to detect
overlap. None of the 435 accepted Medline reports on aspergillosis in immuno-
compromised patients featured A. glaucus or other members of section Aspergil-
lus in their searchable text. In comparison, the roughly predicted number of re-
ports based on a hypothetical 5% ratio of cases [as per Young et al. (9)] is 22;
that is, approximately the level of reporting found for A. niger. This crude pre-
diction ignores the possible tendencies for interestingly rare causal agents to
be disproportionately frequently reported and for highly prevalent opportunists
to be treated as too quotidian to report except in novel cases or in series. Such ten-
dencies would cause the expected number of reported section Aspergillus cases
to rise. Although species are certainly reported in some omnibus papers without
their names entering the text searchable in Medline, it is presumed that in most
if not all cases in which species identifications are given more than desultory
attention, the names will appear in summary material. There is no obvious reason
why an identification of A. glaucus from invasive aspergillosis should elude ab-
stracting more frequently than one of, for example, A. niger. The Fischer exact
probability that the Young et al. (9) data and the above Medline data on section
Aspergillus case frequencies are drawn by chance from the same population is
⬍0.005. On the other hand, the p value derived from χ 2 testing for A. fumigatus
in a comparison of both data sets is 0.466, indicative of high homogeneity. This
suggests that some or all of the ‘‘A. glaucus group’’ cases recorded by Young
et al. (9) were based on misidentifications.
Eurotium amstelodami Mangin, Anamorph Aspergillus vitis Novobr.

[Synonyms: Aspergillus amstelodami (Mangin) Thom & Church, Aspergillus
hollandicus Samson & W. Gams]. This species has been conclusively demon-
strated as an agent of a cerebral abscess in an otherwise healthy female (12) and
of a leg tumor in a Galápagos tortoise, Testudo elephantopus (13) (Figs. 1 and
2). An Argentinian case of black grain mycetoma consistently grew a rugose-
ascospored Eurotium from both native and stringently surface-disinfected grains
(14). Although identified according to the taxonomy of the day (1939) as E.
chevalieri, now regarded as a smooth-ascospored species (minute roughening is
visible in electron microscopy), the isolate illustrated would most probably be
Ascomycetes 253
Table 4 Aspergillosis Case Reports Involving

Immunocompromised Patients, as Determined by Species
Name Search on Online Medline (August 2000)
Number
Species named of reports Percent
A. fumigatus 299 68.7

A. flavus 74 17.0
A. niger 25 5.7
A. terreus 17 3.9
A. nidulans 5 1.1
A. ustus 5 1.1
Neosartorya fischeri 2 0.5
A. granulosus 1 0.2
A. flavipes ‘‘group’’ 1 0.2
A. ochraceus 1 0.2
A. oryzae a 1 0.2
A. versicolor 1 0.2
Emericella echinulata b 1 0.2
E. quadrilineata c 1 0.2
Neosartorya pseudofischeri d 1 0.2
Total species records e 435
Note: Search term string used: case ⫹ [species epithet] ⫹ (Aspergil-

lus or [teleomorph genus name]) ⫹ (immunocompromis* or immu-
nodefic* or immunosuppress* or compromised or lymphoma or leu-
kem* or HIV or AIDS or steroid* or corticosteroid* or transplant*)
not (mouse or mice or rat or rats or alveolitis or hypersensitivity).
Items in brackets were filled in appropriately for each organism.
Various permutations of this string gave similar results.
a
Byard et al. (159): not actually A. oryzae. (See text for that spe-
cies.)
b
Originally identified as A. nidulans var. echinulatus.
c
Originally identified as A. quadrilineatus. (Anamorph name now
changed to A. tetrazonus.)
d
Originally identified as N. fischeri var. spinosa.
e
Searched epithets giving nil records: amstelodami, avenaceus,
candidus, carneus, chevalieri, clavatus, deflectus, glaucus, herbar-
iorum, hollandicus, niveus, parasiticus, penicillioides, restrictus,
rugulosa, rugulosus, sclerotiorum, sydowii, tamarii, unguis.
254 Summerbell
Figure 1 Conidiophore of Aspergillus vitis, anamorph of Eurotium amstelodami.
Figure 2 Ascospores of Eurotium amstelodami.

Ascomycetes 255
identified as E. amstelodami today. The rough ascospores also have very minute
equatorial crests or ridges, consistent with this species. (See the discussion of E.
chevalieri below.)
A report of A. amstelodami causing human hypopyon corneal ulcer subse-
quent to puncture by a bamboo stick (15) contains no identification details, but
since this specific identification cannot be attempted without at least seeing asco-
spores, this case, in which corneal scrapings were positive for fungal elements,
is tentatively accepted. The original description of Aspergillus montevideensis
Tal. & Mack., a name long considered and now molecularly supported (6) as
a synonym of E. amstelodami, includes a convincing case report describing
the postsurgical eardrum colonization from which the isolate was obtained (16).
The isolate is available in several culture collections [e.g., Centraalbureau voor
Schimmelcultures (CBS)], American Type Culture Collection (ATCC)]. A later
report of causation of human otitis by Wadhwani and Srivastava (17) is not ac-
cepted for reasons mentioned under Fennellia nivea below. A report by Grigoriu
and Grigoriu (18) that the fungus was among the most common agents of nonder-
matophytic onychomycosis in Switzerland must be regarded as dubious. (See the
discussion of that report under Emericella unguis, below, along with the discus-
sion of A. glaucus onychomycosis records.)
An unusual case report by Janke (19) records A. vitis (as A. amstelodami) as
the purported cause of a large, psoriasislike scalp lesion causing diffuse hair loss
in an otherwise healthy woman. The paper’s histologic report states ‘‘in epithelium,
especially of the hair follicle, fungal spores were visible by gram stain’’ (transl. W.
Gams). This species, however, would be expected to form typical Aspergillus tissue
filaments if it were causing infection. If conidia were formed, vesiculate conidio-
phores should also be seen. It is possible dermatophyte substrate arthroconidia or
artifacts were seen in this study rather than A. vitis conidia, especially as the struc-
tures seen are not described. Aspergillus species, apart from A. terreus, do not pro-
duce structures resembling conidia within infected tissue. (Conidia formed in colo-
nizing infections such as aspergilloma are not formed within tissue, but rather after
mycelium breaks through a substrate/air interface.) The reason this seemingly mis-
interpreted case of ‘‘aspergillosis capitis’’ (19) is mentioned is that the present
author was also once sent three successive scalp specimens from a similar case in
which a pronounced scalp lesion on a child gave rise to E. amstelodami each time it
was cultured, even though definitive direct microscopic corroboration of infectious
status could not be found. The unresolved etiology of the case precluded publica-
tion. It might be proposed that E. amstelodami, which saprobically colonizes shed-
skin flakes in house dust, may do the same to a limited extent on lesions in which
sufficient dead keratinous material has accumulated. If so, however, filamentation
and conidiophore formation should again be seen, as mentioned above for infection.
Comparable cases may provide clues as to the origin of such ‘‘scalp aspergilloses.’’
A case involving a heavily outgrowing Penicillium species also repeatedly showed
256 Summerbell
only ‘‘brown, double-walled spores’’ in direct microscopy of recurring ‘‘light

brown crusts’’ on the scalp of a 3-year-old boy (20). The family cat in veterinary
examination had similar crusts, also apparently composed of Penicillium conidia.
No dermatophyte was isolated from either the boy or the cat, nor were filaments
seen in microscopy or, in the boy’s case, in a punch biopsy performed after 6 weeks
of oral ketoconazole therapy appeared ineffective. A further 4 weeks of ketocona-
zole at doubled dosage was considered to have cured the infection. This conidial
crust phenomenon, also mentioned in a previous Penicillium case involving two
brothers (21), as well as in additional unreported cases mentioned in that paper,
may derive from adhesion of environmentally formed conidia from an unknown
source to scalp oils or serous exudate. Also, growth of the fungus in hair products
or skin medicaments, with subsequent transfer of conidia to the scalp by the patient
or caregiver, is possible, but no candidate materials have been identified in case
reports to date. The variants of Munchausen syndrome (attention-seeking construc-
tion of false medical symptoms in oneself or a proxy, usually a child) provide per-
haps the most parsimonious potential explanation, and certainly isolates from such
cases should be fully identified to rule out organisms such as Penicillium roque-
fortii, the blue cheese fungus, and species not growing at body temperature. It is
not clear, however, how Munchausen practitioners would acquire pure Eurotium.
The conspicuous presence of such organisms indoors on moldy cloth, leathers,
papers, or particleboard may provide an opportunity in some cases.
The ecology of A. amstelodami is as discussed above for A. subgenus As-
pergillus section Aspergillus in general.
Description. Colonies grow 7 to 10 mm in diameter in 7 days at 25°C,
at first whitish, but rapidly becoming deeply dusty grey-green to dark green with
conidiation of the Aspergillus anamorph. Ascomata arise as small, yellowish,
spheroidal tufts scattered on surfaces of mature (usually 7–20-day-old) colonies,
sometimes massing densely and turning the colony center yellowish overall. The
colony reverse is pale to yellow-brown.
Ascomata under the microscope are mostly 75 to 150 µm in diameter, glo-
bose, nonostiolate, with thin pseudoparenchymatous peridium. Asci contain eight
lens-shaped ascospores, 4.5–6 ⫻ 3.5–4 µm, rough-walled, each with an equa-
torial furrow flanked by two rough, irregular ridges. Conidiophores are mostly
250 to 350 µm high, with thick, hyaline walls, aseptate, uniseriate (i.e., phialides
directly attached to vesicle), with vesicles globose or nearly so. Conidia are in
dry, upright chains, grey-greenish to brownish in transmitted light, subglobose
to globose, sometimes distinctly barrel-shaped with two conspicuously flattened
ends, finely to distinctly roughened, 3.5–5.5 ⫻ 3.5–5.0 µm.
Eurotium chevalieri Mangin, Anamorph Aspergillus chevalieri Mangin

[Synonym: Aspergillus chevalieri (Mangin) Thom & Church]. Few human or
animal case reports concern this species, and the majority are not definitive. The
Ascomycetes 257
most likely genuine human case examined so far is that of da Fonseca (22), who
grew a pure culture of a Eurotium species from eight separate biopsy-derived
mycetoma grains from a foot infection. The fungus was originally identified as
E. chevalieri according to the concepts of Thom and Church (23), and the detailed
descriptions and illustrations accord well; however, according to da Fonseca, the
isolate was then examined by prominent French mycologist Paul Vuillemin, who,
following M. L. Mangin’s original diagnosis of A. amstelodami as a species with
smooth-valved ascospores, reidentified the smooth-ascospored case isolate as A.
amstelodami. It has been carried forward in subsequent review literature under
this name. In more recent times, E. amstelodami has been neotypified as a rough-
ascospored species [see discussion by Pitt (24)], and da Fonseca’s fungus now
clearly best fits E. chevalieri. Unfortunately, the isolate seems not to have been
preserved for confirmation, but it appears likely that E. chevalieri or a very similar
Eurotium may rarely cause mycetoma.
Most other case literature mentioning this species appears unreliable. Three
cases of cutaneous infection attributed by Naidu and Singh (25) are based only
on the results of outgrowth from a single dermatologic mycology scrape in each
case. These appear to be typical cases of tinea manuum, in which the causal
dermatophyte failed to grow out in an initial evaluation and a surface contaminant
was taken to be an etiologic agent. Confirmation of this interpretation is obtained
from a related paper in which E. chevalieri and a large number of additional
contaminating species are interpreted as skin pathogens by Naidu (26). Implica-
tion of E. chevalieri in otitis by Wadhwani and Srivastava (17) is not accepted
for reasons given under Fennellia nivea below.
E. chevalieri is similar in ecology and morphology to E. amstelodami, but
differs by having strongly pulley-shaped, smooth-valved ascospores with two
prominent equatorial crests (Fig. 3).
Eurotium herbariorum (Wiggers :Fr) Link, Anamorph Aspergillus glau-

cus Link. This species has a fragmentary and often dubious record as a human
and animal pathogen. As indicated in the general remarks on Aspergillus subge-
nus Aspergillus above (pages 245–252), the anamorph name may have frequently
been used for any incompletely identified Eurotium anamorph. There is no record
of human or animal infection by this species that combines adequate establish-
ment of causality with adequate verifiability of identification (e.g., any of: clinch-
ing description, diagnostic illustration, collection deposition, or referral to a rec-
ognized Aspergillus expert).
The classic study on otomycosis by Gregson and La Touche (27) clearly
links a case of this affliction to an isolate identified as ‘‘Aspergillus herbariorum’’
(a nonexistent name combination). Although the development of yellow ascomata
(incorrectly called ‘‘perithecia’’) is mentioned, and in the context of other de-
scriptions, confirms that a Eurotium was examined, no microscopic description
258 Summerbell
Figure 3 Ascospores of Eurotium chevalieri.
is given that could confirm this species or rule out other members of the genus.
This appears to be the most strongly confirmed case involving E. herbariorum
published to date. An authoritative study on keratitis in the region of Mumbai,
India, reported 23 cases involving A. glaucus among 327 cases in which fungal
etiology was confirmed by direct microscopy (28). Three hundred of these cases
were also reconfirmed by reculture from scrapings taken the day after the initial
examination. Unfortunately, no identification information was given, although
differential organisms such as A. fumigatus (43 cases) and A. flavus (36 cases)
were significantly more common than A. glaucus, as would be expected. This
general indication of accuracy suggests that at least group-level identification for
the reported A. glaucus isolates is likely to be correct, although there is no means
of determining whether A. glaucus itself was encountered. An analogous report
from climatologically distinct north India mentions three mycologically unveri-
fiable A. glaucus cases etiologically confirmed either by direct microscopy or
repeated culture (29).
In south Florida, A. glaucus has also been included without mycological
elaboration in a list of fungi isolated from keratitis by Rosa et al. (30). Direct
microscopy was done in this series, but results were not reported, except in gen-
eral statements indicating that fungal elements were seen in connection with
about one-third of the tabulated cases.
One of 13 well-established invasive orofacial aspergilloses in leukemia pa-
Ascomycetes 259
tients is attributed to A. glaucus by Dreizen et al. (31); the lack of case and con-
firmatory identification detail, plus the failure of the laboratory to identify 4/12
aspergilli cultured from the remaining cases (possibly indicating a low level
of familiarity with the genus, as is also suggested by the lack of a name in Euro-
tium for the case isolate) makes this report difficult to accept. (In the present
author’s experience, the great majority of clinically encountered aspergilli are
among the easiest fungi for a relatively experienced laboratorian to identify;
therefore, publication of numerous genus-level identifications for etiologic Asper-
gillus isolates strongly suggests reliance on a mycologically unspecialized labora-
tory.) An A. glaucus strain isolated from cellulitis in an HIV-infected child by
Shetty et al. (32) is not etiologically connected by direct microscopy and is re-
ported as an unsupported identification in a table in which three of seven Aspergil-
lus isolates from cases are not identified to species. A similar pattern is again
found in a report on aspergillosis in children with cancer; an unsubstantiated A.
glaucus isolation from a traumatized skin site (armboard or intravenous site) of
a 16-year-old leukemia patient is reported without explicit evidence of causality
in a survey in which 15 of 66 reported aspergilli remained unidentified (33). The
authors stated that only five of the six skin infections reported were histopatholog-
ically examined, but four of these five showed fungal filaments. Whether or not
the A. glaucus case drew one of the two short straws is not discernible. The report
by Weingarten et al. (34) that two of three successive cases of fulminant nasal
and paranasal sinus aspergillosis in immunocompromised patients were caused
by A. glaucus (the third did not yield a culture) appears highly unlikely to be
correct, given the statistical anomaly posed by the coincidence of rare events and
absence of common events, the incomplete (no Eurotium name) and undocu-
mented identifications, and the highly aggressive nature of the pathogen, which
disseminated and caused death in one case. Since sinuses, like bronchi, pulmo-
nary cavities, and outer ear canals, are classic sites for ongoing A. fumigatus and
A. flavus establishment and for generation of atypical host-adapted isolates, it
seems highly probable that such isolates were misidentified in this report.
A well-established case of a fungal frontal bone lesion in an Indian farmer
(35) gives no written details for identification of A. glaucus as the causal agent,
but includes a nondescript photograph that could represent numerous Aspergillus
species, including host-adapted A. fumigatus, as well as members of Penicillium
subgenus Aspergilloides. No structures resembling the characteristic ascomata of
E. herbariorum are described or depicted. A report of mycotic keratitis by Venu-
gopal et al. (36) is not confirmed by demonstration of fungal filaments in corneal
scrapings; indeed, the authors state that such elements were only seen in 19.6%
of cases attributed fungal etiology. Such diagnostic leaps of faith, justifiable clini-
cally in rapidly progressing eye infections, unfortunately do not provide mycolog-
ical documentation.
Gage et al. (37) diagnosed A. glaucus endocarditis after a man recently
260 Summerbell
given an artificial cardiac prosthesis developed a fever and yielded A. glaucus

in one blood culture, although ‘‘many subsequent blood cultures during this fe-
brile period showed no growth.’’ When the patient recovered without treatment,
they concluded that ‘‘some patients are able to successfully combat Aspergillus
infection on their own.’’ Studies of blood cultures [e.g., Bille et al. (38)], how-
ever, generally reveal, among species isolated only rarely, a number of fungi
with no known pathogenic record that are most likely contaminants. Since asco-
spores are extremely resistant, ascosporic fungi seem particularly likely to survive
disinfection procedures and to contaminate the small plug of skin taken up in all
percutaneous syringe samples, as well as any chemically surface-disinfected or
otherwise inadequately sterilized equipment. Such events may have led to confu-
sion in the past (e.g., a hypothesis that the Thermoascus crustaceus metabolite
cyclosporin was the cause of AIDS, based on several isolations of this ascosporic
fungus from monocyte cultures of AIDS patients) (39). The most probable inter-
pretation of Gage et al.’s case (37), then, is not spontaneous recovery from cardiac
aspergillosis, but rather, single contaminated blood culture plus fever of undiag-
nosed origin. In any event, the identification of A. glaucus in this case is not
substantiated. An earlier, etiologically well demonstrated Aspergillus endocar-
ditis case gave no substantiation for identification of the causal agent as A.
glaucus (40).
An attributed case of A. glaucus otomycosis by Bambule et al. (41) lacks
direct microscopic verification; the authors state that such verification was ob-
tained in some cases, but do not state which. Well-demonstrated mycotic middle
ear infection is attributed to A. glaucus by Wulf (42); the identification is substan-
tiated by a microphotograph that shows globose-vesiculate aspergilla compatible
with A. glaucus or another Eurotium anamorph, although A. fumigatus cannot be
completely ruled out. Vennewald et al. (43) list A. glaucus among 31 cultures
from paranasal sinusitis, but state that only 12 of these cultures connected with
positive direct microscopy and fail to indicate which these were. A connection
by Ponikau et al. (44) with causation of allergic fungal sinusitis cannot be ac-
cepted for reasons given below under A. versicolor. More problematic is the
attribution of five cases of Aspergillus onychomycosis to A. glaucus by Bereston
and Waring (45). These authors confirmed the nondermatophyte onychomycoses
impeccably; however, the assertion that A. glaucus made up 83.3% (five of six)
of sequential nail aspergilloses seen is a profound statistical anomaly compared
to all later surveys (e.g., Refs. 46, 47), in which this species is rare (and poorly
etiologically linked) or absent. The present author’s laboratory evaluated at least
75,000 nail specimens from 1985 to 2000 without elucidating a confirmed or
strongly suspected Eurotium onychomycosis (45; unpublished data). At the same
time, the normally relatively common A. subgenus Nidulantes, section Versico-
lores (⫽A. versicolor group) is absent from Bereston and Waring’s study, sug-
gesting that the Army Medical School credited for identifications may have made
Ascomycetes 261
a systematic error. Since no vouchers were deposited and no identification criteria

were given, this matter cannot be investigated.
A nail biopsy figured by Contet-Audonneau et al. (48: Fig. 10) purports to
show A. glaucus filaments in nail. Cases in this study, however, were not verified
by later repeat isolation. Furthermore, no fungal identification characters were
given. The authors stated that they were able to distinguish dermatophyte from
nondermatophyte filaments in biopsies. They characterized dermatophyte fila-
ments, however, as being ‘‘regular in diameter and sometimes associated with a
mass of arthrospores.’’ This describes dermatophyte filaments from distal-sub-
ungual onychomycosis, but not those from superficial white onychomycosis, in
which irregular and frondose filaments are common (49, 50). The figured biopsy
depicted a superficial infection. Very little research has been done to date around
the distinction of dermatophyte and nondermatophyte onychomycoses in biopsy
slides stained with nonspecific fungal stains, and repeat isolation studies are re-
quired as a gold standard to allow the validation of results. Identification criteria
should be given for such unusual fungi as A. glaucus.
In summary, E. herbariorum is a possible opportunistic pathogen badly in
need of an authoritative case report. Based on the ‘‘where there’s smoke there’s
fire’’ principle, it is likely a small but significant number of opportunistic myco-
ses, especially keratitis cases, are indeed caused by Eurotium species, although
clearly the probability of involvement of the thermotolerant and previously etio-
logically substantiated E. amstelodami is much greater than that of the relatively
uncommon and less thermotolerant E. herbariorum/A. glaucus, for which no fully
credible report of pathogenicity appears to exist.
Like other members of its genus (see general discussion of A. subgenus
Aspergillus section Aspergillus above), E. herbariorum is adapted to grow in
conditions of low water activity. Carefully identified isolates have mainly been
from cereals, spices, dried fruits, and other low-water-activity food products (51).
at first whitish, but rapidly becoming deeply dusty grey-green to dark green with
conidiation of the Aspergillus anamorph. Ascomata arise as small, yellowish,
spheroidal tufts on surfaces of mature (usually 14 or more days old) colonies,
often massing densely amid orange to reddish sterile hyphae. The colony center
then becomes yellow-orange to reddish, and progressively more reddish with age.
The colony reverse is pale orange or deep reddish-brown.
Ascomata under the microscope are mostly 100 to 150 µm in diameter,
globose, nonostiolate, with thin pseudoparenchymatous peridium. Asci contain
eight lens-shaped ascospores, 6–7 ⫻ 5–6 µm, smooth-walled, each with a shal-
low equatorial furrow, sometimes with small ridges or flattened crests (Fig. 4).
Conidiophores are mostly 500 to 700 µm high, with thick, hyaline walls, aseptate,
uniseriate (i.e., phialides directly attached to vesicle), with vesicles globose or
nearly so. Conidia in dry upright chains, grey-greenish to brownish in transmitted
262 Summerbell
Figure 4 Ascospores of Eurotium herbariorum.
light, subglobose to globose, sometimes distinctly barrel-shaped with two con-

spicuously flattened ends, spinose, 5.0–6.5 ⫻ 3.0–5.0 µm.
The very similar Eurotium rubrum König et al. is distinguished by its
slightly smaller ascospores, 5.0 to 6.0 µm wide, and by its different growth rates
on specialized diagnostic media, as outlined by Pitt and Hocking (51). The only
slightly less similar E. repens de Bary has ascospores with barely discernible
equatorial furrows, and also has more yellow or orange and less red colony pig-
mentation deriving from colored sterile hyphae. These identifications should be
confirmed by comparison of reference isolates, by experts, or by molecular analy-
sis, and should not be published on the basis of comparison with descriptive
literature alone. Voucher isolates should be deposited in professional culture col-
lections (i.e., collections operated by dedicated, full-time staff ) in connection
with case reports.
Eurotium repens de Bary, Anamorph Aspergillus reptans Samson & W.
Gams. This species has not been documented as an agent of human infection.
It co-occurred with Microascus cinereus in a case of sinusitis, but pathogenic
etiology was established only for M. cinereus (52). Another record from sinusitis
by Vennewald et al. (43) cannot be accepted for reasons given above under E.
herbariorum. For identification, see notes under the description of E. herbari-
orum above.
Ascomycetes 263
Eurotium rubrum König et al., Anamorph Aspergillus rubrobrunneus

Samson & W. Gams. This species has not been conclusively documented as
an agent of human infection. It was reported under the invalid name Aspergillus
sejunctus Bain. & Sart. from keratitis by Shukla et al. (15), but corneal scrapings
were negative for fungal elements. For identification, see the notes under the
description of E. herbariorum above.
Anamorphic Species in A. Subgenus Aspergillus, Section Restricti Gams
et al. (Formerly Aspergillus restrictus Group). The three recognized, closely re-
lated species in this section are particularly specialized osmotolerant organisms,
very common in dry environments such as household dust, but frequently missed
because of their poor growth on ordinary environmental fungal isolation media.
Accurate environmental numbers are obtained with high-osmoticum media such
as dichloran 18% glycerol agar. These species are seldom seen in the medical
mycology laboratory as contaminants, but are occasionally fortuitously isolated
from exposed materials such as skin or respiratory secretions. As outlined below,
they have never been convincingly recorded as opportunistic pathogens. Indeed,
the general ecological picture of an opportunistic mold pathogen is of a plurivor-
ous, enzymatically versatile, thermotolerant, generalist decomposer isolated from
a wide variety of habitats, at least some of which are osmotically similar to animal
tissue (i.e., high water activity). The specialized Restricti do not fit this picture
well. No group of fungi can be ruled out a priori as opportunists, but in reference
to such specialized fungi, adequately mycologically and etiologically documented
case reports, preferably backed up by deposition of case isolates in a recognized
culture collection, must be especially strongly recommended.
Aspergillus {aff. Eurotium} restrictus G. Smith. Many if not all reports
attributing pathogenicity to this slow-growing osmophilic species are specious
or unconfirmed. An attribution of endocarditis by Mencl et al. (53) in a case also
mentioned by Pospisil et al. (54) and Resl et al. (55) is based on an isolate that
is clearly recognizable from the description and photograph of Mencl et al. as a
typical dysgonic A. fumigatus with reduced conidiophores. The authors admirably
confirmed this by showing the isolate doubled its growth rate at 37°C on Czapek–
Dox medium, whereas the growth rate of A. restrictus itself at 35°C is only about
25% of its optimal growth at 25° (56). [An optimum of 30° and maximum of
40.5° was shown by Smith and Hill (57) in a medium of higher water activity;
A. restrictus does not grow at all at 37° in standard taxonomic studies on Czapek’s
agar (51).] Similarly, a report of A. restrictus aspergilloma by Estrader et al. (58)
appears to be based on host-adapted A. fumigatus; smooth, spherical conidia 2 to 3
µm in diameter were seen rather than the elongated 4 to 6 µm conidia of A.
restrictus. The patient had previously been precipitin-positive against A. fumiga-
tus but had a single negative test at the time the purported A. restrictus was
264 Summerbell
isolated. Possibly a false-negative test was involved. A report of ostensibly sero-

logically confirmed A. restrictus aspergilloma by Tamaki et al. (59) was based
on using the case isolate, not a standard A. restrictus strain, as representative of
that species for antigen extraction. The authors appear merely to have shown that
the case isolate, perhaps itself an A. fumigatus, had a stronger precipitin reaction
with the patient’s serum than did an extract of a heterogeneous A. fumigatus
isolate (which gave a negative result). No identification characters were adduced
for the isolate involved. A report of A. restrictus onychomycosis in Schönborn
and Schmoranzer (60) cannot be confirmed. Although these authors state they
tried to obtain confirmatory repeat samples for onychomycoses when possible,
there is no mention of which cases were confirmed in this way. Furthermore, the
authors reported a number of nondermatophyte onychomycosis ‘‘cases’’ in which
direct nail microscopy was negative, suggesting that they were not always obser-
vant of standard confirmatory criteria. (See also the discussion under A. conicus
below.)
It may be noted that A. fumigatus produces a well-known ribotoxin, restric-
tocin (61, 62), which received its name because it was extracted from a medically
isolated strain misidentified as A. restrictus and so deposited in the ATCC (ATCC
34475 ⫽ NRRL 2869). This illustrates the frequency and strong scientific influ-
ence of this common identification error. The identification of ATCC 34475 has
since been corrected by ATCC.
Description. The colonies are slow-growing (6 to 12 mm in 7 days),
dense, often heaped, powdery with sporulation, and dark green, with the reverse
pale or dark green.
Conidiophores are mostly 75 to 200 µm high, with smooth, hyaline walls.
The vesicles are narrowly hemispherical, 6 to 12 µm in diameter [10 to 18 µm
on the high-osmoticum Czapek-yeast extract 20% sucrose (51)], bearing uniseri-
ate (i.e., with phialides directly attached to vesicles) fertile elements (Fig. 5).
Conidia are in dry, strongly coherent chains, often remaining in discernible paral-
lel columns in wet mounts, at first nearly cylindrical, then barrel-shaped to ellip-
soidal at maturity. They are rough-walled, deep green in color, mostly 4.0 to 5.0
(sometimes to 6.0) µm ⫻ 3.0 to 3.5 µm.
There is no growth at 37°C on most media, and no growth at 42°C on any
medium.
This fungus may easily be distinguished from all variants of atypical, host-
adapted A. fumigatus by temperature testing. A. fumigatus almost always grows
at 45°C; its optimum temperature is 40–42°C (51).
Aspergillus {aff. Eurotium} caesiellus Saito. This species strongly re-

sembles A. restrictus, but grows more rapidly on certain specialized media, such
as Czapek’s agar (7) and Czapek-yeast extract 20% sucrose agar (63). Unlike A.
restrictus, it grows weakly at 37°C on Czapek-yeast extract agar. An isolate from
Ascomycetes 265
Figure 5 Aspergillus restrictus.
an aspergilloma was identified by Otčenášek et al. (64) as A. caesiellus. It is

described as nonsporulating on Sabouraud agar and with very rare production of
mostly aberrant conidiophores on Czapek–Dox agar. The few A. caesiellus iso-
lates known in collections, however, sporulate on all media tested, and do so
heavily on malt extract agar (7, 63), which is nutritionally similar to many general
growth and sporulation media used in medical mycology laboratories. The case
isolate also had a growth rate around half that of A. caesiellus, and a ferruginous
brown to brown-black reverse, in contrast to the pale to deep green reverse typical
of A. caesiellus. As the principal author was also involved in the demonstrable
misidentification of dysgonic A. fumigatus as A. restrictus (see above), it appears
possible that the identification of this case isolate as A. caesiellus was a similar
error, despite the exact coincidence of the isolate’s conidial size range with that
given by Raper and Fennell (7) for A. caesiellus. Because of prolonged host/
fungus contact, aspergilloma is one of the most predictable sources of aberrant
266 Summerbell
and poorly and/or atypically sporulating A. fumigatus isolates. Host-adapted A.

fumigatus isolates strongly resembling the isolate of Otčenášek et al. (64), with
restricted colonial growth and aberrantly long conidia, are described by Raper
and Fennell (7, p. 244).
Aspergillus {aff. Eurotium} conicus Blochwitz. Although considered a
dubious taxon and a probable synonym of A. restrictus by Pitt and Samson (63),
this species has recently been revalidated by molecular study (6). It was reported
without identification details or credit by Jones (65) from a well-substantiated
case of postsurgical fungal endophthalmitis. As the fungus was extracted from
the eye 23 days after its implantation, it is unlikely to have been a dysgonic form
of A. fumigatus; this form generally results from prolonged growth in the host.
The assigned identity of the fungus, however, cannot be verified, and the absence
of comment on the unusual attribution, plus the lack of acknowledged expert
confirmation for the highly esoteric identification, suggests a low degree of my-
cological awareness. Microphotographs of A. conicus in Raper and Fennell (7,
p. 230), could easily engender misidentification of an A. fumigatus with smaller
than usual aspergilli, as well as other Eurotialean fungi with small, vesiculate
conidiophores.
Aspergillus {aff. Eurotium} penicillioides Speg. Maršálek et al. (66) re-
corded a case of aspergillosis, first pulmonary and then disseminated, which they
stated was caused by a member of the A. restrictus series, most probably A.
penicillioides. Their excellent description of the isolate, however, reveals it as a
host-adapted A. fumigatus. In particular, its accelerated growth at 45°C and its
prolonged absence of conidiation on all media rule out the section Restricti en-
tirely. The isolate was clearly quite aberrant, with small, cerebriform colonies
on Sabouraud agar at 20° and 37°C, and its identification [by G.A. de Vries of
Centraalbureau voor Schimmelcultures (CBS)] was not unreasonable, given the
paucity of descriptions of such A. fumigatus isolates at the time. Regrettably the
isolate was not preserved in CBS. A case from a lung lesion in a roe deer was
attributed to A. penicillioides by Fragner et al. (67), but was also based on an A.
fumigatus isolate, as evidenced by a growth rate at 24°C, approximately tenfold
that of the former species. Figures depict conidial heads typical of A. fumigatus.
The conidial structures of A. penicillioides are shown in Fig. 6.
2. Emericella Berk. & Br.

Ascomata are globose cleistothecia, yellow, reddish, or brown, usually formed
after more than 1 week of colonial growth on fungal sporulation media [e.g.,
potato dextrose agar, Leonian’s agar (4)] and seen as characteristically colored
tufts on the colony surface, often more dense near the colony center. Peridium
is surrounded by masses of hülle cells, which are seen as subglobose to globose
Ascomycetes 267
Figure 6 Aspergillus penicillioides.
cells up to 25 µm in diameter with thick, hyaline cell walls occupying most of

the cellular volume. Asci are eight-spored, round, and evanescent. Ascospores
are oblate, with equatorial crests, and often with a distinct species-specific color.
Corresponding anamorphs and evolutionarily radiating anamorphic species are
in Aspergillus subgenus Nidulantes, sections Nidulantes, Versicolores, and Usti,
with biseriate conidial heads. Formerly the species included below were consid-
ered members of the Aspergillus nidulans, A. versicolor, and A. ustus series (7).
Species with a Known Teleomorph (which may not form in every culture)
Anamorphs in Aspergillus Subgenus Nidulantes, Section Nidulantes Gams et al.
(Formerly Aspergillus nidulans Group)
Emericella nidulans (Eidam) Vuill.; Anamorph Aspergillus nidulans
(Eidam) Winter, nom. cons. This species is a well-known agent of serious
opportunistic infection (11, 68, 69) (Figs. 7 and 8). Although much less common
268 Summerbell
Figure 7 Emericella nidulans, conidiophore, conidia and sheath of hülle cells at margin
of an ascoma.
as an infectious agent than A. fumigatus, A. flavus, and A. terreus, it may poten-

tially cause a similar range of infections. A relatively large number of reports
are in connection with chronic granulomatous disease (70), where it was deter-
mined by case review to be more virulent and more difficult to treat than A.
fumigatus. Causation of mycetoma in tropical areas is also regularly seen (71).
Another specific association is with guttural pouch disease of the horse (72). In
human nasal sinusitis, this fungus is relatively rare, but may be confirmed in
direct microscopy if present when cleistothecia are produced in situ (73). It also
causes a variety of marginal opportunistic mycoses such as otomycosis (27). In
ecology, it is a widely distributed soil fungus, particularly in the tropics and
subtropics, and is also a prominent colonizer of decomposing plant debris, includ-
ing seeds and grains (74). In secondary metabolism it produces the poorly water-
soluble hepatic carcinogen sterigmatocystin, as well as penicillin (8).
Ascomycetes 269
Figure 8 Emericella nidulans, ascospores.

at first white, but rapidly becoming deeply dusty bluish-grey-green with conid-
iation of the Aspergillus anamorph. Ascomata in macroscopic examination arises
as small, yellowish, spheroidal tufts scattered on surfaces of mature (usually 7- to
20-day-old) colonies, sometimes massing densely and turning the colony center
yellowish overall.
Ascomata in microscopy are 100 to 150 µm in diameter, globose, nonostio-
late and surrounded by a thick layer of subglobose to globose, hyaline, thick-
walled hülle cells. (See genus description.) Asci contain eight vinaceous red, lens-
shaped ascospores, 3.8–6 ⫻ 3.5–4 µm, each with two parallel, flat, equatorial
ridges approximately 1 µm wide, giving the spore a Saturn-shaped appearance
in side view and a fried-egg appearance in face view.
Conidiophores are 75 to 100 µm high, with thick brown walls. They are
aseptate, and biseriate (i.e., bearing metulae and phialides). Conidia are in dry,
270 Summerbell
upright chains, usually massed into short, upright columns, greenish in transmit-
ted light, subglobose to globose, distinctly roughened with verrucose ornamenta-
tion, and 3 to 3.5 µm in diameter.
Although homothallic in pure culture, in subspecific population structure
this species appears to outcross to a small extent in nature (75). There is therefore
no evidence of extended clonal populations, and diverse genetic subtypes may
occur among relatively closely related isolates. There are a number of heterokar-
yon incompatibility subgroups within A. nidulans, and parasexual recombination
is largely—but not completely—restricted to within the genetic boundaries of
these groups (76).
Emericella dentata (Sandhu & Sandhu) Horie, Anamorph A. nidulans
var. dentatus Sandhu & Sandhu. The original ex-type isolate of this species
was, according to its collectors Sandhu and Sandhu (77), ‘‘isolated from diseased
human fingernails.’’ No argument was made, however, nor any evidence given
for a causal connection between the isolated organism and the infection. The
species is distinguished from E. nidulans by the conspicuously dentate (toothed
or starlike) morphology of the two equatorial crests on its ascospores (Fig. 9).
Emericella echinulata (Fennell & Raper) Horie, Anamorph A. nidulans
var. echinulatus Fennell & Raper. Until recently this species was considered
a variant of E. nidulans. It was obtained from disseminated infection of a child
with chronic granulomatous disease (78).
Figure 9 Emericella dentata, ascospores.

Ascomycetes 271
Figure 10 Emericella echinulata, ascospores.
Description. The description is the same as for E. nidulans, except that

the valves of the ascospores are distinctly ornamented with small spines (Fig. 10).
Emericella quadrilineata (Thom & Raper) C.R. Benj., Anamorph As-
pergillus tetrazonus Samson & W. Gams (Synonym: Aspergillus quadrilineatus
Thom & Raper). This species has been well documented from fungal sinusitis
in an immunocompromised patient (79). A report of skin infection in two Indian
sheep (80) is unsubstantiated and appears to be based on fortuitous isolation. In
nature E. quadrilineata is a soil fungus.
Description. Colonies grow 35 to 65 mm in diameter in 7 days at 25°C.
They are at first white, but rapidly become dull greyish-green with conidiation
of the Aspergillus anamorph. The reverse is yellow-brown. Ascomata in macro-
scopic examination arise as small, yellowish, spheroidal tufts scattered on sur-
faces of mature (usually 7–20-day-old) colonies, sometimes massing densely and
turning the colony center dull yellowish.
272 Summerbell
Figure 11 Emericella quadrilineata, ascospores.
Ascomata in microscopy are 125 to 150 µm in diameter, globose, nonostio-

late, and surrounded by a thick layer of subglobose to globose, hyaline, thick-
walled hülle cells (see genus description). Asci contain eight vinaceous red, lens-
shaped ascospores, 4.5–6 ⫻ 3.5–4 µm, each with two narrow equatorial crests
flanked by two smaller ridges, giving the spore a distinctive appearance of pos-
sessing four crests (Fig. 11).
Conidiophores are 40 to 200 µm high, with thick, smooth, brown walls.
They are aseptate and biseriate (i.e., bearing metulae and phialides). Conidia
radiate en masse to columnar, in wet mount appearing greenish, globose, finely
roughened, and 2.5 to 3.5 µm in diameter.
Emericella rugulosa (Thom & Raper) C.R. Benj., Anamorph Aspergil-
lus rugulovalvus (Thom & Raper) Samson & W. Gams apud Samson & Pitt
(Common Synonym: Aspergillus rugulosus Thom & Raper). This species has
been definitively linked to mycotic abortion in cattle in the Midwest by Knudtson
and Kirkbride (81).
In nature, E. rugulosa is a soil fungus of warm soils, especially tropical
and grassland, and composts.
Colonies grow 10 to 15 mm in diameter in 7 days at 25°C. They are at
first white, but rapidly become grey-green with conidiation of the Aspergillus
anamorph, then yellowish to purple-brown with accumulating hülle cells. As-
Ascomycetes 273
comata arise as small, spheroidal tufts, at first yellowish to brownish, then scat-
tered on surfaces of mature (usually 7–20-day-old) colonies, sometimes massing
densely and turning the colony center yellowish to purple-brown overall. The
reverse is usually pale to intense orange-brown to red-brown.
Ascomata in microscopy are seen as cleistothecia, 100 to 200 µm in diame-
ter, globose, with thick reddish walls, surrounded by subglobose to globose yel-
lowish, thick-walled hülle cells. (See genus description.) Asci contain eight red-
brown, lens-shaped ascospores, 5–6.5 ⫻ 3.0–4.5 µm, with heavily roughened
walls and two parallel, flat, equatorial ridges approximately 1 µm wide (Fig. 12).
Conidiophores are 60 to 100 µm high, with thick, brown, smooth walls.
They are aseptate and biseriate (i.e., bearing metulae and phialides). Conidia are
in dry, upright chains, usually massed into short, upright columns, subglobose
to globose, distinctly roughened, and 3 to 4 µm in diameter.
Figure 12 Emericella rugulosa, ascospores.

274 Summerbell
Figure 13 Emericella unguis, Aspergillus unguis conidiophores.
Emericella unguis Malloch & Cain, Anamorph Aspergillus unguis

(Émile-Weil & Gaudin) C.W. Dodge. This species is very similar to A. nidu-
lans except that spiny, erect, rough-walled spicular hyphae, also called setae by
some authors, generally emerge from the colony surface (Figs. 13 and 14). The
species also grows only 20 to 40 mm in 7 days at 25°C, as opposed to 35 to
65 mm for A. nidulans. Fertile cleistothecia are not seen in most isolates. This
uncommon organism has been reported as causing onychomycosis by Schönborn
and Schmoranzer (60), but this report cannot be accepted, for reasons given in
the discussion of A. restrictus above. Likewise, a report by Grigoriu and Grigoriu
(18) stating that A. unguis was the third most common agent of nondermatophyte
onychomycosis obtained in a Lausanne, Switzerland, clinic seems unlikely, given
that the authors’ stated confirmation criteria do not include repeated isolation.
Also, no identification characters are given, the statistical anomaly of this rare
organism appearing prominently is not discussed, and some of the other fungi
Ascomycetes 275
Figure 14 Emericella unguis, spicular hyphae.
listed as prominent agents of onychomycosis are common contaminants that

rarely cause this disease. The original report of A. unguis onychomycosis by
Émile-Weil and Gaudin (82), however, is unequivocal, as the fungus was illus-
trated sporulating in direct microscopy of clinical material and the isolate was
preserved as an ex-type of the species.
Anamorphic Species in A. Subgenus Nidulantes, Section Versicolores
Gams et al. (Formerly A. versicolor Group).
Aspergillus {aff. Emericella} versicolor (Vuill.) Tirab. This fungus ap-
pears to cause opportunistic onychomycosis, usually in elderly patients (83, 84).
It may, however, be found to do so less commonly than the closely related A.
sydowii when the two species are methodically distinguished (47). This distinc-
tion may be rendered difficult in a few cases by an intermediate blue-green color
form that may grow from infected nails. Because of the red reverse pigmentation
276 Summerbell
and exudate associated with this form, it is tentatively included under A. sydowii
in the present work pending molecular resolution.
Like Aspergillus glaucus, A. versicolor is a name that may sometimes be
loosely used to signify the former Raper and Fennell (7) group concept rather
than the species as recognized currently. Therefore, unless colony characters,
especially conidial mass color, are specified, it may be difficult to know that this
species per se has been identified. For example, a review of agents of onycho-
mycosis by Torres-Rodrı́guez and López-Jodra (85, p. 128, Fig. 9) depicts a nail-
infecting ‘‘A. versicolor’’ isolate that is clearly ‘‘greyish-turquoise’’ on Sabour-
aud agar [colors 24 D-E 3-4 in the Methuen color monograph (86)]. This is well
within the published Methuen color range for typical A. sydowii isolates (87),
but is not normally consistent with A. versicolor, whether live or in photos. Com-
pare similar published color photos of A. sydowii on Sabouraud agar in St. Ger-
main and Summerbell (88, p. 77) and Summerbell (89, p. 233), in contrast to quite
different photos of A. versicolor on pp. 76 and 236, respectively, of the same works.
Conidial mass color is the main feature distinguishing these species; Pitt (87)
recommends Czapek yeast extract (CYA) medium as best for clear separation.
Non-onychomycosis case reports involving A. versicolor are very rare, and
some are inadequately evidenced. An isolate from an intracranial lesion in an
otherwise healthy male (90) appears etiologically well connected and well iden-
tified (by R. Vanbreuseghem), despite atypical descriptive notes mentioning
slightly roughened conidiophores and smooth conidia (features which, if taken
at face value, would indicate A. flavus). A record from an etiologically well-
documented mycotic osteomyelitis of the sacrum by Liu et al. (91) does not
substantiate the identification of A. versicolor in any way; a microphotograph
claiming to represent the culture appears to show only filaments. The identifica-
tion is uncredited. Fungal names are repeatedly misspelled in the paper, giving
an impression of unfamiliarity with mycology. A record from a localized lung
infection of a 16-year-old leukemia patient contains no verification either of spe-
cies identification or causality. In particular, no direct microscopic result is men-
tioned for lung lobe tissue resected in handling the case, and the patient, who
had been treated with amphotericin B and itraconazole, was negative for fungus
at autopsy (33). A case of keratitis was well documented by Anderson et al. (92).
No identification characters were given, but Duke University, the site of the study,
was a major mycology center at the time and a likely source of complex identifi-
cations that were given in connection with other cases in the same paper. A recent
case report connecting A. versicolor to nodular dermal lesions in an Argentinian
patient treated with corticosteroids was likewise etiologically well attested, but
included no details supporting the species identification (93). A somewhat similar
case, however, involving a single subcutaneous granuloma in the lip of a horse
was accompanied by extensive descriptive notes confirming A. versicolor (94),
probably not contradicted by an uncharacteristic microphotograph used as an
illustration. A recent record by Ponikau et al. (44) connecting A. versicolor with
Ascomycetes 277
allergic fungal sinusitis (AFS) cannot be accepted. In that study, all fungi growing
from loosened mucus in nasal washings were interpreted as ‘‘colonizing the mu-
cus’’ and ‘‘associated with AFS.’’ It was noted, however, that all healthy control
volunteers were also ‘‘colonized’’ by similar organisms. Clearly, the majority
of ‘‘colonizers’’ were trapped propagules from air. The list of species given is
recognizably a list of common air spora in the area in which the study was done—
including species not growing at or near body temperature—mixed with a small
proportion of isolates probably originating in genuine infections. ‘‘Aspergillus
versiforme,’’ also mentioned in the same publication, is a name that cannot be
traced to a known Aspergillus species and may be based on a verbal error. An
isolation from otomycosis is mentioned by Yassin et al. (95). Unfortunately, even
though the authors did direct microscopy on each specimen, they did not publish
the results or mention an interpretation policy (e.g., ‘‘isolation of an organism
was considered significant if compatible elements were seen in direct micros-
copy’’). Their results are therefore uninterpretable, particularly for infrequently
isolated species not elsewhere well confirmed as agents of otomycosis.
Natural and anthropogenic habitats of A. versicolor appear to be similar to
those of A. sydowii, q.v. Its principal mycotoxin is the hepatotoxin and carcinogen
sterigmatocystin.
Description. Colonies are moderately slow-growing, 1 to 1.5 cm after 7
days, beginning whitish but soon characteristically dusty grey-green, emerald
green, orange-brown, or pinkish-brown (the first two colors based on masses of
conidia, the last two strongly influenced by mycelial color), typically with a pale
colony reverse.
Conidiophores are mostly up to 500 (occasionally to 700) µm high, with
thick, hyaline (clear) walls. They are aseptate and biseriate (i.e., bearing metulae
and phialides) (Fig. 15). Reduced uniseriate conidiophores resembling structures
of the genus Penicillium are also frequently seen, and may predominate in some
cultures. These structures may be shorter than 25 µm. Conidia are in dry, upright
chains, usually massed into short, upright columns, greenish in transmitted light,
subglobose to globose, distinctly roughened with verrucose ornamentation, and
2 to 3.5 µm in diameter. Uncommonly, colonies form subglobose, thick-walled
hülle cells similar to those seen in E. nidulans.
Aspergillus {aff. Emericella} granulosus Raper & Thom. This species

was well causally connected to a case of disseminated infection in a heart trans-
plant patient (96). It is otherwise mainly known as a soil fungus.
Description. The colonies are moderately slowly growing (2.0 to 3.5 cm
in 7 days). They are very irregularly granular and tufted in texture, beginning
whitish but soon characteristically pale olive to purple-brown, with the reverse
dull yellow to red-brown.
Conidiophores are up to 300 µm high, with thick, hyaline (clear) walls.
They are aseptate and biseriate (i.e., bearing metulae and phialides), with ovoidal
278 Summerbell
Figure 15 Aspergillus versicolor.
vesicles (Fig. 16). Conidia are blue-greenish, subglobose to globose, rough-

walled (verruculose), and 3.5 to 5.5 µm in diameter. Globose to subglobose hülle
cells are abundant, often scattered across the colony surface in conspicuous
clumps.
This species is distinguished from the far more common A. versicolor
mainly by its formation of conspicuous clumps of hülle cells in a faster-growing
colony and by its larger conidia.
Aspergillus {aff. Fennellia} janus Raper & Thom. This species was
reported from a patient who had keratitis subsequent to ocular puncture by a twig
(97). No direct microscopy confirming fungal filaments in the cornea was done.
Moreover, no characters confirming the fungal identification were given. The
infection responded to antifungals. This suboptimal evidence is all that links this
fungus to human and animal pathogenicity so far. A. janus is similar to A. versi-
color but is distinguished by: (1) the production, especially on Czapek’s agar,
Ascomycetes 279
Figure 16 Aspergillus granulosus.
of two distinct types of aspergilli, one tall (2000 to 2500 µm long), with clavate
vesicles and white conidia, and the other shorter (300 to 400 µm long), with
ovoid vesicles and dark green conidia, as well as some heads of mixed character,
and (2) regular production on Czapek’s agar of masses of cream to yellow hülle
cells at 24°C. According to Raper and Fennell (7), it varies considerably ac-
cording to the exact media and temperatures used in identification, and is best
identified under the conditions outlined in their monograph. Whether any of its
distinctive features might be produced on any media commonly used in medical
mycology has not been investigated. A recent molecular study indicates that it
is not in fact closely related to A. versicolor, despite its phenotypic similarities (6).
Aspergillus {aff. Emericella} sydowii (Bain. & Sart.) Thom & Church.
This fungus is primarily significant in medical mycology as an agent of opportu-
nistic onychomycosis (46, 47), usually in the elderly patient. It is an abundant
airborne contaminant and colonizer of poorly stored medical specimens, so attri-
280 Summerbell
butions of other opportunistic infectious capacities to it need to be reviewed with

caution. Some early reports of causation of onychomycosis [e.g., those of da
Alecrim and Vital (98) and Schönborn and Schmoranzer (60)] are inadequately
documented or dubious (e.g., a case reported in the latter paper in which fungal
elements were not detected in direct microscopy). Well-confirmed ocular keratitis
was reported by Shukla et al. (15). Identification details were not given, but the
identity of the fungus was confirmed by the Commonwealth Mycological Institute
(now Commonwealth Agricultural Bureau International; CABI) and the strain
was conserved. On the other hand, a keratitis record by Prasad and Nema (99)
lacks direct microscopic verification. Also, the mycology in this paper is sup-
ported only by its figures, none of which purports to show A. sydowii. The figures
presented include a nondescript image of aspergillus heads that appears identi-
cally twice, once labeled A. flavus and the second time Curvularia lunata, while a
photo of C. lunata is labeled A. fumigatus. A pulmonary and pericardial infection
concurrent with systemic staphylococcal infection in a moribund neonate with
adrenal cortical hypofunction was reported by Zimmerman (100). No mycologi-
cal details were given. The identity of the fungus, however, was confirmed by two
general bacteriology laboratories. This unverifiable record or unacknowledged
references to it have appeared in many reviews over the past 45 years [e.g., Rip-
pon (11)], firmly establishing A. sydowii as a systemic opportunist.
The species’ most common indoor habitat may be damp structural material,
especially papered walls and wallboard, but it also occurs in soils, on seeds, and
on many types of decaying litter, foodstuffs, fabrics, and insect cadavers (74).
Its principal secondary metabolites, sydowinin and related compounds, are of un-
certain toxigenic significance (8).
Description. Colonies are moderately slow-growing, attaining 1.5 to 2
cm in 7 days. They begin whitish but are soon characteristically navy blue to
blue-green, typically with beads of red-brown exudate on the colony surface and
with a similarly reddish-brown colony reverse.
Conidiophores are up to 500 µm high, with thick, hyaline (clear) walls.
They are aseptate and biseriate (i.e., bearing metulae and phialides) (Fig. 17).
Reduced uniseriate conidiophores resembling structures of the genus Penicillium
are also frequently seen, as in A. versicolor (Fig. 18). Conidia are in dry, upright
chains, usually massed into short, upright columns, blue-green in transmitted
light, subglobose to globose, distinctly roughened with verrucose ornamentation,
and 2 to 3.5 µm in diameter.
Aspergillus {aff. Emericella} varians Wehmer. A. varians is very simi-

lar to A. versicolor (q.v.) except that it has significantly larger conidia (3.8 to 5
µm in diameter rather than 2 to 3.5 as in A. versicolor). It also has conidiophores
on malt extract agar distinguishably formed in two size classes, tall and short.
A. versicolor may have variability in its conidiophores but no such regular strati-
Ascomycetes 281
Figure 17 Aspergillus sydowii, full-sized conidiophore.
fication. An isolate linked with some hesitation to A. varians has been a validly
attributed causality of a case of onychomycosis by Torres-Rodrı́guez et al. (101).
A later publication by the same research group (84) refers to this isolate as A.
versicolor.
Anamorphic Species in A. Subgenus Nidulantes, Section Usti Gams et al.
(Formerly A. ustus Group).
Aspergillus {aff. Emericella} ustus (Bain.) Thom & Church. This spe-
cies rarely causes human or animal disease, but some cases of pulmonary, dis-
seminated, or primary cutaneous infection in immunocompromised patients have
recently been well documented (102–107). A. ustus formed part of a mixed fun-
gal community growing in grossly visible patches on burn eschar that had been
coated with therapeutic emulsion. The depth of penetration of the organism was
not determined, but there was no evidence of penetration into living tissues (108).
282 Summerbell
Figure 18 Aspergillus sydowii, reduced conidiophore.
Endocarditis connected with a prosthetic cardiac valve has been well demon-
strated (109). Causation of onychomycosis has been suggested by Walshe and
English (46), and probably occurs but has not yet been well confirmed [i.e., by
successive repeat isolation, rather than the relatively unreliable (110) counting
of positive inoculum fragments employed by Walshe and English (46)]. A report
by Wadhwani and Srivastava (17) alleging causation of otitis must be discounted
for reasons mentioned in the discussion of Fennellia nivea below.
In nature, A. ustus is most common in soils and on seeds, especially grains
and peanuts (111). Its characteristic secondary metabolites, austamide, austdiol,
austins, and austocystins, are considered significant mycotoxins (8).
Description. The colonies are moderately rapidly growing (30 to 50 mm
in 7 days). They are velvety to slightly floccose, beginning whitish but soon
characteristically greyish-brown, typically with a yellow to yellow-brown colony
reverse and yellowish soluble pigment.
Conidiophores are 75 to 350 µm high, with thick brown walls. They are
aseptate and biseriate (i.e., bearing metulae and phialides) (Fig. 19). Conidia are
in dry, upright chains, usually seen as radiating or forming short columns, brown-
Ascomycetes 283
Figure 19 Aspergillus ustus, conidiophore.
ish in transmitted light, globose, strongly roughened, and 3 to 4.5 µm in diameter.

A minority of colonies will form elongate, irregular, thick-walled ‘‘squashed ba-
nana’’ hülle cells (Fig. 20).
Aspergillus {aff. Emericella} deflectus Fennell & Raper. This species
has been linked in two publications (five cases) to disseminated infection in dogs,
mostly in the notoriously Aspergillus-susceptible German shepherd breed (112),
but also in one case in a springer spaniel (113).
Isolates from nature have mainly derived from soils in areas of tropical to
mediterranean climate.
Description. Colonies grow restrictedly (1.0 to 1.5 cm in 7 days), begin-
ning whitish, but soon characteristically greyish drab, reverse yellow to dull
orange-red.
Conidiophores are mostly 40 to 50 µm long, with thick, red-brown walls.
They are aseptate and biseriate (i.e., bearing metulae and phialides), with small
284 Summerbell
Figure 20 Aspergillus ustus, hülle cells. From Laboratory Handbook of Dermatophytes,

by J. Kane et al., 1997, Star Publishing Company, Belmont, CA (see Ref. 89). Used with
permission.
vesicles characteristically abruptly bent downward, giving a ‘‘briar pipe appear-

ance’’ (Fig. 21). Conidia are in dry chains, more or less columnar, brownish in
transmitted light, globose, rough-walled, and 3 to 3.5 µm in diameter. A minority
of colonies will form elongate, irregular, thick-walled ‘‘squashed banana’’ hülle
cells.
3. Fennellia Wiley & Simmons

Ascomata are globose cleistothecia, yellow and surrounded by masses of rounded
to elongate hülle cells. Asci are eight-spored, globose, and evanescent. Asco-
spores are hyaline to pale yellow, lens-shaped, smooth or rough-walled, with a
sometimes inconspicuous equatorial groove, and sometimes also with inconspicu-
ous crests. Anamorphs and evolutionarily radiating anamorphic species are mem-
bers of Aspergillus subgenus Nidulantes sections Flavipedes and Terrei, formerly
considered the A. terreus and A. flavipedes series (7). They have biseriate conidio-
phores with fertile elements arising from the upper portions of the vesicle.
Ascomycetes 285
Figure 21 Aspergillus deflectus.
Species with a Known Teleomorph (Which May Not Form in Every Culture)
Anamorphs in Aspergillus Subgenus Nidulantes Section Flavipedes Gams et al.
(Formerly A. flavipes Group)
Fennellia flavipes Wiley & Simm., Anamorph Aspergillus flavipes
(Bain. & Sart.) Thom & Church. Barson and Ruymann (114) reported a fatal
A. flavipes infection in a teenaged leukemic bone marrow transplant patient. It
began as a palmar skin infection after brief contact with an armboard during
catheter placement. No confirmatory identification details or mycological credit
were noted. Apart from this reference to A. flavipes per se causing disease, there
are also a small number of references to members of the ‘‘A. flavipes group’’
doing so. Roselle and Baird (115) received an identification of ‘‘A. flavipes
group’’ from the Centers for Disease Control (CDC) in Atlanta, for a fungus
well demonstrated as causing osteomyelitis in the lumbar vertebrae subsequent
286 Summerbell
to surgical evacuation of an intracerebellar hematoma. Such ‘‘group’’-level iden-

tifications were occasionally received from CDC at the time. [Compare Weiss
and Thiemke (102).] Also, a patient with a history of tuberculosis who had previ-
ously been successfully treated for aspergillosis developed an aspergilloma; A.
flavipes group was isolated from sputum (116). A photograph of a clearly biseri-
ate Aspergillus is depicted in this publication. The conidiophores shown, how-
ever, are not over 50 µm long, as opposed to ⬎150 µm for typical A. flavipes,
so an atypical isolate may be indicated. An Aspergillus isolate from respiratory
secretions may always be incidental, regardless of the patient’s underlying condi-
tion (117), and even when obtained as a heavy growth, so the significance of the
isolation in this case is not entirely certain. The patient gave a positive antibody
response to A. fumigatus antigen in counterimmunoelectrophoresis studies.
F. flavipes is predominantly a soil fungus, especially in warmer parts of
the globe.
Description. The colonies are moderately slowly growing (15 to 35 mm
in 7 days). They are deeply powdery with dense conidiophores, characteristically
orange-grey to dull pinkish-buff, ‘‘with the overall coloration resulting from the
off-white spore masses against a background of brownish conidiophores’’ (7).
The reverse is pale to pale yellow-brown.
Ascomata are seldom formed in culture and are not described in detail here.
[See Wiley and Simmons (118).] Conidiophores are mostly 150 to 450 (⫺800)
µm high, with smooth to slightly roughened, mostly yellow to light brown walls,
biseriate (i.e., bearing metulae and phialides), with metulae arising from the upper
1/2 to 3/4 of the subglobose vesicle, tightly packed together toward an apical
concentration (Fig. 22). Conidia are in dry, short, upright columns, hyaline, whit-
ish en masse, globose, smooth, and 2 to 3 µm in diameter. In the submerged
mycelium of some isolates, conidia of a second type, aleurioconidia, are found
attached to hyphae by short pegs. These conidia, which may be sparsely or
densely produced, are refractile, globose to ovoid, and 4 to 6 µm long. Hülle
cells, if present, are elongate, sometimes branched and swollen, with heavy walls.
Fennellia nivea (Wiley & Simm.) Samson, Anamorph Aspergillus ni-

veus Blochw. A male with poorly controlled diabetes who had had a pulmonary
lesion caused by Aspergillus flavus surgically removed 5 years earlier was re-
ported by Seabury and Samuels (119) to have developed a second lesion caused
by A. niveus, which was also surgically treated. No mycological characters were
given to allow verification of the unusual identification, nor was a mycologist
credited. A. niveus was also reported as an agent of otitis by Wadhwani and
Srivastava (17). The cases in this paper are poorly attested, and otitis appears to
be ascribed to all fungi grown or seen in direct microscopy, including smut telio-
spores that could only have formed on infected plants. An Aspergillus conidio-
phore shown in direct microscopy of ear material, seemingly in connection with
Ascomycetes 287
Figure 22 Fennellia flavipes CBS 260.73, conidiophore apex. Apparent dark color of
vesicle is an artifact of staining.
the report of A. niveus, has a subglobose vesicle with metulae all around and an
untapered stipe attachment point. It may be A. candidus or A. ochraceus. A. niveus
has spathulate to hemispheral vesicles with only the upper 1/2 to 2/3 bearing
metulae. The vesicle tapers gradually into the stipe in many of its conidiophores.
A record from sinusitis by Vennewald et al. (43) cannot be accepted for reasons
given above under E. herbariorum.
F. nivea is predominantly a soil fungus, especially in tropical to midtemper-
ate parts of the globe (74).
Description. Colonies are moderately slow-growing (20 to 30 mm in 7
days). They are deeply powdery with dense conidiophores, characteristically
white, sometimes at least in part pale yellow or cream, with or without beads of
yellowish or reddish exudate, and with yellow-brown, red-brown, dark green, or
nearly black reverse colors.
Ascomata are seldom formed in culture and are not described in detail here.
[See Wiley and Fennell (120).] Conidiophores are mostly 100 to 600 (⫺1000)
µm high, with smooth hyaline walls. They are biseriate (i.e., bearing metulae
and phialides), with metulae arising from the upper 1/2 to 2/3 of the subglobose
vesicle, and tightly packed together toward an apical concentration (Fig. 23).
Conidia are in dry, short, upright columns, and hyaline. They are white to dull
whitish-buff en masse, globose or subglobose, smooth, and 2 to 3.5 µm in diame-
ter. In the submerged mycelium of some isolates, conidia of a second type, aleuri-
288 Summerbell
Figure 23 Fennellia nivea, conidiophores.
oconidia, are found attached to hyphae by short pegs. These conidia, which may
be sparsely or densely produced, are refractile, globose to ovoid, and 3 to 5 µm
long. Hülle cells, if present, are present as tufted, yellow masses of globose to
elongate, thick-walled cells.
Anamorphic Species in A. subgenus Nidulantes, Section Flavipedes Gams
et al. (Formerly the Aspergillus flavipes group).
Aspergillus {aff. Fennellia} carneus Blochw. This fungus was isolated
on several successive occasions from a patient described only as having pneu-
monia (121). No direct microscopic examination of either fluids or tissues was
reported. Possibly an allergic bronchopulmonary colonization was involved, al-
though incidental sources (e.g., contamination due to continuous exposure to an
environmental source) cannot be completely ruled out. In nature, this fungus
mostly derives from tropical soils (122).
Ascomycetes 289
Figure 24 Aspergillus carneus. conidiophore.
Description. The colonies are moderately slow-growing (15 to 30 mm in

7 days). They are powdery, beginning whitish, but soon characteristically pinkish
to pinkish-brown, sometimes orange-brown. The reverse is pale or tinged with
yellow or brown.
Conidiophores are mostly 80 to 200 µm high, with smooth walls that are
hyaline or faintly brownish. The vesicles are clavate to nearly spherical, bearing
biseriate (i.e., with metulae and phialides) fertile elements (Fig. 24). Fertile ele-
ments may also occasionally be irregularly disposed along the conidiophore (74).
Conidia are in dry chains, spherical, smooth, and mostly 2.5 to 3 µm in diameter.
In the submerged mycelium, a conidia of a second type, aleurioconidia, may be
found attached to hyphae by short pegs. These conidia, which may be sparsely
produced, are refractile, globose to ovoid, and 4 to 6 µm long.
290 Summerbell
Anamorphic Species in A. Subgenus Nidulantes, Section Terrei Gams et

al. (Formerly Aspergillus terreus Group).
Aspergillus {aff. Fennellia} terreus Thom. This species is one of the five
prominent and regularly encountered opportunistic fungi in the genus Aspergillus,
along with A. fumigatus, A. flavus, A. nidulans, and A. niger. It and A. nidulans,
however, are generally much less commonly encountered than the other three
species listed. A. terreus causes the full range of known aspergilloses, including
pulmonary and disseminated aspergillosis in the immunocompromised patient
(69, 123), allergic bronchopulmonary aspergillosis (124), otomycosis (27, 125),
onychomycosis (47, 49, 126), and various other infections (69). In the immuno-
compromised patient, it appears to cause higher morbidity and to be significantly
less responsive to amphotericin B therapy than other aspergilli (127). The occur-
rence of aleurioconidia in histopathology may facilitate rapid diagnosis when the
pathologist is aware of their significance (128). A. terreus may cause serious in-
fections in both birds (69) and dogs (129, 130). In nature and the anthroposphere,
A. terreus is a fungus of composts, soils, seeds, and foodstuffs (74, 124). It pro-
duces as major mycotoxins the mutagen patulin, the nephrotoxin citrinin, and the
neurotoxin citreoviridin (8).
Description. The colonies are moderately rapidly growing (25 to 65 mm
in 7 days). They are deeply powdery with dense conidiophores, and characteristi-
cally medium sandy brown. The reverse is pale to pale brownish.
Conidiophores are 100 to 250 µm high, with smooth, hyaline walls that
are straight or gently undulate (Fig. 25). They are aseptate and biseriate (i.e.,
bearing metulae and phialides), with metulae arising from the upper one-half to
two-thirds of the subglobose vesicle and conspicuously appressed against one
another in a nearly parallel orientation. Conidia are in dry, upright columns, in
wet microscopic mounts hyaline to slightly yellow in transmitted light, globose
to ellipsoidal, smooth, and 2 to 2.5 µm in diameter. In the submerged mycelium,
conidia of a second type, aleurioconidia, are found attached to hyphae by short
pegs. These conidia, which may be sparsely or densely produced, are refractile,
globose to ovoid, and 4 to 6 µm long.
Host-adapted (dysgonic) isolates of A. terreus may have few or no aspergil-
lary heads and produce only aleurioconidia, yielding a colony reminiscent of the
filamentous morph of Blastomyces dermatitidis (89, 131). Colonies will be whit-
ish, dense, and often radially furrowed, as with dysgonic A. fumigatus.
Anamorphic Species Currently in A. subgenus Circumdati, Section Candidi
Gams et al., but with Greater Biological Affinities to Teleomorph Genus Fennellia
(Formerly the Aspergillus candidus Group).
Aspergillus {aff. Fennellia} candidus Link. Recent molecular studies

(6) have shown that this fungus and its close relatives are more closely related
Ascomycetes 291
Figure 25 Aspergillus terreus conidiophore.
to the teleomorph genus Fennellia than to Petromyces and Neopetromyces, as

was long thought.
This species was well documented as an agent of brain granuloma in a man
who had injured his head in a fall from a horse (132). A direct microscopically
confirmed case of sphenoid sinus infection (133) contains no confirmatory identi-
fication information; however, the identity of the organism is emphasized as a
key feature of the study. As relatively few hyphomycetes are easier to identify
than A. candidus, this identification is accepted as likely correct. A necrotizing
coinfection by A. candidus and A. niger beginning in the ear of a leukemia patient
was documented by Falser (134). Five case records from otomycosis by Yassin et
al. (95) are regarded as unsubstantiated for reasons mentioned under A. versicolor
above. On the other hand, an otomycosis case registered by Gregson and La
Touche (27) is well attested, even though an unusually dark colony reverse made
the authors cautiously label the isolate ‘‘A. candidus sp.’’ Well-confirmed cases
of onychomycosis have been documented (135, 136), as well as cases problemati-
cally attested from direct-microscopic-positive single specimens yielding heavy
292 Summerbell
Figure 26 Aspergillus terreus aleurioconidium.
outgrowth (46, 137). A fungus identified as A. candidus was repeatedly cultured

from the sputum and needle biopsy of a patient with a pulmonary cavity by
Iwasaki et al. (138), but could not be traced in pathology examinations after the
affected portion of lung was resected. Photographs of the fungus isolated are not
typical of A. candidus, but may show diminutive heads known from this species,
as illustrated by Raper and Fennell (7, p. 348).
A. candidus is mainly known from warmer soils and from seeds, especially
grains (74). Major secondary metabolites are terphenyllin and derivatives, as well
as chlorflavonin (139).
Description. The colonies are moderately slow-growing (10–25 mm in
7 days). They are densely powdery and characteristically white to pale cream.
The reverse is pale to moderate yellow.
Conidiophores are often 200 to 500 (but up to 1000) µm high, with smooth
to finely roughened, hyaline walls. They are aseptate, attaching abruptly to a glo-
bose vesicle, which is biseriate (i.e., bearing metulae and phialides), with metulae
all around the perimeter in a radial orientation (Figs. 27 and 28). Conidia are
found in dry, upright chains. They often radiate or mass into two or more short
columns per head, in wet microscopic mounts. They are grey-brown, globose,
strongly roughened, and 3.5 to 4.5 µm in diameter. Sclerotia, if formed, are red-
purple to black.
A. candidus can be distinguished from the similarly whitish A. niveus by
Ascomycetes 293
Figure 27 Aspergillus candidus, full-sized conidiophores.
its fertile elements covering the whole vesicular surface, as opposed to only the
upper half to two-thirds in A. niveus. Subsurface aleurioconidia can also often
be found to confirm an identification of A. niveus.
4. Neopetromyces Frisvad & Samson

The teleomorph is formed as hard sclerotioid bodies that mature as ascostromata
containing one to two ascomata. Ascostromata are globose, subglobose, or elon-
gate, 400 to 600 µm in diameter, and pale yellow, with an outer layer of thick-
walled angular to subglobose cells and an inner layer of thin-walled cells. Asco-
mata develop as subglobose to globose chambers within the ascostromata, and
are 155 to 300 µm in diameter. Asci are eight-spored, globose, and evanescent.
Ascospores are oblate, subglobose to broadly ellipsoidal, with spiny walls and a
small equatorial ridge, 4.5–5.5 ⫻ 3.5–5.8 µm. The description is based on N.
294 Summerbell
Figure 28 Aspergillus candidus, reduced conidiophores.
(Petromyces) muricatus (Udagawa et al.) Frisvad & Samson (140, 141). Ana-
morphs are members of Aspergillus subgenus Circumdati section Circumdati,
formerly considered the A. ochraceus series (7) (excluding the anamorph of Pe-
tromyces alliaceus Malloch & Cain). They have biseriate conidiophores. The sole
species forming the teleomorph has no record as a human or animal pathogen.
Anamorphic Species in A. subgenus Circumdati, Section Circumdati Gams
et al. (Formerly the Aspergillus ochraceus Group).
Aspergillus {aff. Neopetromyces} ochraceus Wilhelm. This species has
been well documented as an agent of one case of allergic bronchopulmonary
aspergillosis (142) and in one case of suppurative otitis media (142a). A ‘‘sulfur
yellow’’ Aspergillus described as ‘‘probably A. ochraceus’’ was well docu-
mented from keratitis in rural Bangladesh by Williams et al. (143). Wierzbicka
et al. (144) isolated and correctly identified A. ochraceus on a single occasion
Ascomycetes 295
from the sputum of a neutropenic leukemia patient with a cavitating pulmonary

lesion that responded to antifungal drugs. As was shown by Staib et al. (117),
who recorded numerous fortuitous isolations of aspergilli from uninfected AIDS
patients, such isolations are not necessarily significant. Wierzbicka et al. (144)
stated that serum antibodies precipitated in vitro against antigens of both A. fumi-
gatus and A. ochraceus, a finding more likely consistent with a cross-reaction
than with a dual infection. (No cross-absorption steps were mentioned.) The case
report thus neither fully substantiated A. ochraceus infection nor ruled out A.
fumigatus. Bovine abortion was attributed to A. ochraceus by Muñoz et al. (145);
however, the well-described and illustrated organism is clearly A. terreus. Para-
nasal sinusitis caused by A. ochraceus was alleged by Bassiouny et al. (146);
however, no direct microscopic examination was recorded in this case, and fungi
such as Penicillium melinii with no record of human or animal pathogenicity
were recorded from other inadequately documented cases.
In nature and the anthroposphere, A. ochraceus is a soil fungus, especially
in warm regions, and is also commonly isolated from seeds, coffee beans, de-
caying insects, and indoor carpets. It produces a highly economically significant,
nephrotoxic grain-contaminating mycotoxin, ochratoxin A (8).
Description. The colonies are moderately rapid-growing (45 to 55 mm
in 7 days). They are deeply powdery with dense conidiophores and characteristi-
cally chalky yellow to pale ochraceous yellow-brown, with the reverse pale to
brownish.
Conidiophores are up to 1500 µm high, with granular, pale yellow-brown
walls, attaching abruptly to a globose to subglobose vesicle, which is biseriate
(i.e., bearing metulae and phialides), with metulae all around the perimeter in a
radial orientation (Fig. 29). Conidia are in dry, upright chains, often massing into
two or more short columns per head, in wet microscopic mounts hyaline. They
are globose to subglobose, smooth or finely roughened, and 2.5 to 3.5 µm in
diameter. Some colonies form pinkish to purple, irregular, rock-hard sclerotia up
to 1 mm in diameter.
Aspergillus {aff. Neopetromyces} sclerotiorum G.A. Huber. This spe-

cies was well documented as an agent of an onychomycosis by Feuilhade de
Chauvin and de Bièvre (147). A record from sinusitis by Vennewald et al. (43)
cannot be accepted for reasons given above under E. herbariorum. A. scleroti-
orum is mainly a tropical soil fungus.
Description. The colonies are moderately rapid-growing (40 to 55 mm
in 7 days), with velvety to floccose white mycelium mostly covered with light
yellow conidial masses and white to buff sclerotia. The colony reverse is usually
light yellow.
Conidiophores are mostly 400 to 1200 and sometimes as tall as 2000 µm
high, with rough yellow or brownish walls, attaching relatively abruptly to a
296 Summerbell
Figure 29 Aspergillus ochraceus.
globose vesicle, which bears biseriate fertile elements (i.e., metulae and phialides)
in larger heads, with these elements all around the perimeter in a radial orientation
(Fig. 30). Conidia are in dry, radiating chains, smooth to finely roughened, glo-
bose, and mostly 2.5 to 3.0 µm in diameter. The sclerotia are white to buff, mostly
1 to 1.5 mm in diameter.
This species must be distinguished from A. ochraceus, which often has no
sclerotia but may have pale pink to purple sclerotia, distinct from the creamy
sclerotia of A. sclerotiorum. A. sclerotiorum is also distinct in conidial color from
A. ochraceus. The former is pale yellow, whereas the latter is a deeper color,
described as ranging through wheat, ochraceous, buff, or amber-yellow (122).
5. Petromyces Malloch & Cain

The teleomorph is formed as hard sclerotioid bodies that mature as ascostromata
containing 1 to 8 ascomata. Ascostromata are ovate to elliptical, 1000–3000 ⫻
Ascomycetes 297
Figure 30 Aspergillus sclerotiorum.
500–700 µm, at first whitish, but soon maturing grey and then black, with an
outer sclerenchymatous layer composed of thick-walled angular to subglobose
cells. Ascomata develop as globose chambers within the ascostromata. Asci are
eight-spored, globose, and evanescent. Ascospores are ellipsoidal, with smooth
walls and an equatorial furrow scarcely visible in young spores only, 5.5–9 ⫻
5.0–7.0 µm. The description is based on P. alliaceus. (See below.) Anamorphs
are now considered members of Aspergillus subgenus Circumdati section Flavi
(8), based on biochemical and genetic evidence; they were previously placed in
the A. ochraceus group by Raper and Fennell (7).
Species with a Known Teleomorph, and with Anamorphs in Aspergillus

Subgenus Circumdati Section Flavi Gams et al. (Formerly Aspergillus flavus
Group).
298 Summerbell
Petromyces alliaceus Malloch & Cain, Anamorph Aspergillus alliaceus

Thom & Church. This species was reported as the agent of a chronic otitis
externa subsequent to surgery (148). It was isolated on two separate occasions,
but direct microscopy was not recorded, leaving the organism’s role in the disease
process in question. A record from sinusitis by Vennewald et al. (43) cannot be
accepted for reasons given above under E. herbariorum.
The species is known from nature in soils, especially those of deserts and
grasslands, and may also grow from root crops in the onion and garlic family,
Alliaceae. It produces ochratoxin A and B and kojic acid as major secondary
metabolites (141).
Description. The colonies are rapid-growing (60 to 70 mm in 7 days),
with velvety to floccose white mycelium covered sparsely at maturity with cream
to yellow-gold conidial masses and dark grey to black sclerotia, the latter often
formed in concentric accumulations. The colony reverse is pale tan to yellow-
brown.
Conidiophores are a mixture of intergrading large and small types, stipes
as short as 40 and as long as 2000 µm high, with smooth, hyaline walls, attaching
relatively abruptly to a globose to pyriform vesicle, which bears biseriate fertile
elements (i.e., metulae and phialides) in larger heads and uniseriate elements
(phialides only) in smaller heads, with these elements all around the perimeter
in a radial orientation (Fig. 31). Conidia are in dry, radiating chains. They are
hyaline, smooth, subglobose to ovoid, and mostly 3.0 to 3.5 µm in diameter.
Sclerotia/ascostromata are ovate to elliptical, 1000–3000 ⫻ 500–700 µm, white,
then grey with white tips, then black. Production of ascospores is seldom seen
and requires 3 to 10 months of incubation (7, 149).
This species must be distinguished from members of section Circumdati,
such as A. ochraceus and A. sclerotiorum, which have pale to pink or purple
colored—not black—sclerotia, and conidia in paler shades of yellow.
Anamorphic Species in A. subgenus Circumdati, Section Flavi Gams et al.
(Formerly the Aspergillus flavus Group).
Aspergillus {aff. Petromyces} flavus Link. A. flavus has essentially
caused every known variant of aspergillosis, in particular: (1) pulmonary, dissem-
inated, and other systemic infections in immunocompromised, especially neutro-
penic patients; (2) rare, idiopathic, systemic, localized, and occasionally progres-
sive infections in apparently immunocompetent patients; (3) chronic colonizing
infections, mainly of predisposed hosts (e.g., allergic bronchopulmonary aspergil-
losis), mostly in long-term asthmatic and cystic fibrosis patients (150), pulmo-
nary aspergilloma (fungus ball) in lungs of persons with pre-existing cavitation,
chronic mycotic sinusitis, and otomycosis (outer ear canal surface infestation);
and (4) opportunistic mycoses of especially vulnerable body sites (e.g., ocular
infections subsequent to traumatic introduction, otomycosis, onychomycosis,
Ascomycetes 299
Figure 31 Petromyces alliaceus, conidiophore.
dialysis-related peritonitis, and endocarditis). In comparison with A. fumigatus,

however, it generally causes a higher proportion of infections of sinuses and ear
canals and a lower proportion of pulmonary infections in proportion to the overall
frequency of significant isolation of both fungi. This trend arises possibly because
its larger conidia are not as effective as the very small conidia of A. fumigatus
at penetrating deeply into the lungs, but may impact more effectively in more
exposed airways. In places such as Sudan, in which A. flavus is notoriously preva-
lent, however, this trend is scarcely noticeable, and the species overwhelmingly
causes most aspergillosis.
Aspergillus flavus is a cosmopolitan compost-degrading fungus as well as
a general colonizer of warm soils. Some of its variants, as well as closely related
species such as Aspergillus parasiticus, have particular relationships as seed
pathogens and degraders of plants such as Zea mays, maize corn. Extensive notes
on sites of isolation are given by Domsch et al. (74). A. flavus produces the
300 Summerbell
Figure 32 Aspergillus flavus.
notorious hepatic mutagens in the aflatoxin B series as well as cyclopiazonic

acid. As a toxin producer it may be a major cause of hepatic cancer as well as
occasional outbreaks of acute liver toxicosis, especially in countries in which its
levels in peanut and maize products are not stringently regulated.
Description. The colonies are rapidly growing (mostly 50 to 70 mm in
7 days), deeply powdery-granular, yellow-green to green, sometimes with visible
white marginal or central mycelium, and the reverse is pale to brown. Atypical,
host-adapted colonies, degenerated after prolonged growth in the patient, may
be deeply floccose and white with a greenish cast imparted by scattered conidio-
phores in the aerial mycelium.
Conidiophores are often around 400 to 800 (but up to 2500) µm high.
The stipes are hyaline, with characteristic granular wall roughening (must be
distinguished from adherent bacteria or fat droplets on smooth-stalked species)
Ascomycetes 301
(Fig. 32). The walls are generally parallel, but with a slight funnel-like expansion
at the apex supporting the globose to subglobose vesicle. The aspergillary heads
of both biseriate (i.e., bearing metulae and phialides) and uniseriate (phialides
only) character are often found, with larger heads generally more complex. Metu-
lae or phialides are attached around the whole vesicle perimeter or at least the
apical three-quarters in a radial orientation. Conidia are in dry chains. They are
radiate, green, finely roughened or rarely smooth, globose to subglobose, and 3.5
to 5.0 µm in diameter. Although common in isolates from the environment, scle-
rotia are rare in clinical isolates except from nail and skin, but when seen are
stony-hard, usually red-brown to black, roundish, mostly 400 to 800 µm in diam-
eter.
Specific polymerase chain reaction (PCR) primers for identification or di-
rect detection of A. flavus have been published in recent years. For example,
primer pairs based on genes related to aflatoxin biosynthesis (151) detect only
A. flavus and A. parasiticus. Sandhu et al. (152) devised a specific 28S rDNA-
based probe for A. flavus to be used after PCR amplification of clinical specimens
or unknown fungal cultures using universal fungal 28S primers. Primers based
on alkaline protease genes for detection of A. flavus were published by Tang et al.
(153). Walsh et al. (154) found that single-strand conformational polymorphism
analysis could distinguish among 18S rDNA-based PCR products from A. flavus,
A. fumigatus, and selected other medically important fungi.
In subspecific population structure A. flavus consists of two genetically
isolated groups, one (group I) with considerably more genetic variation than the
other (group II) (155). Although the species is largely clonal, there is some evi-
dence of past or ongoing recombination in population genetics statistical tests.
A. flavus should be distinguished from the closely related A. parasiticus,
which differs by having mostly or entirely uniseriate aspergilla, strongly rough-
ened conidia, and a more somber color described as ‘‘dark yellow-green’’ by
Samson et al. (111) as opposed to ‘‘green’’ for A. flavus (111, p. 54, Table 3),
and ‘‘dark olive or deep dark green’’ by Klich and Pitt (122) as opposed to ‘‘olive
green, olive or parrot green’’ [color names from Kornerup and Wanscher (86)].
The distinction is best made after having seen authentic cultures of each species.
A. parasiticus produces aflatoxins of the B and G series—the latter are never
produced by A. flavus—and does not produce cyclopiazonic acid. A. parasiticus
has little record as a human and animal pathogen, but this may be due to inatten-
tion; that is, failure to distinguish it from A. flavus.
Aspergillus {aff. Petromyces} oryzae (Ahlb.) Cohn. The name A. oryzae

was originally based on domesticated A. flavus-like strains used in soy fermenta-
tion processes. In morphological characters, however, these domesticated isolates
intergraded with some wild isolates, and descriptions of A. oryzae such as that
of Domsch et al. (74) tended to include both domesticated and similar wild iso-
302 Summerbell
lates. In contrast to A. flavus as classically defined, A. oryzae tended to have more

brownish-green conidial masses, a more floccose colony, longer conidiophores
(up to 4000 to 5000 µm in length), and larger conidia, mostly 5 to 6 µm in
diameter. Genuine A. oryzae isolates do not produce aflatoxins (111). Recent
population genetics studies have shown that they are closely similar to one of
16 multilocus genotypes discovered in a preliminary survey of A. flavus genetic
diversity (155). Some isolates considered A. flavus are highly genetically similar,
and in terms of normal biological taxonomic criteria, A. oryzae is not regarded
as a separate species. It is maintained as a regulatory category for aflatoxin-free
koji molds and as an acknowledgment of the differentiating effects of domesti-
cating selection (155). At the time of this writing, however, it is not at all clear
that any wild isolate, no matter how superficially similar, can be included under
the name A. oryzae. Certainly any isolate given this name today should at the
very least be tested using sensitive techniques such as high-powered liquid
chromatography to ensure no aflatoxin is produced, regardless of apparent
morphological similarity. Ideally, genetic similarity with the A. oryzae char-
acters demonstrated by Geiser et al. (155) should be shown. A. oryzae has a
relatively extensive track record of medical cases that are clinically indistin-
guishable from cases attributed to A. flavus (69). Except possibly for the small
number of papers documenting cases connected to soy fermentation processes
(e.g., 156, 157), these case reports should all be considered to apply to A. flavus.
It should be noted that the genetic diversity now revealed within A. flavus makes
many previously used defining features for A. oryzae invalid or in need of re-
evaluation. For example, Gordon et al. (158) ‘‘confirmed’’ an alleged A. oryzae
isolate from meningitis in an injection drug addict by showing that patient serum
reacted with ‘‘A. oryzae antigen’’ but not with A. flavus antigen in immunodiffu-
sion tests. (It reacted with both antigens in the more sensitive counterimmuno-
electrophoresis technique used in the same study.) The authors did not state if
the A. oryzae antigen was from a known domesticated reference isolate or from
the case isolate. In any event, even if a reference isolate was correctly used, the
case isolate to which antibodies were directed may have simply been from an
A. flavus group I isolate (see A. flavus subspecific population structure above)
genetically close to A. oryzae, whereas the reference A. flavus isolate may have
been from group II or another genetically relatively distant subgroup of A. flavus.
In hindsight, it can be firmly stated that identity with A. oryzae was not demon-
strated.
Still less, then, was such an identity demonstrated in case reports such as
that of Byard et al. (159), where a ‘‘greenish-yellow’’ A. flavus–like isolate from
sinusitis in Canada was called A. oryzae because it was seen to form only uniseri-
ate conidiophores! Although A. oryzae as an A. flavus subtype is not expected
to differ significantly in pathogenicity from the species as a whole, documentation
Ascomycetes 303
of this pathogenicity in a way that conforms to current standards will require

sophisticated research methodologies.
Aspergillus {aff. Petromyces} avenaceus G. Smith. This species was
well demonstrated from chronic invasive sinusitis in an otherwise healthy Suda-
nese immigrant to the United States by Washburn et al. (160). No identification
characters were given, but the identity of the fungus was confirmed by K. B.
Raper. Curiously, despite the rarity of the organism, the highly interesting source
of isolation, and the examination by one of history’s foremost Aspergillus authori-
ties, the isolate appears not to have been conserved. This naturally raises a ques-
tion about whether or not it was sufficiently typical to be identified with full
confidence and treated as an authentic isolate.
In nature the uncommon fungus has been isolated from legume seeds, corn-
meal, and soil. It produces the distinctive secondary metabolite avenaciolide, and
unlike other members of the section Flavi, makes no ochratoxins, aflatoxins, cy-
clopiazonic acid, or kojic acid (141).
Description. The colonies are rapid-growing (35 to 55 mm in 7 days).
They are deeply granular, with conspicuous black sclerotia. Conidia are yellowish
to dull olive green. The reverse is pale to dirty pink.
Conidiophores are often around 400 to 600 (but up to 1000) µm high, with
smooth-appearing [finely roughened when seen dry, fide Raper and Fennell (7)]
hyaline walls, attaching relatively abruptly to a globose or oblate vesicle without
the funnel-like apical stalk expansion seen in A. flavus and close relatives (Fig.
33). The vesicle bears biseriate fertile elements (i.e., metulae and phialides), with
metulae all around the perimeter in a radial orientation. Conidia are in dry radiat-
ing chains. They are hyaline, smooth, ellipsoid, mostly 4.0–5.0 ⫻ 3.2–4.0 µm,
and occasionally as long as 6.5 µm. Sclerotia are black.
Aspergillus {aff. Petromyces} tamarii Kita. This fungus was reliably
documented from an ulcerating cutaneous infection on an eyelid subsequent to
implantation in an otherwise healthy woman via the bristle of a toothbrush being
used to brush the eyebrows (161).
This fungus in nature colonizes seeds, soils, and various other substrata
(74). Like the closely related A. flavus, it is an important food spoilage organism,
especially in warm areas (51). Its major mycotoxin is cyclopiazonic acid. Unlike
A. flavus, it produces no aflatoxin.
Description. The colonies are rapid-growing (55 to 70 mm in 7 days),
deeply granular, and olive-brown to yellowish-brown. The reverse is pale to
yellow-grey.
Conidiophores are often around 600 to 1500 (but up to 2500) µm high,
with strongly roughened, hyaline walls, attaching with a slightly flaring, funnel-
like expansion to globose vesicle (Fig. 34). Heads with biseriate fertile elements
304 Summerbell
Figure 33 Aspergillus avenaceus.
(i.e., metulae and phialides) and uniseriate heads (phialides only) may both be
found, in both cases with fertile elements all around the perimeter in a radial
orientation. Conidia are in dry, radiating chains, globose to subglobose, with
thick, heavily roughened walls, mostly 5.0 to 8.0 µm in diameter. Sclerotia, if
formed, are red-purple, brown, or black.
This fungus is very similar to A. flavus in morphology but is distinguished
by its considerably browner conidial masses and the thick, heavily roughened
walls of its conidia.
Anamorphic Species in A. Subgenus Circumdati, Section Nigri Gams et al.
(Formerly the Aspergillus niger Group).
Aspergillus {aff. Petromyces/Neopetromyces} niger van Tieghem. This
species is arguably the least virulent of the regularly seen opportunistic aspergilli,
and is also an abundant contaminant both of body surfaces and laboratories. It
Ascomycetes 305
Figure 34 Aspergillus tamarii.
is most commonly significantly isolated as an agent of otomycosis (27, 69, 162),

but also as an agent of aspergilloma and allergic bronchopulmonary aspergillosis
(69, 163). In immunocompromised patients, pulmonary infections or coloniza-
tions may occur, and are often characterized by oxalosis, which is extensive pro-
duction of microscopically conspicuous oxalic acid crystals in sputum (164, 165).
Oxalosis may also distinguish A. niger in some cases of invasive otomycosis in
compromised patients (166). Other infections occurring in immunocompromised
patients include disseminated and primary cutaneous infections. A bizarre lep-
rosy-like cutaneous infection of an Egyptian farmer with no known predisposing
conditions was immaculately documented in 1967 (167), and a similar primary
cutaneous infection was reported in a Swedish metalworker in 1965 (168). To
our knowledge no similar cases have been reported since. As with many relatively
weak opportunists, A. niger may cause dialysis-related peritonitis, endocarditis,
306 Summerbell
and other invasions of particularly vulnerable sites (69). Onychomycosis may be

caused, although this is rare and the fungus is a constantly occurring nail contami-
nant (89, 169). The rare condition proximal subungual onychomycosis can rarely
be caused by A. niger (170).
In nature and anthropogenic habitats, A. niger colonizes soils, seeds, root
crops, spices, composts, and many different foodstuffs (51, 74, 124). Its character-
istic secondary metabolites, naphtho-T-pyrones and malformins, were not consid-
ered to be significant mycotoxins by Frisvad and Thrane (8).
Description. The colonies are rapid-growing (45 to 70 mm in 7 days) and
deeply granular, with conidiophores dense at the colony center and scattered at
the margins. They are characteristically fuscous black (i.e., black with a dirty
brownish cast), and the reverse is pale to yellow. Atypical, poorly sporulating
colonies or colonies on sporulation-suppressive media such as bacteriological
brain-heart-infusion-blood agar may have conspicuously yellow mycelium.
Conidiophores are often around 500 (but up to 3000 µm) high, with smooth,
hyaline to pale yellow walls. They are aseptate, attaching abruptly to a globose
vesicle, which is biseriate (i.e., bearing metulae and phialides), with metulae all
around the perimeter in a radial orientation (Fig. 35). Conidia are in dry, upright
chains, often radiating or massing into two or more short columns per head, in
wet microscopic mounts. They are grey-brown, globose, strongly roughened with
small, irregular ridges and bars of dark material, and are 3.5 to 4.5 µm in diameter.
In subspecific population structure this species appears to be largely or en-
tirely clonal, and can be genetically typed using techniques similar to those used
for A. fumigatus (171).
Aspergillus {aff. Petromyces/Neopetromyces} japonicus Saito (Synonym:

A. aculeatus Iizuka). This species has black conidia and superficially resembles
A. niger. Its aspergilla, however, are uniseriate, and its conidia are purple-black
en masse, a color quite distinct from the dirty brownish-black of A. niger. Micro-
scopically, the roughening on the conidia consists of evenly spaced spines, not
irregular ridges and bars as in A. niger. A. japonicus appears to have no track
record as far as being a pathogen of humans and animals, but has been taken
into some review literature because causation of otitis was attributed by Wadh-
wani and Srivastava (17). These records are not accepted. (See the discussion
under Fennellia nivea above.) A. japonicus was also mentioned under the syn-
onym A. aculeatus as a possible agent of a mysterious, diphtheria-like tongue
and throat condition in Nigeria (172). The proposed etiology appears to be based
on isolation of the species in culture from tongue scrapings and vomitus of a
single patient examined only once, without direct microscopic demonstration of
any fungal elements in apparently infected, darkened areas of the tongue. It ap-
pears likely that the fungus had simply contaminated some food recently eaten
Ascomycetes 307
Figure 35 Aspergillus niger.
by the patient. A. japonicus in nature is a soil and leaf litter decay fungus, mostly
from the tropics, but occurring in the temperate zone occasionally as an invader
of grapes and rarely as a medical specimen contaminant. [For a description see
Klich and Pitt (122) and de Hoog et al. (69).]
6. Neosartorya Malloch & Cain

Ascomata are globose cleistothecia, mostly 150 to 500 µm in diameter, and white,
with a thin, fragile peridium composed of two to three layers of pseudoparenchy-
matous cells. Asci are eight-spored, globose, and evanescent. Ascospores are lens-
shaped to nearly spherical, hyaline, with two equatorial crests and no or various
types of ornamentation on the valves. Anamorphs are members of Aspergillus
subgenus Fumigati section Fumigati, formerly considered the A. fumigatus series
(7). They have uniseriate conidiophores.
308 Summerbell
Species with a Known Teleomorph, and with Anamorphs in A. Subgenus

Fumigati Section Fumigati Gams et al. (Formerly Aspergillus fumigatus Group).
Neosartorya pseudofischeri Peterson, Anamorph Aspergillus thermomu-

tatus (Paden) Peterson. This recently recognized fungus was mentioned in
case reports under a variety of names, mostly names of former varieties of Neo-
sartorya fischeri, prior to its description. Fortunately, isolates from most cases
were obtained by Padhye et al. (173) and preserved in ATCC after being correctly
reidentified as this species. The fungus is known, then, to cause osteomyelitis,
endocarditis, keratitis, and bronchopulmonary colonization (173). In nature and
anthropogenic habitats, it is apparently uncommon, but when found typically oc-
curs on seeds and on cellulosic manufactured materials such as papers and matches
(124). Secondary metabolites include tryptoquivaline and trypacidin (124).
Description. The colonies grow approximately 60 mm in diameter in 7
days at 25°C. They are whitish to cream, and granular. Ascomata are abundantly
formed as small, white to cream, spheroidal tufts massed on the colony surface.
The colony reverse is pale.
Ascomata under the microscope are hyaline cleistothecia mostly 150 to 500
µm in diameter. They are globose and non-ostiolate, with a thin pseudoparenchy-
matous peridium. Asci contain eight lens-shaped ascospores, 5–6 ⫻ 4–4.5 µm,
each with an equatorial furrow flanked by two broad, irregular ridges, and each
with valves ornamented with irregular triangular ridges (Fig. 36). Conidiophores
are seldom seen at 25°C, often formed sparsely at 37°C, and then mostly 150
to 500 µm high, with thick, often somewhat undulate, hyaline walls, aseptate,
uniseriate (i.e., phialides directly attached to vesicle), with vesicles mostly pyri-
form. Conidia are in dry, upright chains. They are pale blue-green, ellipsoidal,
apparently smooth to finely roughened, and 2.5–3 ⫻ 2–2.5 µm.
The otherwise similar N. fischeri has an anastomosing reticulum and lacks
the protruding triangular vertices of N. pseudofischeri (Fig. 37). N. spinosa has
numerous isolated short spines, while N. glabra has smooth valves. The charac-
teristic ascospores of these species are illustrated in connection with previously
used N. fischeri varietal names by Samson et al. (174). The complex relationships
of the many Neosartorya species now described as well as their relatives in the
section Fumigati is analyzed using β-tubulin sequences by Varga et al. (175).
A case of disseminated infection in a bone marrow transplant patient was
attributed to N. fischeri by Lonial et al. (176). According to D. Sutton (personal
communication, March 2001), who provided the identification in that case, the
organism was provisionally identified as N. fischeri var. spinosa, currently called
N. spinosa, but the possibility of the recently described N. pseudofischeri was
not ruled out. Many authors, including the present one, have identified N. pseudo-
fischeri as N. spinosa prior to the description of the former as a separate species
(177). While this manuscript was in press, Sutton sent the Lonial et al. isolate
Ascomycetes 309
Figure 36 Neosartorya pseudofischeri CBS 109512, ascospores.
Figure 37 Neosartorya fischeri, ascospores.

310 Summerbell
Figure 38 Aspergillus fumigatus.
to CBS, where it was confirmed as N. spinosa by R. A. Samson and deposited

as CBS 109511. A myeloma patient with pneumonia attributed to N. fischeri is
described by Chim et al. (178). The identification is unsubstantiated at the species
level, but well verified at the genus level, including demonstration of recognizable
cleistothecia in infected tissue.
Anamorphic Species in A. Subgenus Fumigati Section Fumigati Gams et
al. (Formerly Aspergillus fumigatus Group).
Aspergillus {aff. Neosartorya} fumigatus Fres. (Synonym: Aspergillus
phialoseptus Kwon-Chung). A. fumigatus is the best known and most commonly
seen of the filamentous fungal opportunistic pathogens in most parts of the world
(11, 68, 69). It causes opportunistic disease in the severely immunocompromised
patient, usually beginning from a primary infection in the lungs but also occasion-
ally in other sites, such as the nasal sinuses. Systemic and deep tissue infections,
Ascomycetes 311
often limited to a single site such as a lung lobe, may be caused in people who are
apparently immunocompetent or only weakly immunocompromised. AIDS pa-
tients, although relatively rarely affected unless secondarily neutropenic due to
cancer or immunosuppressive therapy (e.g., ganciclovir), may develop unusual as-
pergillosis presentations, such as primary cutaneous lesions (179). A. fumigatus also
causes various chronic colonizing infections of people with certain predispositions.
For example, it is the major agent of chronic bronchopulmonary aspergillosis, a
disease featuring an exacerbated allergic response to aspergilli growing on upper
respiratory surfaces, mostly in long-term asthmatic and cystic fibrosis patients
(150). Other colonizing infections include pulmonary aspergilloma (fungus ball)
in the lungs of persons with pre-existing cavitation, chronic mycotic sinusitis, and
otomycosis (outer ear canal surface infestation) (11). The species also causes the
full spectrum of opportunistic mycoses of specially vulnerable body sites (e.g.,
ocular infections subsequent to traumatic introduction, otomycosis, onycho-
mycosis, dialysis-related peritonitis, and endocarditis). It causes many animal in-
fections, most notably pulmonary infections of birds—it is, for example, a well-
known risk for penguins in zoos (180)—as well as potentially fatal infections of
dogs, especially the notably susceptible German shepherd breed, in which it often
becomes established as an invasive mycotic sinusitis.
In nature and anthropogenic habitats, it is a thermotolerant compost organ-
ism, growing in self-heating decaying vegetation, warm soils, and warm building-
related habitats, especially organically enriched (e.g., with bird dung) warm venti-
lation ducts or ledges, as well as indoor composters and potted plants (74, 124).
It also commonly invades seeds, especially grains, and warmth-dried spice and
smoking materials. Its principal mycotoxins include gliotoxin, verruculogen, fu-
mitremorgens, fumitoxins, and tryptoquivalins (8).
Description. The colonies are fast-growing (70 to 85 mm in 7 days at
25°C). They are powdery and dull blue-green with a whitish margin. The reverse
is pale to slightly greenish.
Conidiophores have phialides directly attached (uniseriate) over the upper
two-thirds of the surface. They are greenish, with clavate vesicles and smooth
stipes. Conidia are borne in interconnected dry chains cohering above conidio-
phores as compact upright columnar masses. When seen individually, they are
subglobose, finely roughened, and 2.5 to 3.0 µm in diameter.
Most isolates grow well at 45°C; a few have maximum growth temperatures
around 43°C.
A common host-adapted ‘‘dysgonic’’ (conidiation-impaired) variant usu-
ally lacks conidiation on initial outgrowth. It is typically isolated from long-term
colonization habitats such as aspergillomas, or bronchopulmonary, nasal sinus,
or outer ear colonizations. It can be distinguished preliminarily by good growth
at 45°C, and dense, matted, whitish colonies with radial folds. Conidiation can
often be reinitiated by growing colonies on modified Leonian’s agar (4) at 37°C
312 Summerbell
and making subcultures from the small conidial tufts or faint haze of surface
conidiation that may develop. When formed, conidiophores, are often aberrant
and strongly reduced in size, sometimes with only one to five phialides. Com-
pletely nonsporulating isolates may be identified by exoantigen (181) or specific
molecular studies. (See below.)
The difficulty of detecting invasive aspergillosis by culture has led to exten-
sive development of molecular direct detection methods. Many of these involve
amplifications using primers detecting all aspergilli or a wider range of fungi
followed by posttreatment with nested PCR, specific probes, restriction digests,
sequencing, or single-strand conformational studies to yield species-level identi-
fications. These techniques have predominantly utilized ribosomal DNA amplifi-
cations, especially the 18S subunit (154, 182–186), the large subunit (152, 187),
and the internal transcribed spacer region (188). In a few cases, ribosomal primers
specific to A. fumigatus have been used directly (189, 190). In other cases, they
are used in tandem with general primers (186, 187). Random primers have been
designed specifically for A. fumigatus (191) or for A. fumigatus and closely re-
lated Neosartorya species (192). The strategy of relatively broad-based amplifi-
cation for various aspergilli followed up by additional analysis for species deter-
mination has also been pursued using alkaline protease (153) and mitochondrial
(193) primers. A. fumigatus-specific and Aspergillus genus-specific primers based
on the aspergillopepsin PEP gene (194) have been published. In addition, primers
based on an 18-KD immunoglobulin E-binding protein (195) specific to A. fumi-
gatus are available. In the latter study, the authors’ claim that their primers also
amplify A. restrictus DNA appear to be based on A. fumigatus isolates misi-
dentified under that name. (See discussion under A. restrictus above.) Their tech-
nique is highly specific to A. fumigatus, not amplifying even the closely related
Neosartorya fischeri. Recently a plate-hybridization technique (196) and a quan-
titative Light Cycler PCR protocol (197) specific for A. fumigatus have been
reported.
A. fumigatus is a predominantly or entirely clonal organism (198) that may
be biotyped using various molecular techniques (171). Patients with colonizing
infections may carry several genotypes, while patients with invasive aspergillosis
generally are infected by a single genotype (124).
Investigators carrying out epidemiological studies on A. fumigatus need to
keep certain ecological possibilities in mind that have not always been considered
in published studies.
1. Even in a small focus, A. fumigatus growing in a hospital or other envi-
ronment need not consist of a single genotype. It is very common for
conducive fungal habitats to support mosaics of multiple genotypes of
the same species. Therefore if a small sample size of environmental iso-
lates contains only genotypes different from the one found in an in-
fected patient, this does not mean the patient was not infected by a local
Ascomycetes 313
(e.g., in-hospital) source. Typical hospital habitats such as accumula-

tions of bird dung around air intakes are stable habitats of the kind most
likely to support a well-developed community of various strain types.
The presence in a hospital of any genotype of A. fumigatus, whether
the same as that obtained from the patient or different, suggests that
the possibility of a local source for the patient’s isolate requires full
investigation. A genotype that constitutes only a small fraction of the
local A. fumigatus population may—through greater virulence or by
chance alone—be the one that infects one or more patients.
2. As a largely clonal organism, A. fumigatus may manifest as the same
genotype in two different sites by coincidence. Elucidating the same
genotype from two sources (e.g., a patient and a nearby hospital air
duct) does not necessarily mean that the two isolates are connected.
More complete sampling might show that local outdoor air, the pa-
tient’s house, or the clothing or hair of visitors might be just as probable
a source of the same genotype as the air duct.
These possibilities indicate that epidemiological testing with A. fumigatus
is fundamentally a probabilistic problem. For investigating whether or not an
environmental source is linked to a patient isolate, local environmental isolates
must be studied in sufficient detail to ensure that the full biodiversity has been
covered in order to prevent false-negative associations. As in all ecological stud-
ies, the diversity of different types present should be studied until adequate cov-
erage has been attained. The percent–coverage statistic outlined by Gochenaur
(199) is one means of addressing this question. To prevent false-positive associa-
tions, more distally obtained environmental data need to be obtained or generated
through de novo studies. This constitutes an essential control study. It needs to
be shown that the probability of the patient being exposed to a given genotype
in the local environment (e.g., in the hospital) is significantly greater than the
risk of exposure in the miscellaneous exterior environments in which the patient
or his or her contacts (not to mention any unsterile environmental materials; e.g.,
foods, tap water, or flowers) may have been present. Of course, a patient who
is infected by a genotype making up only 3% of hospital isolates but 57% of lo-
cal outdoor air isolates may still in reality have been infected from the in-hospital
source. This link cannot be conclusively demonstrated by epidemiological geno-
typing studies, however.
7. Hemicarpenteles Sarbhoy & Elphick

Anamorphic Species in A. Subgenus Clavati Section Clavati Gams et al.
(Formerly Aspergillus clavatus Group).
Aspergillus {aff. Hemicarpenteles} clavatus Desm. This species is ap-
parently of relatively low virulence. It has been reliably reported from a case of
314 Summerbell
endocarditis by Opal et al. (200). Two case records from otomycosis by Yassin
et al. (95) are regarded as unsubstantiated for reasons mentioned above under A.
versicolor. A. clavatus is widely distributed in nature, especially in the tropics,
and is common in these areas on decaying foodstuffs, especially grains (122). It
is also common in settings connected with the brewing industry (111).
Description. Colonies are moderately fast-growing (30 to 45 mm in 7
days at 25°C), and are granular or deeply and evenly powdery with a dense felt
of long conidiophores. They are dull blue-green with a whitish margin. The re-
verse is uncolored to pale yellow-brown.
Conidiophores are mostly 500 to 1000 µm long, with phialides directly
attached (uniseriate) over the surface of dramatically elongated vesicles up to
250 µm long, and mostly 40 to 60 µm wide (Figs. 39 and 40). Stipes are smooth.
Conidia are ellipsoidal, smooth, and 3.0–4.5 ⫻ 2.5–4.5 µm.
Figure 39 Aspergillus clavatus, 10X objective.

Ascomycetes 315
Figure 40 Aspergillus clavatus, 40X objective.
Aspergillus {aff. Hemicarpenteles?} clavatonanica Batista, da Silva

Maia & Alecrim. This fungus, which resembles A. clavatus but has short, re-
branching conidiophores, was isolated and described from a painful paronychia-
like inflammation in a patient whose work involved washing materials by hand
(201). Microscopic examination of purulent exudate revealed ‘‘rare and small
spherical bodies’’ considered to be fungal ‘‘spores,’’ but the nails themselves
contained no discernible fungal elements. The isolation was not repeated and the
infection was successfully treated with iodide. Clearly this interesting study did
not demonstrate etiology, and no parallel case has been reported since.
B. Penicillium Link :Fr.

Penicillium species are among the most common decomposers in nature. They
are closely related to Aspergillus species but in general are less thermotolerant
316 Summerbell
and are most prominent ecologically in cooler areas, although they are by no
means absent in the tropics. The majority of species grow poorly or not at all at
37°C in vitro, and in keeping with this, are not reported from human and animal
disease. The prominent exception is Penicillium marneffei, which, like other ther-
mally dimorphic fungi probably adapted to infect animals as a means of perenna-
tion, has evolved a particulate assimilative phase specialized for growth and dis-
persal in host tissues and fluids. This particulate phase, analogously with that of
Coccidioides immitis, is ontogenetically unique in the fungal kingdom, despite
its superficial similarity to fission cells of Schizosaccharomyces, and indicates an
independent evolutionary path to thermally dimorphic facultative pathogenicity.
Penicillium is an amalgam of anamorphs corresponding to two prominent
teleomorphic genera, Eupenicillium and Talaromyces. These will be dealt with
separately. The Penicillium species in this chapter are presented in a conventional
arrangement according to their taxonomic subgenera and teleomorph affinities.
The arrangement can be seen at a glance in Table 5.
Table 5 Natural (Teleomorph-Based) Groups of Penicillium Species

as Arranged in This Chapter
Penicillium
Teleomorph subgenus Species
Eupenicillium Aspergilloides Penicillium citreonigrum

P. decumbens
P. spinulosum
Furcatum P. citrinum
P. janthinellum
P. melinii
P. oxalicum
Penicillium P. brevicompactum
P. chrysogenum
P. commune
P. cyclopium
P. expansum
P. griseofulvum
Talaromyces Biverticillium Talaromyces emersonii (P. emersonii)
T. flavus (P. dangeardii)
T. thermophilus (P. dupontii)
P. marneffei
P. piceum
P. purpurogenum
P. rugulosum
P. verruculosum
Ascomycetes 317
The anamorphs in general are characterized by possessing solitary and un-

divided, or more commonly brushlike and multiply rebranched, septate and erect
or divaricate conidiogenous stipes with a cluster of phialides at the terminus of
each branch. Apices may be slightly vesiculate, but not generally more than three
times the diameter of the subtending branch. By contrast with Aspergillus, Peni-
cillium lacks foot cells anchoring the stipes in substrate hyphae, and forms young
phialides on an apex sequentially, not synchronously. Tips of phialides formed
below the fertile branch apex are bent to bring their alignment closer to that of
the uppermost phialides, giving rise to subparallel clusters. By contrast, phialides
in most species of Paecilomyces tend to splay widely apart, forming divergent
clusters. Phialides are typically bottle-shaped (ampulliform) in Eupenicillium
anamorphs, with an extended ‘‘neck’’ (collula) in a few species. Talaromyces
anamorphs have distinctive phialides that are lanceolate, meaning ‘‘long
spearhead-shaped,’’ also called acerose, meaning ‘‘sharp.’’ These Talaromyces-
type phialides have a long basal zone of nearly parallel-sided morphology before
narrowing abruptly to a neck at the apex. Conidia in all Penicillium species form
in dry, interconnected chains. They may be smooth but are often roughened, and
are in shades of blue, green, grey-brown, and grey (most typically sage-green,
glaucous, or blue). Similar fungi with pink, purple, or white conidia in intercon-
nected chains are in other genera, often Paecilomyces. Similar fungi with conidia
in slimy heads or unconnected imbricate (overlapping like roof slates, tiles, or
shingles) chains are in taxonomically distant genera such as Clonostachys.
1. Identification Techniques
Penicillium species are among the most difficult hyphomycetous fungi to identify
morphologically, and the means to identify most molecularly are at this writing
in a very preliminary state of development. Many species do, however, readily
yield to rationalization if the special growth conditions outlined by Pitt (87, 202)
are adhered to. The system involves a special inoculation pattern (three points,
one at the halfway point of each of three radii dividing the 85-mm plate into
three equal 120° sectors) and technique (mashing inoculum in a separately con-
tained small quantity of agar medium prior to use in order to adsorb dry conidia
into wet material and prevent the formation of satellite colonies on inoculated
plates), use of CYA, malt extract agar (MEA), and 25% glycerol nitrate agar
(G25N) at a standard 25°C, and CYA at 37° and 5°. All incubations are carried
out for 7 days, and then colony diameters are measured at 90° to the petri plate
radius across the original inoculation point, which is now the colony center. Some
members of Penicillium subgenus Penicillium may require additional inoculation
onto creatine sucrose agar (111), and especially for some atypical isolates may
require the preparation of mycotoxin profiles (111). None of the necessary media
or techniques is standard in biomedical laboratories, and apparently etiologically
significant Penicillium species are best sent for professional expert identification.
318 Summerbell
Morphological analysis is rendered easier by several procedures. The heavy

burden of conidial production, which can completely prevent visualization of fine
structures, is best avoided by taking material from areas near the colony margins
that are not yet so heavily sporulated. The present author prefers to use a flat-
ended inoculating tool or ultrafine forceps to cut a narrow (⬃1 mm or narrower),
parallel-sided radial slice out of the marginal region of heavily sporulating colo-
nies, being careful not to crush the structures. Then the slice is laid on its side
in a drop of 95% ethanol (to extract hydrophobic materials and minimize air
bubble formation) so that the conidial stipes tend to lie flat along the slide. Regu-
lar laboratory slide mounting medium is added (e.g., lactic acid–cotton blue,
lactophenol–cotton blue, detergent/water mixtures). The coverslip is angled onto
the material to flatten it so that any excess agar is squeezed off to one side rather
than over the surface of the fungal material.
Several learned-by-experience morphological characters make preliminary
Penicillium characterization agonizing for the novice and relatively easy for the
developing expert. First, the branching pattern must be analyzed as to whether
conidiophores are unbranched, the monoverticillate condition, or whether phi-
alides are supported on a single order of short supporting branches (metulae,
meaning ‘‘between-structures’’; singular, metula), the biverticillate condition, or
on an even more complex branching system. Metulae are often more or less
symmetrically arranged, especially in P. subgenus Biverticillium, the group re-
lated to teleomorphs in Talaromyces. The next most complex branching condi-
tion, terverticillate, features a second order of branches called rami (singular ra-
mus, meaning simply ‘‘branch’’) that support the metulae. Most commonly there
is a main ramus and side ramus, which may be curved to more or less parallel
the main ramus. Occasionally there are more rami, or rami that are splayed at
wider angles. The common household penicilla such as P. chrysogenum are ter-
verticillate. Finally, a few Penicillium species may have quaterverticillate peni-
cilli, where rami support two intervening orders of branches, ramuli and metulae.
There is an agonizing ‘‘grey area’’ intermediate between some monoverti-
cillate Penicillium species placed in P. subgenus Aspergilloides and a so-called
divaricate type of biverticillate Penicillium seen in some members of P. subgenus
Furcatum. Monoverticillate species with long unbranched penicilli arising from
the agar surface are easy to pigeonhole correctly, but those like P. decumbens
with short stipes arising from aerial mycelium may be more difficult. Likewise,
members of P. subgenus Furcatum with three or more metulae in a verticil are
easy to recognize, but those such as P. janthinellum with a mixture of metulae
and solitary side branches are more difficult. Here the necessary distinction is
between species that regularly make true metulae; that is, groups of two or more
short, terminal, phialide-bearing branches arising at an acute angle to one another
to form a somewhat compacted cluster (subgenus Furcatum), and groups that
simply produce short, solitary penicilli, often arising at 90° angles to an aerial
Ascomycetes 319
branch (subgenus Aspergilloides). For the most intractably intermediate cases,

the distinction tends to lie in finding the true metulae uncommonly to commonly
(subgenus Furcatum), as opposed to finding such structures very rarely in slide
mounts (generally subgenus Aspergilloides).
Similar intermediacy may occur between members of the subgenera Furca-
tum and Penicillium, in which structures with metulae may be interspersed with
more complex terverticillate structures. This problem is encountered frequently
because the common P. chrysogenum is one of the most micromorphologically
variable members of subgenus Penicillium. In dealing with this grey area, the
best practice is to look through the microscopic preparation to detect any typical
terverticillate structures that are present. If even a small number of such structures
is formed, the isolate is normally a member of subgenus Penicillium, no matter
how much the remaining microscopic material may suggest subgenus Furcatum.
The initially agonizing decision about whether or not any biverticillate
structures seen should be determined as typical of P. subgenus Biverticillium or
P. subgenus Furcatum has been treated in detail by Pitt (87). The subgenus Fur-
catum tends to feature phialides much shorter than their supporting metulae,
whereas in subgenus Biverticillium the lengths of these structures are approxi-
mately equal. Also, subgenus Furcatum species seldom have more than five met-
ulae per stipe, while subgenus Biverticillium species usually have more than five.
Subgenus Furcatum species, as typical osmotolerant penicillia, grow fairly well
on the concentrated G25N medium, with colony diameters 10 mm or more in 7
days at 25°C. Subgenus Biverticillium members grow less than 10 mm on G25N.
Finally, the initially agonizing decision between ampulliform phialides in
Eupenicillium anamorphs and lanceolate phialides in Talaromyces anamorphs
(P. subgenus Biverticillium) has been conventionally expressed in the generic
description above. As an additional aid to the user, the author adds his own obser-
vation that if the phialides of Penicillium species were scaled up as identically
shaped bottles, the phialides of the Eupenicillium clade would make plausible
beer or wine bottles within the historical range of normal sizes and shapes, apart
from being somewhat too curvaceous. Talaromyces type phialides, however,
would generally appear to be impractically tall, narrow bottles that would be in
great danger of toppling over. This imagery expresses a conspicuous regularity
in length–width ratio that is sufficiently variable that a more precise, statistical
expression would be difficult to formulate in a useful way.
2. Unidentified Penicillium in Opportunistic Pathogenesis

The difficulties in identification of Penicillium, and the historically weak link
between Penicillium taxonomists and the biomedical community (still evident in
the exclusion of most mycological literature from Medline) have meant that many
cases of non-P. marneffei Penicillium infection have been attributed to unidenti-
320 Summerbell
fied species. A detailed review is not appropriate here, but the scope of such
infections includes keratitis (28), onychomycosis (203, 204), localized pulmonary
infection (205, 206), duodenal colonization as a side effect of carbenoxolone and
cimetidine therapy (207), dialysis-related peritonitis (208, 209), and endocarditis
(210, 211). Two cases of nasal disease with turbinate invasion in dogs were well
confirmed (four more were partially evidenced) by Harvey et al. (212); a photo-
graph shows a fungus that could be Penicillium citrinum, P. chrysogenum, or a
close relative of either. Disseminating Penicillium invasive sinusitis in a cat has
also been established (213). There are a number of older papers linking—in some
cases—well-documented mycoses to penicillia identified with now untraceable
species concepts [e.g., a well-documented urinary bladder colonization linked in
1911 to a ‘‘penicillium glaucum or common mold’’ (sic)] (214) that produced
yellow exudate in culture (suggesting, in light of current knowledge, P. citrinum
or P. chrysogenum, the first of which has been explicitly reported from urinary
tract colonization). The present author has not attempted to mine all these papers
for currently relevant information.
Many of the reports of Penicillium sp. in opportunistic mycosis suffer from
the same epistemologic problems as reports with named species [e.g., lack of
confirmatory direct microscopy in otomycotic, sinus, pulmonary, and ocular (215)
infections and lack of compatible direct microscopy ⫹ confirmatory later re-
isolation in onychomycosis]. In any case, it is scarcely possible to correlate two
successive Penicillium isolations from nails when they have not been identified
to species, since the isolations may simply be of two different species. Also,
many Penicillium sp. reports lack published substantiation for the genus-level
identification. Penicillium sp. identifications without substantiation or with non-
specific characterization may well be token misidentifications of host-adapted A.
fumigatus or Paecilomyces variotii, or isolates of Aspergillus section Versicolores
with unusually high proportions of simplified, Penicillium-like phialides, espe-
cially on inappropriate media such as Sabouraud agar. For example, an uncharac-
terized ‘‘Penicillium sp.’’ from olecranon bursitis that was resistant to amphoteri-
cin B but susceptible to ketoconazole in vitro (216) would be strongly suspected
to be a polyene-resistant Hypocrealean Paecilomyces such as P. lilacinus. On
the other hand, a ‘‘Paecilomyces sp.’’ depicted by Leigheb et al. (217) from
a toe abscess (negative for direct microscopic signs of mycosis) is clearly an
etiologically insignificant Penicillium. It should be noted that P. lilacinus was
often called Penicillium lilacinus prior to the mid-1970s.
3. Identified Penicillium in Cases Suggestive of Pathogenesis

Special mention must be made of the review by Pitt (218), in which 10 non-P.
marneffei Penicillium isolates from biomedical sources more or less suggestive
of clinical significance were identified by a world authority on the taxonomy of
Ascomycetes 321
the genus. Although none of the cases appears to have been published (meaning
that insufficient detail is available to establish or refute etiology), the list of fungi
given may provide some indication of possible emerging opportunistic pathogens.
Most are members of P. subgenus Biverticillium and the related teleomorph genus
Talaromyces. Some of the records, such as a Penicillium piceum isolate listed
only as having ‘‘human nail’’ as a source, seem unlikely to be significant. All of
the isolations but two are from normally contaminated bodily materials, such as
respiratory secretions.
4. Eupenicillium F. Ludwig
Anamorphic Species in Penicillium Subgenus Aspergilloides Dierckx.
Penicillium {aff. Eupenicillium} citreonigrum Dierckx. This species
may be linked to the teleomorph Eupenicillium euglaucum, as proposed by Stolk
and Samson (219); however, the matter requires molecular clarification.
In 1929, Talice and Mackinnon (220) meticulously described a case of
repeated cultivation of a Penicillium species from clumps of mycelium expecto-
rated by a 58-year-old, tuberculosis-free pulmonary disease patient in Uruguay.
The species, described as Penicillium bertai n. sp., was synonymized with P. ci-
treonigrum by Pitt (202) on the basis of its detailed characterization, even though
a representative culture no longer exists. The case was diagnosed as bronchopul-
monary mycosis caused by the Penicillium, although fungus ball was not ex-
cluded. (No X rays were reported.) No similar cases have been reported since,
but since few Penicillium isolates from human disease are identified to the species
level, this absence of reports may not be significant. The description of a species
with bright orange-yellow soluble pigment is certainly incompatible with P. de-
cumbens, the only other member of P. subgenus Aspergilloides well linked to
human disease. The case isolate was described as growing weakly at 37°C, a
property compatible with some isolates of P. citreonigrum.
This species in nature is a soil and compost fungus.
Colonies on CYA grow 20 to 27 mm in 7 days. They are mostly velvety
in texture, often with some yellow mycelium and with greenish-grey conidiation.
The reverse is usually bright yellow, or occasionally yellow-brown, with typically
yellow-soluble pigment. Colonies are on MEA 22 to 26 mm, velutinous or floc-
cose, with whitish and/or yellowish mycelium and greenish-grey conidiation and
with pale to red-brown reverse, sometimes with yellow to brown soluble pigment.
G25N colonies are 11 to 14 mm in diameter. Growth occurs at 5°C but may only
be microscopically visible after 7 days. Growth at 37°C is nil or colonies up to
10 mm are formed.
Conidiophores often arise from aerial hyphae. The stipes are mostly 60 to
100 µm long, smooth-walled, and typically monoverticillate, but with a minority
with two or three metulae in some isolates, typically lacking an apical vesicle
322 Summerbell
Figure 41 Penicillium citreonigrum.
(Fig. 41). The phialides are ampulliform and mostly 5 to 12 µm long. The conidia
are globose to subglobose, smooth or with finely roughened walls, and 1.8 to
2.8 µm.
The description of P. bertai was based on a purely monoverticillate isolate.
P. citreonigrum isolates with metulae are most readily distinguished from P.
citrinum by their significantly faster growth on MEA. The much more blue-tinted
conidial color of P. citrinum on CYA is also unmistakable for those who have
seen each species several times.
Penicillium {aff. Eupenicillium} decumbens Thom. A case of pulmo-
nary fungus ball caused solely by this Penicillium species in an otherwise healthy
Japanese farmer was elegantly demonstrated by Yoshida et al. (221). Confirma-
tory characters for the identification were not given, but a mycological reference
center was credited. P. decumbens has relatively few look-alikes that grow at
Ascomycetes 323
37°C, so this identification is accepted. Alvarez (222) described a case of apparent

disseminated infection, evidenced by four positive blood cultures, in a gay man
with AIDS and fever. Amphotericin B resolved the fever. The culture was identi-
fied by the M. Rinaldi mycology laboratory at the University of Texas Health
Science Center.
In nature, this fungus is mainly found in various types of decaying plant
material, a niche that extends both to soil and to foodstuffs (51). The majority
of records are from soil (74).
Colonies on CYA grow 20 to 30 mm in 7 days. They are mostly velvety
in texture. They are dull greyish-green, and the reverse is pale to olivaceous or
yellow-brown. Colonies on MEA are similar but 25 to 40 mm. G25N colonies
are 11 to 16 mm in diameter. Growth occurs at 5°C but may only be microscopi-
cally visible after 7 days. Colonies at 37° may be 5 to 20 mm.
Conidiophores mostly arise from aerial hyphae. The stipes are mostly 20
to 60 µm long, smooth-walled, monoverticillate, and often lacking an apical vesi-
cle but sometimes possessing one approximately twice the diameter of the stipe
(Fig. 42). The phialides are ampulliform and mostly 8 to 11 µm long. The conidia
are ellipsoidal to subglobose, smooth-walled, and 2.5–3.0 (-4.0) µm.
As mentioned above, this is one of a minority of monoverticillate Penicil-
Figure 42 Penicillium decumbens CBS 230.81.

324 Summerbell
lium species that grows at 37°C in vitro, a distinction that may accord with its
occasional isolation from opportunistic infection.
Penicillium {aff. Eupenicillium} spinulosum Thom. This monoverticil-

late Penicillium species was the subject of two bizarre case reports in an otherwise
apparently well-worked study on fungal ulcerative keratitis by Anderson et al.
(92). Both patients involved were listed in both individual case reports and dis-
cussion as being infected by ‘‘Penicillium sp.’’ that is, ‘‘genus Penicillium,
species undetermined,’’ a common laboratory report. Legends under the figures
depicting the infected eyes, however, expanded this to ‘‘Penicillium spinulo-
sum.’’ None of the other species names in the paper was contracted to a two-
letter abbreviation; furthermore, the absence of similar case reports in the ensuing
40⫹ years has made the arrival of two independent P. spinulosum keratitis pa-
tients at the same clinic in a single week in 1957 seem increasingly incredible.
Analytic parsimony suggests that Penicillium sp. was the correct identification,
with P. spinulosum perhaps deriving from unwarranted later rationalization of
the abbreviation ‘‘sp.’’ P. spinulosum does not grow at 37°C in vitro, and is
therefore not among the more likely Penicillium species to have caused a keratitis.
As an additional oddity, one of the eyes infected by Penicillium sp. was alleged
to have had both direct microscopic and cultural evidence of coinfection by a
fungus named only as ‘‘a corn smut.’’ At the time, however, the culturable bud-
ding yeast state of Ustilago maydis, the corn smut, was not distinguishable from
dozens of other smut anamorphs in vitro. The report yields no clue as to what
observations led to this identification; possibly a Ustilaginalean anamorph in the
recently recognized genus Pseudozyma, a group commonly present in outdoor
air, was seen. No further cases of smutted human eyes have been recorded in
subsequent decades. In light of all these anomalies, Anderson et al.’s (92) reports
of P. spinulosum keratitis do not merit continued uncritical inclusion in review
literature. ‘‘Fusidium terricola,’’ coisolated by Anderson et al. (92) from the
second case of ‘‘P. spinulosum’’ keratitis, is discussed below under Acremonium
implicatum.
Senturia and Wolf (223) mentioned that P. spinulosum was ‘‘known to
cause otomycosis’’ in a study of its drug susceptibilities, but the isolate used in
testing came from a source ‘‘other than ears,’’ presumably environmental. No
etiologic or mycologic details were given, nor were any cases described. This
study is mentioned only because it has occasionally been cited subsequently as
if it were a record of pathogenicity.
A well-demonstrated, severe case of bronchopulmonary mycosis was as-
cribed to P. spinulosum by Delore et al. (224). The authors’ description of ‘‘fructi-
fications with simple verticils, unbranched; spherical, smooth conidia 3 to 4 µm
in diameter; and rather restricted, greenish-grey, velutinous colonies’’ (English
translation by the present author) rules out P. spinulosum, which has heavily
Ascomycetes 325
echinulate conidia and rapid growth. The description is, however, entirely com-
patible with the expected organism from the infection in question, a host-adapted
A. fumigatus.
Anamorphic Species in Penicillium Subgenus Furcatum Pitt.
Penicillium {aff. Eupenicillium} citrinum Thom. A necrotizing P. citri-
num pulmonary mass in an immunocompromised lymphocytic leukemia patient
was thoroughly documented by Mori et al. (225). Another unidentified Penicil-
lium species not growing at 37°C was also cultured from the same lesion, but
may have been incidental. The case was also summarized in a later review of
human Penicillium infections (226). A neutropenic leukemia patient was well
demonstrated by Mok et al. (227) as having pulmonary and pericardial P. citrinum
infection. A report by Gilliam and Vest (228) exquisitely documenting a chronic
renal infection caused by P. citrinum in an otherwise healthy man gives no identi-
fication details, but credits an authoritative reference laboratory, the U.S. Depart-
ment of Agriculture Northern Regional Research Laboratory in Peoria (NRRL).
An influential paper by Jones et al. (229) about Fusarium keratitis incidentally
mentions a severe keratitis case attributed to P. citrinum, but gives no confirma-
tory clinical or identification information. The general quality of the paper and
the well-known mycological skill of one coauthor (G. Rebell) make the veracity
of this report highly probable, though not formally verifiable. A total of nine
corroborating cases of P. citrinum keratitis from Nigeria are well documented
etiologically and mycologically by Gugnani et al. (230, 231). A well-substanti-
ated case of continuous ambulatory peritoneal dialysis (CAPD)-related peritonitis
attributed to Penicillium sp. (232) includes a photo of a typical P. citrinum-like
penicillus. The accompanying description supports a likely identification of P.
citrinum, despite some anomalies (e.g., a statement that the typical ‘‘blue-green’’
Penicillium colony had conidia that under the microscope were ‘‘yellow-red’’—
not a spectrally possible color, except under the familiar name ‘‘orange’’). The
authors state that preserved slides were sent to mycologist M. R. McGinnis for
technical description. McGinnis, however, did not originate the expression
‘‘yellow-red’’ (personal communication, September 2000) and a transcription
error may be involved.
P. citrinum is an extremely common soil and vegetation decay organism.
Its principal mycotoxin, citrinin, is a nephrotoxin.
Colonies on CYA grow 25 to 30 mm in 7 days. They are densely velvety
to felt-like, blue-green. The reverse is yellow, yellow-brown, orange-brown, or
less commonly red-brown. Clear to pale yellow to reddish-brown exudate or
yellow-soluble pigment may be produced. Colonies on MEA are 14 to 18 mm,
powdery-granular, and characteristically blue-grey at the margin (but become
more greenish-grey centrally). G25N colonies are 13 to 18 mm in diameter.
Growth usually occurs at 37°C, but does not occur at 5°C.
326 Summerbell
Conidiophores are single, biverticillate, characteristically with a V-shaped

whorl of 3 to 5 nearly equally long metulae with somewhat vesiculate apices
(Fig. 43). Variants with some rami or aerial branches may be seen. The 37°C
growth character helps to confirm some of the more usual isolates as P. citrinum,
provided P. chrysogenum has been otherwise ruled out. The stipes are 50 to 200
µm long and smooth-walled. The phialides are ampulliform, and 7 to 8 (⫺12)
µm long. The conidia are globose to subglobose, smooth to finely roughened, and
2.5 to 3.0 µm.
This is usually a very distinctive species, with its 37° growth, its relatively
broad CYA colonies often with yellow exudate, reverse or soluble pigment, its
significantly smaller MEA colonies with dirty greenish-blue centers and a distinc-
tive marginal zone of faded blue jean blue, and characteristic triads of V-angled,
equal metulae.
Figure 43 Penicillium citrinum.

Ascomycetes 327
Penicillium {aff. Eupenicillium} janthinellum Biourge. This species

was one of four reported by Sandner and Schönborn (108) as part of a mixed
association overgrowing heavily ointment-treated burn eschar, as described above
under A. ustus. It is not clear that such a record, although medically interesting,
constitutes pathogenicity. A case of bronchiolitis obliterans organizing pneumo-
nia (BOOP), ‘‘a nonspecific response to pulmonary injury characterized by intra-
luminal infiltration of the terminal airways,’’ was seen in a man who had appar-
ently inhaled a large quantity of P. janthinellum conidia from a fungal mat on
contaminated juice (233). Although the patient grew the fungus from bronchial
lavage and biopsy, there was no evidence of germination or tissue invasion. This,
then, was not an invasive or colonizing disease, but rather an organic dust reac-
tion. P. janthinellum is described by Pitt (87, 202).
Penicillium {aff. Eupenicillium} melinii Thom. Isolation of this species

from a chronic sinusitis patient was reported by Bassiouny et al. (146). The sig-
nificance of the isolation was not confirmed by direct microscopy. The identifica-
tion also was not documented except by a nonspecific microphotograph.
Penicillium {aff. Eupenicillium} oxalicum Currie & Thom. This spe-

cies was conclusively demonstrated from posttraumatic keratitis and subsequent
endophthalmitis by Rodrı́guez de Kopp and Vidal (234). E. Piontelli of la Uni-
versidad de Valparaiso performed the identification, and a compatible description
and photographs were published.
This fungus is a widely distributed vegetation decay and soil organism,
with a strong but by no means exclusive affinity for preharvest maize corn (Zea
mays) (87). Its principal mycotoxin is secalonic acid D.
Colonies on CYA grow 35 to 60 mm in 7 days. They are densely velvety,
sometimes wrinkled, usually heavily conidial, and dull green. The colony beneath
conidia and the colony reverse are yellow, salmon, orange, orange-brown, or
pinkish-brown. Exudate is clear or lacking. Colonies on MEA are 20 to 50 mm
and deeply powdery, with a dense layer of conidia colored as on CYA. Heavily
conidial masses in this species form continuous sheets of parallel conidial chains,
and have a distinctive irideous sheen described by Pitt (87) as ‘‘uniquely shiny,
even silky.’’ G25N colonies are 12 to 16 mm in diameter. Growth at 37° exceeds
10 mm. At 5°C, conidia may or may not germinate.
Conidiophores are biverticillate, usually with two nonvesiculate metulae
closely appressed together (Fig. 44). Verticils of three or four metulae may also
be common. Stipes are 200 to 400 µm long and smooth-walled. Phialides are
ampulliform to acerose, and mostly 10 to 15 µm long. The conidia are ellipsoidal
to ovoid, smooth, and distinctively large (3.5–6.5 [⫺7.0]) ⫻ 2.5–4.0 µm.
Anamorphic Species in Penicillium Subgenus Penicillium.
328 Summerbell
Figure 44 Penicillium oxalicum.
Penicillium {aff. Eupenicillium} brevicompactum Dierckx. This fungus

was reported from a lung mass in a neutropenic leukemia patient (235). Although
the patient had concomitant Aspergillus fumigatus cerebral dissemination, the
Penicillium was consistently isolated first by transbronchial biopsy, then at au-
topsy, and appeared to be associated with a distinguishable histopathologic pre-
sentation. No characterization was given, but the fungus was identified by C. K.
Campbell of the Mycology Reference Laboratory, Bristol, England. As with the
apparent Penicillium commune case described below, however, the apparent
causal agent is a fungus never growing at 37°C in vitro, and yet it appeared to
cause infection during a time when the patient had a fever. (A figure for degrees
of fever was not given in this case.) The widely publicized (51, 74, 87, 202)
temperature restrictions on P. brevicompactum growth are not discussed by the
authors. The general possibilities for reconciling this type of apparent contradic-
tion are outlined under P. commune.
Ascomycetes 329
It may be noted that the present case, unlike the Huang and Harris (236)
P. commune case discussed below, featured a contained pulmonary lesion rather
than a disseminated Penicillium infection. In such a case, it might seem logical
that an organism not growing at 37°C or fever temperatures but surviving at those
temperatures might be able to grow, at least in the more exposed parts of the
respiratory tract, during moments of inhalation when surface temperatures drop,
provided no molecules necessary for ongoing metabolism were rendered non-
functional by the temperature fluctuations. The most recent data and mathemati-
cal models indicate, however, that even though exhaled air is only 34.6°C, the
lungs have extensive heat recovery mechanisms so that the middle and lower
portions of the respiratory system remain close to 37°C (237). In the present case,
the author noted that ‘‘the fungi invaded blood vessels and bronchioles, forming
plugs.’’ Since pulmonary blood vessels are the primary respiratory heat source
(238) and are unlikely to drop below body temperature, this suggests that the
invader was in fact a thermotolerant organism, not P. brevicompactum. The stan-
dard of evidence must be raised for an allegation that a fungus not known to grow
at body temperature caused an infection. The gold standard would be professional
culture collection deposition to allow verification of the identity of the case iso-
late, plus differential immunohistochemistry or molecular study to certify the
same organism was present in the tissue. The case report as it stands is not ac-
cepted.
Penicillium {aff. Eupenicillium} chrysogenum Thom (Common Syn-

onym: Penicillium notatum Westling). This fungus has been reported from a
variety of opportunistic infections. A dramatic and well-described case was that
of a necrotizing esophagitis in an AIDS patient (239). No identification criteria
were given for P. chrysogenum, but the identification was credited to reference
mycologist D. W. R. Mackenzie. A necrotizing cavitary lung lesion in a squa-
mous cell carcinoma patient was authoritatively documented by D’Antonio et al.
(240), and the case isolate was deposited in the ATCC as ATCC 201838. A well-
demonstrated report of P. chrysogenum colonization of an aortic valve prosthesis,
with fungal emboli seeded to the kidney, credited identification to the CDC in
Atlanta (241).
Keung et al. (242) attributed fever and generalized follicular and papular
rash in a Texas acute myeloid leukemia patient to P. chrysogenum on the basis
of a single blood culture isolation, and noted resolution of the affliction after
treatment with amphotericin B lipid complex and oral itraconazole. The etiologic
attribution was supported by a strong positive response to a test for antibody to
Penicillium marneffei developed by Yuen et al. (243). The case description, how-
ever, differs markedly from most infections attributed to aspergilli and other non-
dimorphic Eurotialean fungi, and was unlike any infection previously reported
for P. chrysogenum. The observed rapid formation of dispersed skin lesions is
330 Summerbell
more characteristic of organisms producing conidia or particulate assimilative

states in vitro, such as yeasts, dimorphic fungi, or hypocrealean fungi, particularly
Fusarium. Moreover, the serodiagnosis technique of Yuen et al. had been tested
for cross-reaction only with Candida species and Cryptococcus neoformans, at
least at the time of its publication (243). Furthermore, P. marneffei, as an ana-
morph in Penicillium subgenus Biverticillium, which is related to Talaromyces
teleomorphs, need not have a particularly close antigenic relationship with P.
chrysogenum (subgenus Penicillium, related to Eupenicillium teleomorphs), and
Keung et al.’s statement that ‘‘the Penicillium antibody in this patient was highly
positive’’ does not take into account that, for example, anti-A. fumigatus anti-
body is scarcely less likely to react with a P. marneffei antigen than is anti-P.
chrysogenum antibody. The name Penicillium as currently used is not a predictor
of such specificities. The serological reaction used to support the case attribu-
tion is therefore nonspecific and has little evidentiary value in regard to P.
chrysogenum. The identification of P. chrysogenum from the case was confirmed
by the laboratory of M. Rinaldi. Although at present the attribution of the case
symptomatology to this fungus appears poorly substantiated, similar future case
reports or more detail on the specificity of the Yuen et al. (243) serodiagnosis
test could tend to confirm it.
A report of posttraumatic endophthalmitis subsequent to perforation of the
eye by a metal shard (244) featured credible identification (confirmed by K-J.
Kwon-Chung and Penicillium expert K. Raper), but only a partial substantiation
of causality. P. chrysogenum was cultured from the lesion and the patient re-
sponded well to topical amphotericin B, but no direct microscopy was reported,
nor was there any indication of whether or not the fungus grew out from the
cultured surgical swab in a quantity suggestive of a significant source. Seven case
records from otomycosis by Yassin et al. (95) are regarded as unsubstantiated for
reasons mentioned under A. versicolor above. Also, a microphotograph given to
document the identification is uncharacteristic of this species. A record by Prasad
and Nema (99) from keratitis in India is etiologically and mycologically unsub-
stantiated. (See the discussion of this paper under A. sydowii above.) A case
report under the synonymous name P. notatum from etiologically well-connected
sinusitis (245) mentions ‘‘typical rami and phialides’’ in culture, but otherwise
provides no confirmatory characters. The author appears to misinterpret cross
and tangential sections of hyphae in a histopathological slide as conidia and
phialides. This, plus the use of the arcane synonym and the misspelling of
many fungal terms and names make this a dubious report, at least at the species
level.
P. chrysogenum is a soil fungus and decomposer of vegetation, especially
seeds, and also—because of its growth on crumbs, stored foods, and wall cov-
ering papers—an indoor fungus par excellence. Roquefortine C is the major toxic
Ascomycetes 331
secondary metabolite of this species. It also produces meleagrin and penicillin.

It is the main industrial source of the latter.
Colonies on CYA grow 35 to 45 mm in 7 days. They are velvety and dull
blue-green to dull green. The reverse is typically bright yellow, uncommonly
pale or red-brown. Yellow exudate and soluble pigment are usually produced.
Colonies on MEA are 25 to 40 mm and velvety, usually with heavy conidiation,
colored as on CYA, but often with less exudate and soluble pigment. G25N colo-
nies are 18 to 22 mm in diameter. Growth may or may not occur at 37°, but does
occur at 5°C.
Conidiophores are characteristically terverticillate with 1 to 2 rami, but
such penicilli are often mixed with biverticillate or less commonly quaterverticil-
late structures (Figs. 45 and 46). Most penicilli arise from substrate or surface
Figure 45 Penicillium chrysogenum, variation in conidiophores.

332 Summerbell
Figure 46 Penicillium chrysogenum, variation in conidiophores.
mycelia, but an admixture of penicilli arising as aerial side branches may need
to be distinguished from structures borne on rami as part of complex terverticillate
penicilli. This problem is exacerbated in heavily squashed slides. There are often
structures that could be interpreted either as an additional ramus arising one cell
lower on the stipe than the normal rami, or as an extra small biverticillate penicil-
lus diverging one cell beneath the classic terverticillate penicillus above. Stipes
are 200 to 300 (⫺500) µm long and smooth-walled. The phialides are ampulli-
form and 7 to 8 (⫺10) µm long, with a relatively wide and green-stained collula.
The conidia are ellipsoidal to subglobose, smooth-walled, and 2.5–4.0 ⫻ 2.5–
3.8 µm.
Typical isolates of this fungus are relatively easy to identify, particularly
when they show 37° growth. The bright yellow soluble pigment, dull turquoise
conidiation, rapid growth, and terverticillate penicilli with smooth stipes are dis-
tinctive. The greenish-tinted phialidic necks provide a subtle character that be-
Ascomycetes 333
comes increasingly useful with habituation. Oil droplets within stipes or adhering
to their surfaces must not be confused with roughening. Penicillium expansum,
which typically has orange-brown soluble pigment and exudate on CYA, must
be carefully distinguished in colonies that fail to grow at 37°C. Penicillium auran-
tiogriseum, another similar 37°-negative species, usually has finely roughened
stipes, but these may occasionally be smooth. Cultures may have clear or brown
exudate and/or brown soluble pigment.
Colonies of P. chrysogenum on MEA with closed but unsealed lids may
emit an aromatic odor sharply suggestive of pineapple. P. expansum smells of
apple, one of its characteristic substrates, while P. aurantiogriseum mixes the
pineapple odor of P. chrysogenum with a strong earthy-musty volatile.
Penicillium {aff. Eupenicillium} commune Thom [Common Synonym:

Penicillium puberulum Bain. sensu Pitt (202)]. This common food and vegeta-
tion decay fungus was apparently well documented by Huang and Harris (236)
from a disseminated infection in a leukemic patient who had been treated with
prednisone. The fungus was heavily cultured from autopsy specimens heavily
invested with septate hyphae. Identification was credited to C. W. Hesseltine of
NRRL, and a photograph in the paper is consistent with the current concept of
P. commune. This species, however, does not grow at 37°C in vitro, and the
patient had a fever of 41°C at the time the infection was progressing. No similar
cases have been reported since. The reconciliation of these data is scarcely possi-
ble, but one of the following is likely to be correct: (1) autopsy materials were
secondarily overgrown by P. commune so that an original infectious fungus was
suppressed, (2) cultures were contaminated in the laboratory, (3) the fungus was
misidentified, (4) there is an as yet undocumented thermotolerant variant of P.
commune, or (5) P. commune may become thermotolerant under host conditions.
The fourth and fifth possibilities are much less likely than the first three, so P.
commune cannot yet be considered confirmed as an opportunistic pathogen of
humans and animals. The case isolate is not among the 225 NRRL Penicillium
isolates deposited in the ATCC and is not among the cultures listed on the NRRL
website.
Penicillium {aff. Eupenicillium} cyclopium Westl. An interesting and

well-substantiated case of Penicillium invasion of the beak of a macaw, Ara ara-
rauna, was attributed to P. cyclopium by Bengoa et al. 1994 (246). The identifi-
cation is unverifiable; the authors stated which characters they looked at, but did
not give the results of these examinations. A 45-year-old monograph by Raper
and Thom (247) was used for identification, but modern works [e.g., Pitt’s 1979
monograph (202)] were not consulted. This species was synonymized with P.
aurantiogriseum by Pitt (202), and its revival only in context of molecular and
mycotoxin studies (248, 249) shows the difficulty of identifying it correctly.
334 Summerbell
Penicillium {aff. Eupenicillium} expansum Link. This species is re-

ported from keratitis in Nigeria by Gugnani et al. (230, 231). Although etiology
is well documented in these cases, no identification details are given. Since P.
expansum does not grow at 37°C and is easily confused with other members of
Penicillium subgenus Penicillium, this identification must be regarded as uncon-
firmed. P. expansum is most frequently isolated as a pathogen of pomaceous fruits
(apples, pears) (87) and has been isolated in temperate and subtropical areas, but
seldom in the tropics (74).
Penicillium {aff. Eupenicillium} griseofulvum Dierckx. This fungus

was substantiated as extensively colonizing the body of an unidentified neotropi-
cal toucanet (Ramphastidae) that was one of several that died while in the posses-
sion of a European bird fancier (250). Identification characters were given, the
identification was confirmed by experts, and a culture was deposited in the CDC
collection. There must be some doubt about the significance of this isolation, as
the fungus was obtained from autopsy specimens taken an unspecified time after
the bird’s death. P. griseofulvum does not grow at 37°C. The present author can
find no record of the normal body temperature of a toucanet, but it is noted that
many birds have normal body temperatures higher than those of mammals (e.g.,
39–41°C in chickens) and that birds respond to illness with fever as mammals
do. Finally, although only one bird was autopsied, several had died of an apparent
epidemic, and an epidemic connected with a nonthermotolerant common fungus
not otherwise known from avian disease seems unlikely.
A record from a cold-blooded animal, the Seychelles giant tortoise, appears
to be well demonstrated (251). The fungus caused multiple pericardial and kidney
lesions in a zoo tortoise that had previously been injured in a fire.
P. griseofulvum is a very common colonizer of decaying vegetation, includ-
ing grains, foodstuffs, and animal feeds. Its toxic secondary metabolites include
roquefortine C, cyclopiazonic acid, and patulin. It also produces the well-known
antifungal drug griseofulvin.
Colonies on CYA grow 20 to 25 mm in 7 days. They are deeply velvety
to granular in texture, and greyish-green to yellow-green. The reverse is pale to
red-brown or yellow-brown. Clear to pale yellow exudate or red-brown soluble
pigment may be produced. Colonies on MEA are 15 to 25 mm and granular.
G25N colonies are 16 to 22 mm in diameter. Growth occurs at 5° but not at
37°C.
Conidiophores are single or in loose bundles, terverticillate or quaterverticil-
late, and very variable in branching pattern, often occurring as aerial side
branches diverging acutely from an ascending hypha which also has a terminal
penicillus (Fig. 47). The stipes are often undulating. They are greatly variable in
length, but up to 500 µm long and smooth-walled. The phialides are ampulliform,
unusually short and with a very short neck, and mostly 5.5 to 6.0 (⫺6.5) µm
Ascomycetes 335
Figure 47 Penicillium griseofulvum.
Figure 48 Talaromyces emersonii CBS 549.92, ascospores.

336 Summerbell
long. The conidia are ellipsoidal to subglobose, smooth-walled, and 2.5 to 3.5 ⫻
2.0 to 2.5 µm.
Colonies on MEA with closed but unsealed lids may emit an aromatic odor
suggestive of mango.
5. Talaromyces C.R. Benj.

Ascomata are spheroidal gymnothecia characterized by a peridium of loosely to
densely interwoven, thin hyphae, colored yellow, cream, or white. The ascomatal
initials are often helical or clavate. The asci are usually borne in short chains,
with thin walls deliquescing at maturity. Ascospores are usually eight per ascus.
They are yellow or hyaline, ellipsoidal to spheroidal, and usually ornamented with
fine, short spines, but sometimes bearing ridges or remaining smooth.
Species with a Known Teleomorph and with Anamorphs in P. Subgenus
Biverticillium Dierckx.
Talaromyces emersonii Stolk, Anamorph Penicillium emersonii Stolk
[Common Synonym of the Anamorph: Geosmithia emersonii (Stolk) Pitt]. A
purely anamorphic strain of this fungus was repeatedly isolated from a protracted
bronchopulmonary colonization in a 12-year-old cystic fibrosis patient (252). A.
fumigatus colonization preceded this colonization and intermittently co-occurred
with it; however, the itraconazole-susceptible A. fumigatus was more readily
eliminated by therapy than was the more resistant P. emersonii. On the other
hand, episodes of A. fumigatus colonization were symptomatic, whereas P. emer-
sonii colonization was benign or nearly so. P. emersonii was extensively de-
scribed and figured, and the identification was confirmed by Eurotialean authority
R. Samson of Centraalbureau voor Schimmelcultures, the Netherlands. Accord-
ing to Pitt (253), the species shows no growth at 25°C and minimal growth at 30°C.
Growth rates below are for 37°C. The species in nature is a compost and soil
thermophile.
Colonies on CYA at 37°C mostly grow 15 to 25 mm in 7 days. They are
velvety in texture, and pale greyish-yellow to drab yellow-brown with conidiation
overlying white mycelium. The reverse is pale to brown. Clear exudate may be
produced. Colonies on MEA are 45 to 70 mm. They are sometimes granular in
appearance due to heavy ascoma formation; otherwise they are velvety, colored
pale yellow or as on CYA. The reverse is yellow or brown. G25N colonies are
0 to 5 mm. Growth does not occur at 5°.
Ascomata are pale yellow or red- to orange-brown, spherical and covered
by a thin layer of reticulate hyphae surrounded by ‘‘loose wefts of radiating,
branching, twisted yellowish, encrusted hyphae’’ (254). Asci are formed in coiled
chains. They are subglobose to ellipsoidal, and approximately 8 to 11 µm long.
Ascomycetes 337
They contain eight smooth-walled, subglobose to ovoidal ascospores 3.5–4 ⫻

2.7–3.5 µm, sometimes with remnants of a thin, gelatinous coating.
Conidiophores arise from submerged or aerial hyphae. They are biverticil-
late or with one or two rami, or occasionally with more complex branching pat-
terns, including in extreme cases four to five levels of branching. Stipes are 35
to 150 µm long, septate, rough-walled, or rarely smooth (Fig. 49). Phialides are
acerose with an extended 1 to 2 µm collula, and are 8.5 to 10 µm long overall.
Conidia are cylindrical, occasionally ellipsoid at maturity, smooth-walled, and
3.5–5 (⫺10) ⫻ 1.5–2.7 µm. The species grows well at 40°C.
Figure 49 Talaromyces emersonii, conidiophore.

338 Summerbell
Talaromyces flavus (Klöcker) Stolk & Samson, Anamorph Penicillium

dangeardii Pitt. This species was isolated from direct microscopically verified
bovine mycotic abortion by Knudtson and Kirkbride (81) and identified at NRRL.
In nature, it is an abundant and widely distributed fungus of soil and organic
material.
Colonies on CYA mostly grow 18 to 30 mm in 7 days. They are velvety
to floccose in texture, with conspicuous bright yellow mycelium occasionally
showing sparse greenish-grey areas of conidiation and usually yellow floccose,
round gymnothecia. The reverse is yellow or reddish to brown. Colonies on MEA
are 30 to 50 mm, and are sometimes granular in appearance due to heavy ascoma
formation, generally similar to CYA colonies. G25N colonies are 2 to 7 mm.
Colonies at 37°C are 20 to 45 mm. Growth does not occur at 5°.
Ascomata are yellow, spherical, and 200 to 500 µm in diameter, with tightly
interwoven peridium, Asci are formed in chains. The ascospores are yellow,
rough-walled, ellipsoidal, and 3.5–5 ⫻ 2.5–3.2 µm (Fig. 50).
Conidiophores arise mainly from aerial hyphae. They are biverticillate or
occasionally monoverticillate. Stipes are 20 to 80 µm long (Fig. 51) and are
septate and smooth. Phialides are acerose and 8 to 16 µm long. Conidia are el-
Figure 50 Talaromyces flavus CBS 262.78, ascospores.

Ascomycetes 339
Figure 51 Talaromyces flavus CBS 262.78, conidiophore.
lipsoidal to fusiform, smooth or finely roughened, and 2.2–4 ⫻ 2.0–2.5 µm. The
species grows well at 40°C.
Talaromyces thermophilus Stolk, Anamorph Penicillium dupontii Grif-
fon & Maublanc. This species was isolated from three cases of direct micro-
scopically verified bovine mycotic abortion by Knudtson and Kirkbride (81) and
identified at NRRL. In nature, the fungus is primarily a decomposer of compost-
ing materials.
There is no growth at 25°C. Colonies on CYA at 37° are 20 to 35 mm in
7 days. They are floccose or tufted, with whitish to pale pinkish-orange mycelium
producing greenish-grey areas of conidiation. The reverse is usually deep brown.
Colonies on MEA are 15 to 22 mm. The colors are as in CYA colonies except
paler or green on the reverse. G25N colonies are 1 to 3 mm. Growth does not
occur at 5°.
Ascomata are seldom formed except on sterile oat grains at 45°C (254).
340 Summerbell
Figure 52 Talaromyces thermophilus, conidiophore.
They are cream colored, spherical, and 400 to 1300 µm in diameter, with a thick,
tightly interwoven, multilayered peridium. Asci are formed in chains. Ascospores
are ellipsoidal, with irregular ridges more or less longitudinally arranged on the
walls, 3.5–4.5 ⫻ 2.2–3.5 µm.
Conidiophores arise mainly from aerial hyphae (Fig. 52). They are biverti-
cillate, monoverticillate, or occasionally with a ramus, with stipes mostly 5 to
15 µm long and smooth. Phialides are acerose and 5 to 10 µm long. Conidia are
ellipsoidal, smooth or finely roughened, and 3–4 ⫻ 1.8–2.5 µm. Conspicuous chla-
mydospores are often present, often on short lateral stalks, and are 4.5 to 6.5 µm.
Anamorphic Species in Penicillium Subgenus Biverticillium.
Penicillium {aff. Talaromyces} marneffei Segr., Capp. & Sur. This spe-
cies is one of the six virulent, thermally dimorphic, systemic pathogens described
in medical mycology thus far (Figs. 53 and 54). The range of diseases it causes and
its overall presentation are roughly similar to histoplasmosis. It has, however, many
distinctive features, including a distinct endemic range extending eastward from
the Indian state of Manipur through mountainous areas of north Myanmar, the south
Chinese states of Sichuan, Yunnan, Guangxi, and Guangdong, and most notori-
ously, northern Thailand and adjacent areas of Laos and Vietnam. It also appears
Ascomycetes 341
Figure 53 Penicillium marneffei conidiophores.
Figure 54 Penicillium marneffei, fission-yeast-like state produced at 37°C in vitro.

342 Summerbell
to be indigenous in Taiwan. Ecologically it is associated with bamboo rats in the

genus Rhizomys and Cannomys. It appears likely to use intermittent animal infection
in perennation as dimorphic Onygenalean fungi are suspected to do.
Prior to the HIV pandemic, penicilliosis marneffei was seen in only a small
number of patients, mainly immunocompromised, most of whom had acquired
the organism many years previously without experiencing significant symptoms
(255). Since then, however, an enormous upsurge in HIV-infected persons with
penicilliosis has led to hundreds of recorded cases (256, 257). The fungus can
be either focal or disseminated, and in the latter case most commonly infects the
lung, liver, and skin. Dramatic oral papules and ulcers may be seen in dissemi-
nated cases (258). Small, oblong to rounded yeastlike structures are usually seen
in histopathology. In some cases, the fission mechanism of progeny production in
the host can be seen in close examination of histopathological preparations (255).
Colonies on CYA grow 28 to 32 mm in 7 days. They are velvety in texture,
with colonies most prominently showing pale brownish to dull reddish mycel-
ium overlaid by some whitish aerial mycelium. The conidiation is typically light,
sometimes patchy on the colony surface. It is drab green. The reverse is red to red-
brown. Colonies on MEA are 28 to 30 mm, featuring aerial hyphae aggregated in
loose strands (funiculose). They are orange-brown or red, sometimes with few
conidia, but turning dull green where conidia are formed densely enough to be
apparent. Red exudate may be produced, and bright red soluble pigment is charac-
teristically produced, although atypical isolates may not show this feature. G25N
colonies are microscopic. Growth does not occur at 5°. According to Pitt (202),
at 37°C on CYA, ‘‘colonies 2–5 mm produced, low, sometimes slimy, of white
mycelium only.’’ More characteristically, on Sabouraud agar, colonies at 37°C
generally convert to a white, fission yeastlike morph in which hyphal-type apical
growth produces short hyphae that quickly fragment into individual arthroco-
nidia. Sabouraud colonies at 25°C also characteristically show the red soluble
pigment typical of the species.
Conidiophores are biverticillate, arising from surface hyphae or, on MEA,
from ropy strands. Stipes are 70 to 190 µm long and smooth-walled, usually
bearing only 3 to 5 metulae. Phialides are 6 to 8 µm long. The conidia are ellipsoi-
dal, smooth, and 2.5–4.0 ⫻ 2.0–3.0 µm.
An investigation of the subspecific population structure showed that two
predominant DNA endonuclease restriction patterns were distinguishable (259).
Although both types of isolates were found commonly in patients, several isolates
from one animal host, Rhizomys sumatrensis, appeared to be associated with one
type and three isolates from another, Cannomys badius, were associated with the
other.
Penicillium {aff. Talaromyces} piceum Raper & Fenn. This fungus was
reported without identification characters from two cases of mycotic abortion in
an omnibus overview of such infections by Austwick and Venn (260). The text
Ascomycetes 343
strongly implies but does not state outright that all cases had direct microscopi-
cally visible fungal filaments in the placenta, in fetal stomach contents, or in skin.
The pathognomonic significance of filaments in stomach contents, according to
the authors, had been confirmed in previous published and unpublished studies
of experimentally induced mycotic abortions. There is at least a small chance the
isolations were fortuitous, in that one case was ascribed to Polystictus (currently
Trametes) versicolor, a basidiomycetous wood-decay fungus with no pathogenic
record, and according to Austwick and Venn (260) themselves, not growing at
37°C in vitro.
A fungemia case involving P. piceum in an immunocompromised human
has been summarized (261); the case isolate is deposited as CBS 102383.
Colonies on CYA grow 15 to 25 mm in 7 days. They are velvety in texture.
The colonies are dull green in heavily conidial areas and show pale to straw-
yellow mycelial areas elsewhere. The reverse is red-brown or brown. Colonies
on MEA are 18 to 25 mm, often with clear to yellow exudate. The reverse is
yellow to orange. G25N colonies are 4 to 8 mm in diameter. Growth does not
occur at 5°, but usually exceeds 25 mm at 37°C.
Little is known about the organism’s habitat. Some isolates in collections
have come from animal tissues (e.g., the lung of a pig); however, collection rec-
ords lack etiologic indications.
Conidiophores are biverticillate, distinctively including an expanded, vesic-
ulate stipe apex (Fig. 55). Stipes are 15 to 22 µm long, smooth-walled, with
vesicle up to 6 µm in diameter. Phialides are acerose and 7 to 9 µm long. The
conidia are mostly broadly ellipsoidal to subglobose, smooth, and 3.0–3.5 ⫻
2.2–2.5 µm, with columns massing in conspicuously conelike clusters when
formed on relatively large conidiophores within the species’ size range.
Penicillium {aff. Talaromyces} purpurogenum Stoll. Two papers by

Morin et al. (262) and Breton et al. (263) documented the same case of a neu-
tropenic myeloblastic leukemia patient with a pulmonary infection who yielded
this fungus on two successive bronchial lavage specimens, one of which evinced
fungal filaments in direct examination. Previous nasal and stool specimens had
grown the same fungus. Intravenous inoculation of conidia into normal rab-
bits resulted in scattered granulomas containing filamentous fungal growth. The
causal agent was thoroughly described and illustrated; identification was con-
firmed by C. de Bièvre.
Colonies on CYA grow 15 to 30 mm in 7 days. They are velvety in texture.
The colonies are dark green in heavily conidial areas and show yellow or red
mycelial areas elsewhere. The reverse is deep red-purple. Orange or red exudate
and bright red soluble pigment are characteristically produced. Colonies on MEA
are 20 to 35 mm, usually lacking the red exudate and soluble pigment seen on
CYA. The reverse is pale to red-brownish. G25N colonies are 0 to 6 mm in di-
ameter. Growth does not occur at 5°, but usually exceeds 10 mm at 37°C.
344 Summerbell
Figure 55 Penicillium piceum conidiophore.
Conidiophores are biverticillate, with closely compressed metulae and phi-

alides (Fig. 56). Whole verticils of metulae, if measured across their collective
apices, often measure only 8 to 10 µm. Stipes are 70 to 300 µm long and smooth-
walled. Phialides are acerose and 10 to 14 µm long. The conidia are mostly el-
lipsoidal, sometimes subglobose, smooth, or more commonly with finely rough-
ened to heavily warty walls, and are 3.0–3.5 ⫻ 2.5–3 µm.
The rather similar P. verruculosum lacks red soluble pigment on CYA and
has globose, rough conidia. Its conidiophores are not as compressed as those of
P. purpurogenum, and usually measure around 15 µm or more across the apices.
Several other P. subgenus Biverticillium species with red colony reverse are dis-
tinguished by Pitt (87, 202).
Penicillium {aff. Talaromyces} rugulosum Thom. Neuhann (97) cul-
tured this species from scrapings of a corneal ulcer of a man who had been im-
paled by a wood fragment. Confirmation of fungal infection by direct microscopy
was not recorded, but mycotic keratitis was suggested by positive response to
clotrimazole. Characters substantiating the identification were not given (a com-
Ascomycetes 345
Figure 56 Penicillium purpurogenum.
mon shortcoming of ophthalmology literature), but food/water microbiologist H.

Barth from Universität Heidelberg was credited with mycological studies. A fun-
gus isolated from any scraping of surface material could be incidental, so this
record must be regarded as suggestively but not definitively attested. Most iso-
lates of this species do not grow at 37°C in vitro, but some do (87). An attribution
of keratitis to P. rugulosum by Świetliczkowa et al. (264) is also etiologically and
mycologically unsubstantiated; the record is based purely on growth in culture. A
record from sinusitis by Vennewald et al. (43) cannot be accepted for reasons
given above under E. herbariorum.
Descriptions may be found in works by Pitt (87, 202). The fungus is isolated
from soils and rotting vegetal foods.
Penicillium {aff. Talaromyces} verruculosum Peyr. This species has
been definitively documented as an agent of osteomyelitis in a German shepherd
dog by Wigney et al. 1990 (265). The case isolate was preserved in the Common-
346 Summerbell
wealth Science and Industrial Research Organization (CSIRO) (North Ryde,

NSW, Australia) culture collection. The species in nature is a soil fungus.
Colonies on CYA grow 20 to 30 mm in 7 days. They are velvety or slightly
floccose in texture. The colonies are green in heavily conidial areas and show
yellow mycelial areas elsewhere, occasionally also with some orange or red color-
ation. The reverse is orange- to red-brown. Clear or red exudate may be produced,
but no soluble pigment. Colonies on MEA are conspicuously broader, 30 to 45
mm, with the reverse usually paler than on CYA. G25N colonies are 2 to 6 mm
in diameter. Growth does not occur at 5°, but usually exceeds 20 mm at 37°C.
Conidiophores are biverticillate, with relatively divergent metulae, even
occasionally to an angle of 90°, according to Pitt (87), often 15 µm across the
apices (Fig. 57). (See description and discussion of P. purpurogenum.) Stipes are
150 to 250 µm long, smooth-walled, and tend to arise from aerial mycelium.
Phialides are acerose and 7 to 10 µm long. The conidia are mostly globose,
sometimes subglobose, heavily roughened, and 3.0 to 3.5 µm in diameter.
Figure 57 Penicillium verruculosum CBS 624.72.

Ascomycetes 347
C. Polypaecilum
1. Polypaecilum {aff. unknown} insolitum G. Smith
[Synonyms: Scopulariopsis divaricata Yamashita
(Invalidly Published)]
This unusual fungus has mainly been isolated from ears of patients with suspected
otomycosis (265, 266, 267, 268, 269). No case details were given in any of these
studies. More recently, a well-confirmed onychomycosis in an alcoholic patient
with dystrophic legs (a sequel of childhood poliomyelitis) and chronic dermato-
logical problems was shown to have P. insolitum as an apparent sole agent (270).
The identity of the fungus was confirmed by D. Minter at CABI, but the isolate
appears not to have been conserved there.
A fungus producing some similar, aberrant structures was isolated by
Coutelen et al. (271) from a well-confirmed pulmonary fungus ball and invalidly
published as Scopulariopsis insolita. No culture was preserved. In his species
description of P. insolitum, Smith (268) stated that the S. insolita isolate, which
he had not seen, was ‘‘identical’’ to P. insolitum. The isolate studied by Coutelen
et al. (271), which was extensively documented with illustrations, produced not
only the irregular polyphialidic structures apparently typical of P. insolitum, but
also numerous inflated structures suggesting aspergilla of the dysgonic, host-
adapted form of Aspergillus fumigatus. Such structures are not otherwise reported
in connection with P. insolitum. ‘‘S. insolita’’ grew rapidly at 37°C and had
compact, cerebriform colony morphology similar to that of atypical A. fumigatus.
An ad hoc investigation done for the present writing showed that the ex-type
isolate of P. insolitum, CBS 384.61, grows very slowly at 36°C and not at all at
40°C. Since dysgonic A. fumigatus forms various aberrant conidiogenous struc-
tures, and since pulmonary fungus ball is the source par excellence of such iso-
lates, the identity of Coutelen et al.’s isolate must be questioned. Smith (268)
described P. insolitum as producing annellides; however, Yamashita and Yama-
shita (269) and Piontelli et al. (270) published photographs revealing the apparent
annellations as a visual artifact of the conidiation process. Electron microscopy
done by Cole and Samson (272) and De Hoog et al. (69) leaves no doubt that
the conidiogenous cells of the ex-type are phialides. Smith also described the
fungus as grey-brown, in color, but Yamashita and Yamashita (269) showed that
on Sabouraud agar it became greenish, similar to their (regrettably unpreserved)
36 isolates from Japanese otomycosis. The nature of this fungus requires further
investigation. Regrettably, out of all the published isolates reported by various
authors, only the ex-type appears to be available for further studies.
Description. Colonies are 7.5 to 8.5 mm after 7 days at 25°C. They are
velvety and radially wrinkled to heaped, cerebriform, or cupulate. They are dirty
white to pale brownish-grey to pale grey-green, with the reverse beginning pale
348 Summerbell
Figure 58 Polypaecilum insolitum CBS 384.61.
olive and later becoming coffee brown to brownish-black, with yellow soluble
pigment. Conidiophores are erect and multiseptate with thin, smooth walls (Fig.
58). They are irregular in width, with occasional irregularly disposed branches,
and 6.6–47 ⫻ 2–4.2 µm. The main and side branches are terminated by a cluster
of 1 to 5, commonly 2, equal, short phialidic protuberances 2.8–9 (⫺13) ⫻ 1.2–
2.3 µm. The conidia in interconnected dry chains are ellipsoidal to subglobose,
nearly smooth, finely roughened or rugose, and 3.5–5 (⫺7.2) ⫻ 2.4–5 (⫺6) µm.
Chlamydospores are present. They are sometimes abundant, terminal or interca-
lary, thick-walled, smooth, globose to ellipsoidal or slightly curved, and 8–12 ⫻
6–11 µm. The rough, catenulate conidia with connectives indicate that this fungus
is likely of Eurotialean affinity.
D. Paecilomyces Bain. (Eurotialean Part: Olive-Brown

and Greenish Species)
This genus has been revealed as heterogeneous by molecular characterizations,
although some key studies are as yet unpublished. P. variotii is the type species
of the genus and will retain the generic name when other species are split off
into different genera. The Eurotialean affinity of P. variotii has been shown on
Ascomycetes 349
several occasions (e.g., Refs. 6, 273). The discrepancies among the species are
particularly shown in their responses to antifungals (274, 275). The Eurotialean
P. variotii shows the usual susceptibility of members of its order to the polyenes
amphotericin B and natamycin/pimaricin (used in treating ocular infections),
while P. lilacinus shows the usual tendency of Hypocrealean fungi and their
derivatives to be resistant or poorly susceptible to this class of drugs. There are,
however, some commonalities that have made the name Paecilomyces as cur-
rently conceived useful; namely, a tendency for the species in this genus to be
relatively weak opportunists, most characteristically causing infections in the
presence of implanted avascular devices such as artificial heart valves and cor-
neas, Tenckhoff catheters, and shunts; a tendency for any invasion of tissue to
be localized rather than disseminated, except in connection with the most severe
neutropenia; a tendency for yeastlike elements, possibly actually conidia in most
cases, to be reported in histopathology in disseminated infections of apparently
immunocompetent dogs and cats; and a tendency for similar particulate elements
recently well verified as conidia (276) to be seen in histopathology of invaded
human tissues and bodily fluids. Also, the Paecilomyces species seen in medical
mycology share a tendency to associate ecologically with manufactured products,
especially creams, lotions, cosmetics, plastics, and diagnostic materials con-
taining antifungal inhibitors.
1. Unidentified Paecilomyces in Opportunistic Pathogenesis

Clinically important members of the genus Paecilomyces, like Aspergillus spe-
cies, are among the easiest opportunistic molds for moderately mycologically
specialized laboratory staff to identify, especially since the very distinctive P.
variotii and P. lilacinus are overwhelmingly predominant. On the other hand,
mycological trainees invariably find them difficult to distinguish from Penicillium
species, especially members of Penicillium subgenus Biverticillium and related
species currently classified in Geosmithia, as well as Penicillium janthinellum and
similar species with extended collulas. Therefore, at least since the publication of
Compendium of Soil Fungi by Domsch et al. in 1980 (74), there has been an
increasingly clear split between competent mycology generating species-level
reports for Paecilomyces and unreliable mycology generating genus-level reports,
a phenomenon particularly notable in developed countries with good access to
literature and training courses. In Penicillium, the chance of an incorrect genus-
level identification being given even by an unspecialized laboratory is relatively
small; moreover, since individual species are very difficult to identify, a labora-
tory report of Penicillium sp. for a non-P. marneffei isolate is to be expected
unless species identification has been expressly requested. A report of Paecilo-
myces sp., however, from any situation in which the fungus may have been sig-
nificant, at the very least indicates a reporter who is unaware of the drug suscepti-
350 Summerbell
bility differences in this genus, a difference that has been widely written about
since the 1960s. Such reports, then, especially in publications, clearly signal sub-
optimal mycological awareness and must be treated with caution. Review litera-
ture should not accept such reports at face value. For example, a recent ‘‘lung
infection with paecilomyces [sic] species’’ in a child with chronic granulomatous
disease gives no mycological substantiation, nor are fungal elements in histopa-
thology mentioned for the biopsy sample that grew the organism (277). Although
such a report may be well based, it does not as published rule out: (1) growth
of a Paecilomyces species as a respiratory contaminant from a cultured biopsy
(P. variotii in particular is a common contaminant from respiratory sources) or
(2) the common misidentification of a Penicillium species occurring either as a
contaminant or as an etiologic agent. Close reading of a recent report on ‘‘dissem-
inated paecilomycosis’’ of a dog reveals isolation of a fungal colony described
as ‘‘dark green,’’ a color not found in any Paecilomyces species (except the
colony reverse of P. carneus, a species not known as a mammalian pathogen),
but common in Penicillium (278). Possibly the authors may be unconventionally
indicating the vivid olive-brown of P. variotii, which has caused similar cases.
Serum and bone marrow are stated to have tested positive for anti-Paecilomyces
immunoglobulin G in an exoantigen procedure, but as is common with such se-
roidentification reports, the authors do not state if the case isolate or a reference
isolate was used as the source of exoantigens. Use of the case isolate tends to
confirm that the isolate was etiologic, but does not confirm its identification. In
any case, since the genus Paecilomyces is heterogeneous, no single reference
isolate could legitimately be used to confirm or disconfirm genus identification
using such procedures.
As can be seen, the heterogeneity of the genus and the low reliability of
most genus-level reports make a compilation of diseases caused by Paecilomyces
sp. all but uninformative. The present author, therefore, has not attempted such
a compilation, and recommends that since there are now over 50 reports in the
literature based on identified Paecilomyces spp., these more informative reports
should be focused on as the best information sources about the pathogenicity of
these organisms.
Identification (Table 6). Paecilomyces in its current conception includes

fungi forming brushlike clusters of phialides (penicilli) producing conidia in in-
terconnected chains. These structures differ from those of Penicillium species by
being mostly widely divergent (splayed apart) at the tips rather than tending to
bend to form nearly parallel clusters as is typical of Penicillium. In addition,
Paecilomyces phialides typically have an inflated base and an extended, tapered
neck (collula, Latin for ‘‘little neck’’), whereas Penicillium phialides are inflated
into a bottlelike shape at the base, midregion, and usually even close to the apex,
with just the final 1 to 2 (rarely 4 to 5) µm extended as a significantly thinner
Table 6 Laboratory Characters of the Medically Important Paecilomyces, Acremonium, Cylindrocarpon, and Fusarium Species
Species Conidia Typical colony appearance Chlamydospores Special features
Paecilomyces variotii Fusoid to ellipsoidal, in dry, Olive-brown, dusty ⫹ Thermotolerant

connected chains
Ascomycetes
P. lilacinus Ellipsoidal, in dry, con- Dusty vinaceous pink; dusty ⫺

nected chains or cottony
Acremonium alaba- Pyriform; in chains Buff; felty to powdery ⫺ Obligately thermophilic;
mense growth poor at 25°C,
good at 37°C
A. blochii Subglobose; in long chains Whitish; moist with bundles ⫺
or heads of aerial mycelium
A. falciforme Curved, often two-celled, Grey-brown surface, purple ⫹ Long, unbranched, often
in heads reverse; wet-looking with multicelled conidiophores
sparse aerial hyphae or
tufts
A. kiliense Long-ellipsoidal to cylin- Pale greyish-brown, some- ⫹ Colony reverse brown after
droidal, in heads times orange-pink; flat or 3 weeks on Sabouraud
wrinkled; bald or slightly agar
tufted
A. potronii Ovoidal, in heads Pale to pale salmon; smooth ⫺(⫹) Thornlike phialides
to slightly granular
A. recifei Cashew-shaped, in heads Whitish, pale yellow-green ⫹/⫺
or pinkish; smooth to
thinly felted or tufted
A. spinosum Subglobose, finely warty to Pale to orange or red- ⫺ Thornlike phialides
spinulose, in heads or brown; reverse pale to
chains purple-brown; flat to wrin-
kled or ropy
351
Table 6 Continued
352
A. strictum Long-ellipsoidal to cylin- Pale to salmon or pinkish- ⫺

droidal, in heads orange; flat and slimy
to very thinly cottony
Cylindrocarpon Small, ovoid, in chains Pale, with brown to black re- ⫹ Colony small, heaped,
cyanescens verse and blue soluble mostly composed of
pigment; clumped; gla- chlamydospores
brous to velvety
C. destructans Macroconidia 1- to 3- Reddish-brown surface and ⫹
septate; straight or reverse; felty to cottony
slightly curved; mostly
20 to 40 µm, microconi-
dia ⫹
C. lichenicola Macroconidia mostly three- Whitish to pale brown sur- ⫹
to five-septate; straight face, sometimes mixed
with truncate base; with red-purple; reverse
mostly 18 to 40 µm; mi- brown; cottony often with
croconidia-, but some a frothy appearance
underdeveloped one-
celled conidia formed
Fusarium chlamy- Macroconidia rare; three- to Ochraceous surface, car- ⫹ Many phialides ‘‘polyblas-
dosporum five-septate; microconidia mine to red-brown retic,’’ with multiple open-
predominant; spindle- verse; cottony ings on separate toothlike
shaped, mostly one- to protuberances, each giv-
two-celled; ‘‘mesoco- ing rise to a single blasto-
nidia’’ up to five cells conidium
formed (see description) (‘‘mesoconidium’’)
Summerbell
F. coeruleum Macroconidia predominant; Beige to purple surface; ⫹ Phialides monophialidic;
relatively broad and ink-blue reverse; felty long and thin, to 25 µm
blunt; mostly 3 (-5) sep-
tate; microconidia⫺, but
some underdeveloped
Ascomycetes
one-celled conidia formed

F. dimerum Macroconidia predominant, Orange, slimy, flat ⫹ Phialides monophialidic; of-
short (7 to 11 µm long), ten relatively short and
pointed, mostly one- bottle-shaped
septate; microconidia⫺,
but some underdeveloped
one-celled conidia formed
F. incarnatum Macroconidia predominant; Brownish surface; usually ⫹ Many phialides ‘‘polyblas-
mostly 3 (-5) septate; mi- ochraceous reverse; cot- tic,’’ with multiple open-
croconidia sparse; mostly tony ings on separate toothlike
1 (-2) celled, intergrading protuberances, each giv-
with macroconidia, ing rise to a single co-
spindle-shaped nidium
F. napiforme Macroconidia mostly (3-)-5 Whitish to pale purple sur- ⫹ Phialides are monophialides,
septate; microconidia in face; pale to deep pur- subulate (awl-shaped; i.e.,
short chains or heads, plish or vinaceous re- moderately long and ta-
ovoid to turnip-shaped verse; cottony pered), and 14 to 31 µm
long
F. nygamai Macroconidia mostly three- Whitish to pale purple sur- ⫹ Phialides are mostly
septate; pointed; micro- face; pale to deep pur- monophialides, subulate
conidia in short chains or plish or vinaceous re- (awl-shaped; i.e., moder-
heads; ovoid to spindle- verse; cottony ately long and tapered),
shaped with truncate 13 to 40 µm long
bases
353
Table 6 Continued

354
F. oxysporum Macroconidia mostly four- Whitish to pale purple sur- ⫹ (or scarce) Phialides are monophialides,
to-five-septate; pointed; face; pale to deep pur- flask-shaped, (i.e., some-
microconidia in heads; el- plish or vinaceous re- what inflated and short),
lipsoidal to sausage- verse; cottony especially in aerial myce-
shaped lium; 8 to 14 µm long
F. proliferatum Macroconidia seldom seen; Whitish to pale purple sur- ⫺ Phialides are monophialides
pointed; three- to five- face; pale to deep pur- at first, then proliferate as
septate; microconidia in plish or vinaceous re- polyphialides, 11 to 32
long chains or in heads; verse; cottony µm long
club-shaped with truncate
bases
F. sacchari Macroconidia seldom seen; Whitish to pale purple sur- ⫺ Phialides are monophialides
pointed, three-septate; mi- face; pale to deep pur- at first, then proliferate as
croconidia in heads; ellip- plish or vinaceous re- polyphialides; 17 to 30
soidal, ovoid, or sausage- verse; cottony µm long
shaped
F. solani Macroconidia mostly 3 (-5), Whitish to medium brown ⫹ Phialides are monophialides,
septate; relatively broad or red-brown surface; distinctively long and
and blunt; microconidia pale to brown to red- thin, especially in aerial
in heads; ellipsoidal to brown or blue-greenish mycelium, 15 to 40 µm
nearly cylindrical or reverse; felty long
curved
F. verticillioides Macroconidia seldom seen; Whitish to pale purple sur- ⫺ Phialides are monophialides
pointed; three-to-five-sep- face; pale to deep pur- subulate (awl-shaped; i.e.,
tate; microconidia in long plish or vinaceous re- moderately long and ta-
chains (or in heads on in- verse; cottony pered), 11 to 32 µm
appropriate media); club-
Summerbell
shaped with truncate

bases
Ascomycetes 355
collula. These morphological subtleties are often greatly clarified by examining

the color of conidial masses formed on top of the colony. Paecilomyces never
has any blue or dark green color in its conidial masses; the closest it comes is
the golden olive-brown of P. variotii or the bright yellow-green of Paecilomyces
viridis or P. leycettanus. On the other hand, conidial masses of Penicillium spe-
cies are never pink, purple, reddish, or white, as may be seen in Paecilomyces
species. (Very importantly, however, the colors of the mycelium, exudate, and
soluble pigments must be distinguished from the conidial mass colors.) It is im-
portant to note that since Sabouraud agar often represses conidiation in some
members of both genera, this distinction should be made on potato dextrose agar,
modified Leonian’s agar, or an equivalent conidiation-promoting medium. There
are a few common Penicillium species, such as the citrus fruit pathogen, P. digita-
tum (often seen from hospital samples because of consumption of fruit by pa-
tients, staff, and visitors), that are similar in color to P. variotii and must be
distinguished micromorphologically. The clinically isolated Paecilomyces spe-
cies most likely to show some compressed phialidic clusters is P. lilacinus, which
always shows pink conidiation except in a few strongly host-adapted isolates (in
which the pink conidia are present but are too sparse to see en masse) or on
richer Sabouraud agar formulations, where overgrowth of white mycelium and
secretion of brownish reverse pigments may determine the colony’s appearance.
Paecilomyces {aff. Byssochlamys?} variotii Bain. This species is well

established as an opportunist, most commonly in connection with implanted arti-
ficial devices such as catheters, grafts, shunts, and breast implants. Although rare
overall, P. variotii infection is regularly reported from continuous ambulatory
peritoneal dialysis patients (279, 280). Catheter-associated fungemia may also
occur (281). Soft tissue invasions of immunocompromised patients may occur
(e.g., an infection in the heel of an 8-year-old boy with chronic granulomatous
disease) (282). Another case connected with chronic granulomatous disease man-
ifested as multifocal osteomyelitis (283). Endogenous endophthalmitis has been
reported (284). Endocarditis was reported several times in the 1970s and early
1980s (e.g., Ref. 285). Another regular source of etiologically significant isolation
is from sinusitis (286). A significant number of cases of disseminated infection
from dogs without known immunodeficiencies have been reported (287–290).
In some, but not all cases, yeastlike elements (290), sometimes within giant cells
(288, 289), have been prominent in histopathology. Mycelial elements may be
seen in other parts of the body (288, 290), or in some cases (287) are the only
structures seen. In some of these canine cases (288, 290) the etiologic agent was
not identified at the species level, but sufficient information was given to allow
the identification to be reasonably inferred.
A case of onychomycosis featuring linear melanonychia of both hallux nails
showed pigmented conidia in direct microscopy as well as rare filaments, and
356 Summerbell
grew P. variotii heavily in culture (291). No other fungus was grown. The etio-
logic status of P. variotii is thus strongly suggested, although additional isolation
attempts in order to elucidate a possible concomitant dermatophyte would be
ideal in such investigations. P. variotii onychomycosis was alleged by Contet-
Audonneau et al. (48), but the record is not accepted for the reasons mentioned
above in connection with Aspergillus glaucus.
The fungus is also one of the most common contaminants in medical isola-
tions, and is routinely seen from all nonsterile body sites as an insignificant
growth.
In nature P. variotii has been isolated from many types of warm soil and
plant litter (74). The fungus biodegrades many stored and preserved products,
such as foods, ‘‘optical lenses, leather, various chemical solutions, photographic
paper, synthetic rubber, creosoted wood, mouldy cigars and ink’’ (74). An affinity
for oils, cosmetic creams, and pharmaceutical emulsions has been noted (292).
The major mycotoxins produced by P. variotii are patulin and viriditoxin (111).
Description. Colonies are variable in growth rate, mostly fast-growing
(30 to 70 mm in 7 days at 25°C, most often ⬎45 mm), heavily powdery and
yellow-brown to olive-brown. The reverse is pale.
Conidiophores are mostly penicillate; that is, consisting of broomlike
branching structures with whorls of two to seven phialides on most branch tips.
The branches themselves may be solitary terminal or side branches, but are often
grouped in whorls (Fig. 59). Subterminal branches may appear in larger conidial
structures as a distinct whorl of Penicillium-like metulae. The apex of the branch
bearing these metulae in atypical isolates may become so swollen that conid-
ial measurement may be needed to completely rule out an atypical Aspergillus
terreus. [Compare relatively swollen structures depicted by Samson et al. (111,
p. 170)]. Occasional solitary phialides may be seen attached laterally on conidio-
phore branches, something that would be very unusual in Penicillium. Phialides
are mostly divergent, with a basal swelling and a prolonged, thin, tapered to
nearly parallel-sided neck (collula). They are 12 to 20 µm long. Conidia are borne
in interconnected, long, dry chains that tend to an unusually great extent to remain
intact even in squash microscopic mounts. They are smooth-walled, ellipsoidal
to fusiform (spindle-shaped), variable in size, and mostly 3.0–5.0 ⫻ 2.0–4.0 µm.
Chlamydospores are present in submerged mycelium. They are mostly subglo-
bose, with discernibly thick, brownish walls, solitary or in chains, and 4 to 8 µm
in diameter (Fig. 60). Most isolates grow well at 45°C and may grow up to 50°C.
The species should be distinguished from the much less commonly seen
Thermoascus crustaceus, which begins growth as its P. variotii-like Paecilo-
myces anamorph and then forms cleistothecia. It lacks chlamydospores. The pres-
ence of these structures can therefore be used to rule T. crustaceus out in examina-
tions of young P. variotii cultures. The converse should not be inferred (i.e.,
Ascomycetes 357
Figure 59 Paecilomyces variotii, conidiophore.
immature cultures should not be identified as T. crustaceus simply because chlam-

ydospores are not detected).
Paecilomyces {aff. unknown} viridis Ségr. & Sams. P. viridis has been
isolated on several occasions from a systemic infection of Chamaeleo lateralis,
a chameleon from Madagascar (292, 293). It is noteworthy as a dimorphic infec-
tion, in which yeastlike cells are produced in infected tissue, particularly the liver
and the spleen. No filaments are seen in the infected host.
A fungus well connected with endophthalmitis by Rodrigues and MacLeod
(294) in a boy who had been struck in the eye by a nail was identified at the
CDC as P. viridis. A description in the case report, however, gives the colony
color as yellowish-brown, not the bright yellow-green of P. viridis. In addition,
the conidia are described as elliptical and appear in a photograph as fusoid, and
their stated measurements also agree with P. variotii, not with the smaller, glo-
358 Summerbell
Figure 60 Paecilomyces variotii, chlamydospores.
bose to subglobose conidia of P. viridis. P. viridis appears only to be known

from the chameleon infections mentioned above, while P. variotii is a widely
distributed fungus well known as an opportunist of mammals. This case report,
then, clearly applies to P. variotii. This reidentification has been noted previously
by Chandler et al. (295) and is mentioned in a review of Paecilomyces mycoses
by Castro et al. (274).
Description. Colonies are slow-growing (20 mm in 7 days at 25°C), ini-
tially pale, then becoming yellow-green with formation of conidia. The reverse
is yellow to yellow-brown.
Conidiophores are not penicillate but rather verticillate; that is, consisting
of upright stalks giving rise to successive whorls of conidia at nodes near each
septum on the ascending branch. The apex of the conidiophore as well as the
tips of occasional side branches generally terminate with a longer than usual
phialide, usually emerging from a whorl of laterally arranged, shorter phialides.
Ascomycetes 359
Phialides have a globose base and a thin, tapered neck (terminal phialides more
evenly tapered) 4 to 9 µm long. Conidia are smooth-walled, globose to subglo-
bose, and 2.5 to 3.2 µm in diameter. Chlamydospores are absent.
Despite the green conidial color of this species, which led to its inclusion
in this chapter subsection along with P. variotii, the verticillate conidiophores
and the affinity for reptilian disease suggest a closer biological affinity with P.
lilacinus.
III. HYPOCREALES
The best-known Hypocrealean fungi in the clinical laboratory are the Fusarium
and Acremonium species (Table 6). They are anamorphs of teleomorph genera
that are mainly only seen in agricultural, ecological, or biodiversity studies, such
as Gibberella, Nectria, and Bionectria. The order Hypocreales contains three
families that intersect with clinical mycology; namely the Hypocreaceae (com-
mon anamorph Trichoderma, teleomorph Hypocrea), the Nectriaceae (common
anamorphs Fusarium, Cylindrocarpon, teleomorphs Nectria, Gibberella), and the
family Bionectriaceae (common anamorphs Acremonium, teleomorph Emericel-
lopsis). Although typical Acremonia are Hypocrealean, a number of Acremonium
species are unrelated; for example, Acremonium alabamense is the anamorph of
a member of the genus Thielavia of the family Sordariaceae (296). For conve-
nience, and since the relationships of many Acremonium species are not yet well
known, all species known from human and animal disease will be treated in this
chapter.
Closely related to the Hypocreales is the family Clavicipitaceae, often
treated as the order Clavicipitales, within which the best-known genera are the
plant pathogen Claviceps and the insect pathogen Cordyceps. Phylogenetic stud-
ies have shown that this group appears to arise as an evolutionary offshoot within
the family Hypocreaceae, making the latter a heterogeneous evolutionary branch
(or to use the technical adjective from phylogenetic systematics, paraphyletic).
Taxonomists have not yet reached a consensus about whether or not they must
break up such a heterogeneous branch into small branchlets with new names, as
the core cladistic philosophy of Willi Hennig (297) argues; prominent evolution-
ists such as Ernst Mayr (298) and fungal taxonomists such as Seifert et al. (299)
have argued against this. Although a few miscellaneous Clavicipitalean anamor-
phic fungi such as Beauveria bassiana have proved to have some medical impor-
tance as marginal opportunists, the main intersection of this group with medi-
cal mycology appears to be Paecilomyces lilacinus and closely related species.
(Since this is not a book on toxic fungi, the famous toxic effects of Claviceps
purpurea, the ergot fungus, and of its artificially derivativized metabolite LSD
[lysergic acid diethylamide] will not be discussed, although they are certainly
360 Summerbell
considered medically important.) Since the first part of this chapter ended with
Eurotialean Paecilomyces, the next part will begin with the Hypocrealean/Clavi-
cipitalean Paecilomyces. Note that some Clavicipitalean genera, such as Beau-
veria and Engyodontium, are dealt with in Chap. 11 rather than in the current
chapter.
Hypocrealean and related medically important fungi have some common
factors making the above higher-level taxonomic information useful. Members
of this group of fungi have a strong tendency to be resistant to both polyene and
azole antifungal drugs, and have also shown resistance to many developmental
agents, such as echinocandins and pneumocandins (300, 301). Unlike most Euro-
tiales, in which conidiation is stimulated by emergence of the fungus through
the water–air interface, the conidiation of Hypocrealean fungi typically occurs
both in submersion and upon exposure to air. The present author and colleagues
pointed out in a 1988 review (302) that this explained the facility of isolating
these fungi from blood cultures in disseminated infections, a situation quite differ-
ent from that found in connection with aspergilloses. It concomitantly explains
the rapid seeding of these infections to numerous capillary beds, especially in
the skin, a process that gives rise to the widespread ecthyma gangrenosumlike
lesions that characterize disseminated Fusarium infections. Subsequently, several
studies by Wiley Schell, John Perfect, and collaborators have established that
this subaqueous conidiation may readily be seen in in vivo materials, potentially
allowing rapid identification of Hypocrealean infections by differential histopa-
thology (303–305). The pathologist may profitably learn to recognize the typical
phialides and conidia of these fungi in tissues and bodily fluids. Yeastlike states
composed of self-replicating phialoconidia may also uncommonly occur (303).
It should be noted that, as mentioned above, one Eurotialean fungus, P. variotii,
may also form copious phialoconidia in tissue (276). Another common feature
of many of the medically important Hypocrealean fungi is an ecological associa-
tion with growth in fluids and with dispersal in aqueous aerosols. Many, though
not all, of these fungi bear conidia in sticky heads. This makes them less likely
to produce opportunistic infection by contacting the lungs via dry, airborne dust,
but may make them more likely to produce opportunistic infection after exposure
via tap water, for example, or via contact lens cleaning fluid. An additional com-
monality that is noteworthy in veterinary mycology is that whereas Eurotialean
fungi, especially thermotolerant Aspergilli, are notoriously hazardous opportunis-
tic pathogens of birds, Hypocrealean fungi are notoriously hazardous opportunists
of reptiles. (See below.)
Since teleomorphs are rarely seen in connection with Hypocrealean species
in the medical laboratory, they are not discussed for most groups below, although
an exception is made for Neocosmospora vasinfecta, in which the teleomorph
does normally occur in cultures.
Ascomycetes 361
A. Paecilomyces Bain. (Hypocrealean Part—Pink, Purple,

and White Conidial Species)
For a general discussion, see Sec. II. C (Paecilomyces) above.
1. Paecilomyces {aff. Cordyceps} fumosoroseus (Wize)

A.H. Brown & G. Smith
This fungus has been linked to pulmonary infection in a captive giant tortoise
by Georg et al. (13). No identification characters were given. Soil mycologist
W. B. Cooke was credited with identification. Three Paecilomyces species, P.
fumosoroseus, P. lilacinus, and P. marquandii, are primarily distinguished, es-
pecially in keys (74, 292) by shades of pink, vinaceous, or purple conidial mass
color. The normal ‘‘greyish vinaceous’’ color of P. lilacinus on many media
readily suggests the described pinkish color of P. fumosoroseus, so confusion
between these organisms is predictable. The characteristic inability of P. fumosor-
oseus to grow at temperatures above 30°C has not been widely publicized since
the 1957 monograph of Brown and Smith (305). By contrast, P. lilacinus grows
to 38°C (74). As P. lilacinus has emerged as a pathogen since the 1970s, it has
become much more widely known and more likely to be identified correctly;
however, the Georg et al. study was published prior to this emergence. Since P.
lilacinus is a regularly seen pathogen of tortoises and turtles while P. fumosoro-
seus is not, it would be ideal to be able to reconfirm the identification of the iso-
late Georg et al. (13) examined. The isolate, however, has not been conserved in
a collection with a publicly available database.
A disseminated infection attributed to P. fumosoroseus in an Indiana cat
began as a paw lesion before extending to nasal tissue and the liver (306). Pathol-
ogy showed many structures described as pseudohyphae and yeast forms, some
in macrophages. Histoplasmosis was excluded by repeated cultivation of the Pae-
cilomyces. An isolate was deposited in the ATCC and at the CBS, and in both
locations has been reidentified as P. lilacinus (ATCC 52586; CBS 754.96).
P. fumosoroseus is a generalist insect pathogen also known from leaf litter
and other decaying organic substances, including such foods as butter.
Description. Colonies grow 25 to 35 mm in 7 days. They are powdery to

cottony, sometimes producing upright, pink synnemata, especially after extended
incubation, which are white at first and later pale pink. The reverse is pale to
yellow or pinkish.
Conidiophores arise from aerial hyphae or from the substrate. They are
often erect, thin (approximately 2 µm), smooth-walled, and scarcely distinguished
in aspect from ordinary hyphae. They are of indeterminate length, but are usually
up to 100 µm, and are sometimes bundled into erect branched or unbranched
362 Summerbell
synnemata up to 3 cm long and 0.4 cm broad. They are septate, with a terminal
whorl of phialides as well as lateral structures that diverge at the septa and that
consist either of whorls of four to six phialides or of whorls of short side branches,
each bearing a terminal whorl of up to six phialides, or less commonly of single
side branches, single phialides, or mixed whorls containing both phialides and
side branches. Phialides are divergent. They are globose to ellipsoidal at the base,
but with a thin, tapered neck (collula). Overall they are 5.7–8 (⫺18) ⫻ 1–2 µm.
Very inflated phialides on synnemata may be up to 3.5 µm wide. Conidia are
borne in interconnected chains. They are smooth-walled, hyaline to pale pink, el-
lipsoidal to fusiform (spindle-shaped), variable in size, and mostly 3–4 ⫻ 1–2 µm
(taking into account slight variations occurring on different media).
As noted above, the inability of this fungus to grow above 30°C should be
confirmed when an identification is made.
2. Paecilomyces {aff. Cordyceps} javanicus

An endocarditis case involving a porcine aortic valve heterograft and a subse-
quent cerebral embolism was meticulously documented by Allevato and coinves-
tigators (307–309). The fungus was identified by medical mycologist John Rip-
pon and a description was given. This description, however, contained phialidic
and conidial measurements clearly copied from the published description of Sam-
son (292) and did not give the measurements of the isolate actually studied. Sam-
son’s measurements were based on size ranges seen in four isolates studied, and
would not be duplicated to the first decimal place by any individual isolate. The
isolate does not appear to have been deposited in a public collection. As authentic
P. javanicus has only been isolated from insects in the Old World tropics, and
as the description given by Allevato et al. (307) is a generic account that does
not characterize the isolate seen (except in the vague sense that it is presumed
to be concordant with the general description), this report must be regarded as
unconfirmed. Allevato et al. (307) state that P. javanicus ‘‘shows poor to no
growth at 37°C,’’ whereas the monographic study of Brown and Smith (305)
states that growth at this temperature is nil, and even growth at 30°C is restricted.
This suggests that Allevato et al. possessed an isolate growing, albeit poorly, at
37°C, deviating significantly from the authentic isolates of P. javanicus available
in collections.
P. javanicus has been isolated on only a small number of occasions from
parasitized insects, mostly lepidopteran pupae, but also the economically impor-
tant coffee berry borer beetle, Hypothenemus (Stephanoderes) hampei Ferr. (Col-
eoptera :Scolytidae), from Indonesia and Ghana. Although it seems unlikely that
P. javanicus per se has caused disease in a mammal, the description is given for
comparison with similar isolates that may be obtained.
Ascomycetes 363
Description. Colonies are 25 to 30 mm in 7 days, deeply matted, and

somewhat ropy (funiculose). They are white at first, and later cream or with heavy
conidiation pale blue-grey. The reverse is pale to yellow, sometimes with blue-
grey zonation on Czapek agar.
often erect, thin (approximately 2 µm), smooth-walled, and scarcely distinguished
in aspect from ordinary hyphae. They are of indeterminate length, but usually
up to 50 µm. They are septate, with a terminal phialide or whorl or phialides as
well as lateral structures that diverge at the septa and that consist of whorls of
two to three phialides, of single short side branches bearing a terminal whorl of
up to five phialides, or of single phialides. Phialides are divergent, cylindrical at
the base but with a thin, tapered neck (collula), and overall 8–14 ⫻ 2–2.8 µm.
Conidia are borne in interconnected chains. They are smooth-walled, hyaline,
ellipsoidal to fusiform (spindle-shaped), variable in size, and mostly 4.0–7.4 ⫻
1.0–1.8 µm (taking into account slight variations occurring on different media).
Growth is ‘‘normal at 24°C, restricted at 30°C, and nil at 37°C’’ (305). It is very
important that case isolates identified as this species and deviating by showing
growth at 37°C be deposited with a professional culture collection.
3. Paecilomyces {aff. Cordyceps} lilacinus (Thom) Samson

(Common Synonym in Older Literature:
Penicillium lilacinum)
This fungus has a very distinctive set of roles in opportunistic disease, mediated
by such factors as moderately low virulence (304) and an unusual, multiply resis-
tant drug response pattern (274, 310). In the immunocompromised patient, infec-
tions often consist of localized cutaneous or subcutaneous soft tissue lesions,
classified by Heinz et al. (304) as ‘‘indolent.’’ In a major nosocomial outbreak
connected with the use of contaminated skin lotion in the oncology and bone
marrow transplant units of a Swiss hospital, nine affected patients had cutaneous
lesions, but only one with a very low leukocyte count (0.13 ⫻ 10 9 L⫺1 ) went on
to develop dissemination leading to death (310). On rare occasions, soft tissue
infections may also occur in immunocompetent hosts (e.g., a reported infection
complicated with prepatellar bursitis) (311). Catheter-related fungemia is occa-
sionally reported, sometimes with lung nodules or other indications of dissemi-
nated establishment in tissues (312, 313). Leukemia patients have also presented
with invasive sinusitis caused by P. lilacinus (314), and there has been one case of
keratitis progressing to endophthalmitis after surgical intervention in a lymphoma
patient (315). A lung abscess in an immunocompetent patient has been demon-
strated (316).
Another major association of P. lilacinus is with ocular infections in the
364 Summerbell
otherwise healthy patient. Both keratitis (317) and endophthalmitis—usually

postsurgical (65, 318)—have been repeatedly reported. Typically, some cases of
the latter have been connected with contaminated fluids; for example, a bicarbon-
ate neutralizing solution supplied by a manufacturer of intraocular lens implants
(318, 319). P. lilacinus cases are unusual in keratitis in responding poorly to
pimaricin, the most common polyene drug of choice, which is often effective
against related fungi such as Fusarium solani (229).
Chronic mycotic sinusitis caused by this organism has been demonstrated
(320). In addition, there is one well-demonstrated case of onychomycosis attrib-
uted to P. lilacinus (321). It should be noted that this cycloheximide-tolerant
fungus is one of the most common contaminants from nails in the dermatologic
mycology laboratory, and cases of onychomycosis must be stringently confirmed
with later consistent repeat isolation. In the published case, this was done on four
separate occasions, along with demonstration of compatible fungal elements in
direct nail specimen microscopy. A review by Castro et al. (274) lists two earlier
studies with inadequately confirmed allegations of onychomycosis caused by P.
lilacinus. P. lilacinus onychomycosis was alleged by Contet-Audonneau et al.
(48), but the record is not accepted for reasons mentioned in connection with
Aspergillus glaucus above.
In veterinary mycology, P. lilacinus has a long and extensive history as an
agent of opportunistic infections in reptiles, including turtles and tortoises (322,
323) as well as crocodiles (324).
Mammals may also rarely be afflicted (e.g., an armadillo) (325). As men-
tioned above, a disseminated infection attributed to P. fumosoroseus in a cat with
no known immunodeficiencies (306) was based on a fungus later reidentified as
P. lilacinus.
This fungus has been isolated from many soils and decaying vegetative
materials, as well as from parasitized insects. In the anthroposphere it colonizes
stored nuts and root crops as well as various moist materials, such as the plastics
and biomedical solutions mentioned above, with only small amounts of organic
material and often with inhibitory additives. In soil it is often remarkably tolerant
of exposure to diverse fungicides (74). It is a strong keratin decomposer.
Description. Colonies are 25 to 35 mm in 7 days. They are generally moder-

ately floccose. They are white at first, but soon dusty pinkish to drab vinaceous with
conidiation. The reverse is often pale, frequently vinaceous on malt extract agar
according to Samson (292), and sometimes brownish on Sabouraud agar.
Conidiophores are variable (Figs. 61–63). The most highly differentiated
forms—not always found, especially on Sabouraud and other inappropriate me-
dia—arise from the substrate, and are often erect, relatively thick (3 to 4 µm),
and 400 to 600 µm long, with yellowish or pale purple-brown, roughened stalks,
often bearing several whorls of terminal and subterminal phialides and as well
Ascomycetes 365
Figure 61 Paecilomyces lilacinus, penicillate conidiophore.
as side branches also bearing whorls of phialides. In the most highly devel-
oped cases these structures may be arranged in compact, Penicillium-like heads
composed of a compact whorl of short metulae, sometimes with ramuslike side
branches below. In many isolates, however, more typical Paecilomyces structures
are more commonly seen, with conidiophores bearing several successive whorls
of phialides, short, phialide-bearing side branches, and mixtures of phialides and
short side branches. Less differentiated conidiophores that are thin and have
smooth walls may also be seen and may predominate in some isolates. These
conidiophores again tend to have successive whorls of phialides and/or side
branches, and may also bear single phialides at some septa. Phialides are diver-
gent or with a degree of compacted alignment reminiscent of Penicillium, ellip-
soidally to cylindrically inflated at the base but tapering to a thin neck (collula).
Overall they are 7.5–9 ⫻ 2.5–3 µm. Conidia are borne in interconnected chains.
They are smooth-walled or finely roughened, hyaline, pinkish-purple in mass, el-
lipsoidal to fusiform (spindle-shaped), and 2.5–3 ⫻ 2–2.2 µm. Chlamydospores
are absent.
366 Summerbell
Figure 62 Paecilomyces lilacinus CBS 430.87, verticillate conidiophore.
This species rather uniformly grows to 38°C (74). P. marquandii isolates

investigated so far have not grown at 37°C in vitro (74); therefore, temperature
tolerance may be helpful in separating these species. As always in biology, how-
ever, the possibility of an atypical organism should not be completely ruled out.
P. marquandii—with its violet color, yellow colony reverse, chlamydospores,
and absence of colored or rough-walled conidiophores—is easily distinguished
from P. lilacinus using other characters where indicated. It should be noted, how-
ever, that P. lilacinus isolates with little or no formation of large, colored, rough
conidiophores are not uncommon from medical isolations. Some degree of in
vivo degeneration similar to that seen with A. fumigatus may occur, causing an
attenuation of the formation of distinctive characteristics.
4. Paecilomyces {aff. Cordyceps} marquandii

(Massee) Hughes
Harris et al. (326) reported this fungus from a dermal lesion in the leg of an
immunosuppressed renal transplant patient. The tissue invasion with compatible
Ascomycetes 367
Figure 63 Paecilomyces lilacinus, simple conidiophore.
fungal elements was well demonstrated, and the fungus identification was cred-
ited to CDC. Since this species does not grow at 37°C in vitro, however, and
since this report comes from a historical period in which the otherwise rather
similar P. lilacinus was not yet widely known in medical mycology, this identifi-
cation must be questioned. No characteristics of the isolate are mentioned by
Harris et al., and the isolate is not deposited in a culture collection with a publicly
accessible database.
This fungus is an insect pathogen and a parasite of mushrooms, especially
Hygrophoraceae, as well as a commonly isolated soil fungus.
Description. Colonies are 18 to 25 mm in 7 days. They are powdery or
floccose, occasionally with small synnemata to 1 cm long. They are white at first
but soon violet to dark vinaceous brown. The reverse is characteristically strongly
yellow, sometimes with yellow soluble pigment.
often erect, thin (circa 2.5 to 3 µm), smooth-walled, and 50 to 300 µm. They are
septate, with a terminal whorl of phialides, often also in longer conidiophores
with lateral structures that diverge at the septa and that consist of whorls of either
two to four phialides or short side branches, each bearing a terminal whorl of
368 Summerbell
up to four phialides, or less commonly of single side branches, single phialides,

or mixed whorls containing both phialides and side branches. Single phialides
not associated with conidiophores may be seen on aerial hyphae. Phialides are
divergent and cylindrical to ellipsoidal at the base but with a thin, tapered neck
(collula), and overall are 8–15 ⫻ 1.5–2 µm. Conidia are borne in interconnected
chains. They are smooth-walled or finely roughened, hyaline, broadly ellipsoidal
to fusiform (spindle-shaped), and 3–3.5 ⫻ 2–2.2 µm. Chlamydospores are usu-
ally present in submerged mycelium. They are globose to ellipsoidal and approxi-
mately 3.5 µm in diameter. According to Brown and Smith (305), the species
does not grow at 37°C. See the discussion under P. lilacinus.
B. Acremonium
This group of very simply structured anamorphic fungi is one of the most hetero-
geneous groups of organisms currently retained within a single genus. The phylo-
genetic affinities of many members of the genus remain unknown as of this writ-
ing, but major components of the genus appear to be in the orders Hypocreales,
Sordariales, and Microascales (296). The type species, A. alternatum, is in the
Hypocreales.
Most Acremonium species appear to be completely nonpathogenic, and
many of those with some opportunistic potential are of very low virulence. They
have a relatively strong tendency to cause limited infections even in severely immu-
nocompromised patients (304), in contrast to the many disseminated infections
attributed to related Fusarium species. Nonetheless, some serious infections do
arise. Drug responses may be variable, but many species appear highly resistant
to most antifungals, with amphotericin B being the most effective overall (327).
As noted above, poor antifungal response is common in Hypocrealean fungi. In
vitro–in vivo correlation studies have not been done, and there are several ac-
counts of successful treatment of A. falciforme with itraconazole (see below) even
though its in vitro minimum inhibitory concentration (MIC) has been measured
as 32 µg/ml (i.e., is highly resistant) (69).
1. Unidentified Acremonium in Opportunistic Pathogenesis

Clinical cases caused by Acremonium species have been thoroughly reviewed by
Fincher et al. (328) and Guarro et al. (327). Neither review critically examined
documentation of organism identifications, and both replicate some definite or
likely errors (e.g., the Acremonium roseogriseum identifications discussed be-
low). Acremonium isolates from mycetoma are frequently identified to species,
especially since the 1970s, and it is now clear that A. falciforme, A. recifei, and
A. kiliense are the regularly recurring (though overall very uncommonly seen)
agents. In general, other Acremonium opportunism, while relatively rare, is regu-
Ascomycetes 369
larly seen in connection with keratitis, endophthalmitis, catheter-related perito-

nitis, and soft-tissue infection or mycotic arthritis of immunocompromised or
physically traumatized patients, and fungemia or disseminated infection of
neutropenic and other severely immunocompromised patients. Disseminated in-
fections are relatively rare compared to those caused by Fusarium species.
Acremonium spp. are regular causes of superficial white onychomycosis (50).
There are a few cases of meningitis or brain lesion, mostly involving known
major barrier breaks (e.g., intravenous drug use, spinal anesthetic administration).
As with other relatively weak pathogens that are environmentally common, a
few cases of infection of high-vulnerability sites, such as prosthetic heart valves,
are known (327). Again, however, these infections appear to be less common
than those caused by other environmentally common fungi of marginal virulence,
such as Paecilomyces species. In terms of overall documented incidence, the
most significant Acremonium infection is keratitis. A brief, useful review of this
manifestation has been provided by Kennedy et al. (329).
As the number of medical centers able to identify common opportunistic
fungi increases, and as molecular verification techniques become more commonly
used, genus-level identifications of Acremonium species in connection with case
reports become increasingly suspect. A number of recent potentially significant
case reports give no genus- or species-level identification information [e.g., recent
cases of keratitis after excimer laser photorefractive keratotomy (330) and mixed
connective-tissue disease-related infectious esophagitis (331)]. The importance of
providing this information may be dramatically seen in cases in which it is present.
For example, a recent case report on a severe necrotizing arm tissue infection in a
corticosteroid-treated patient (332) ascribes the infection to ‘‘Acremonium sp.’’
Photographs, however, vividly depict the Scedosporium state of Pseudallescheria
boydii. A parallel ‘‘Acremonium sp.’’ case from previous literature is discussed
under Fusarium verticillioides below. As with the identification ‘‘Paecilomyces
sp.,’’ ‘‘Acremonium sp.’’ in the context of a published case report is currently a
confession of inadequate microbiology. Lack of documentation leaves several po-
tentially important biomedical assertions of uncertain significance (e.g., an asser-
tion that Acremonium may cause systemic mycosis in dogs) (333). Of course, how-
ever, Acremonium sp. will and should be the common laboratory report for
acremonia isolated from cases in which they are judged to be insignificant, since
species identification by any technique is a nontrivial exercise in this genus.
2. Identification
The distinction of Acremonium species from other fungi is considered in detail
by Gams (334, 335).
Acremonium species may be readily identified morphologically by their
relatively long, narrow phialides (often remarkably long and narrow), which are
370 Summerbell
formed singly or in very simple branching structures, and which bear small,
mostly unicellular (often bicellular in A. falciforme) conidia either in sticky heads
or less commonly in chains. Hyphae are notably thin, usually under 2.5 µm, and
hyphae over 4 µm in diameter are generally not found. In Fusarium, hyphae are
generally relatively broad and diameters over 2.5 µm are common. The colonial
growth rate of Acremonium species is generally under 35 mm in 7 days at 25°C
(often under 20 mm), whereas Fusarium species that could potentially be con-
fused generally grow over 50 mm in 7 days, and in most cases exceed 60 mm.
Verticillium species may be superficially Acremonium-like. The more com-
plex species have phialides mostly in verticils; that is, in radiating structures
arranged like the spokes of a wheel or the supports of a wind-inverted umbrella.
Frequently, tall conidiophores may be seen with successive layers of such verti-
cils, reminiscent of the branching pattern of a spruce or of an ornamental Norfolk
pine. In the more simply structured Verticillium subgenus Prostrata, now divided
into several genera, phialides tend to be narrow and solitary as in Acremo-
nium, but colonies are compact, cushionlike, and deeply, densely woolly. By
contrast, Acremonium colonies are generally flat to very thinly cottony. Verti-
cillium species may sometimes have characters seldom or never found in Acre-
monium, such as crescent-shaped conidia or stalked, often dark-pigmented,
multicelled, irregularly rounded chlamydospores (dictyochlamydospores), some-
what reminiscent of Epicoccum conidia in shape and associated with substrate
mycelium.
Sagenomella species, which are not uncommon as laboratory contaminants,
were distinguished from Acremonium by Gams (335) because they formed inter-
connected chains of conidia (conidial chains connecting adjacent conidia with
small bridges of cell wall material) in the manner of Paecilomyces rather than
unconnected chains, as may be seen in some Acremonium and Fusarium species.
Some Sagenomella species have teleomorphs in Sagenoma, indicating biological
relationship with the order Eurotiales, not Hypocreales.
Some Acremonium sequences possibly facilitating identification of some
medically important species have been deposited, and ribosomal restriction frag-
ment patterns are given for representative isolates of some species by De Hoog
et al. (69). No species, however, has yet been studied molecularly in detail.
3. Acremonium alabamense Morgan-Jones Anamorph

of Thielavia terrestris (Apinis) Malloch & Cain
This species was well demonstrated as causing a fatal cerebral infection of an
intravenous drug user (336). No identification characters were given, but identi-
fication was credited to mycological expert M. R. McGinnis.
The anamorph was described from Alabama pine litter by Morgan-Jones
(337). Isolates producing the teleomorph are most frequently isolated from warm
soils (e.g., in tropical areas or sun-heated pastures) or from herbivore dung.
Ascomycetes 371
Description. This fungus grows poorly at 25°C; the description is based

on 37°C growth. Colonies are 76 to 87.5 mm in 7 days. They are floccose to
felty, eventually powdery with conidial production, and usually whitish to pale
ochraceous on the surface, becoming greyish-buff near the center. The reverse
is yellowish-buff near the periphery and reddish-brown near the center.
Conidiophores consist mainly of solitary phialides (Fig. 64). Phialides are
awl-shaped to nearly cylindrical. They are 8–25 ⫻ 1–1.5 (basal width) µm with
a thickened ring at the apex and occasionally with percurrent proliferation. Co-
nidia are borne in chains or slimy heads. They are smooth-walled, hyaline, ob-
ovate, clavate, or pyriform with a truncate base, and 3–6 ⫻ 2–3 µm. Chlamydo-
spores are not formed.
Figure 64 Acremonium alabamense.

372 Summerbell
A. alabamense grows well at 50°C, unlike the rather similar Acremonium

thermophilum W. Gams & Lacey.
4. Acremonium {aff. unknown} atrogriseum (Panasenko)

W. Gams
This species was reported from keratitis by Read et al. (338). Although etiol-
ogy was well demonstrated, the identification of the organism was completely un-
substantiated. Discussion ostensibly outlining ‘‘differentiation from other, more
common fungal organisms’’ was inappropriately restricted to the genus level
without considering species identification. Particularly as this species is readily
confused with known opportunistic black Scopulariopsis anamorphs (and in fact
was originally erroneously described as such an anamorph), this report is not
accepted. The genus-level discussion mentioned did not consider Scopulariopsis.
Two unpublished cases apparently involving this fungus were recently
noted by de Hoog et al. (69). The fungus in nature is mostly from agricultural
soils or crop roots.
Description. Colonies grow 10.5 to 12.5 cm in 7 days. They are powdery
and slightly floccose at the center, ranging from pale ochre-brown to brownish-
black, according to the degree of conidiation. The reverse is reddish-brown to
brown.
Conidiophores arise from submerged mycelium or on prostrate hyphal bun-
dles. They are solitary or in nearly verticillate clusters of up to four, often on
short lateral side branches from thick-walled, brown supporting hyphae. Phialides
are flask-shaped with a somewhat inflated base and a long, thinly tapering apex.
They are 8–18 ⫻ 2–3.5 (basal width) µm, with a short, pale collarette at the
apex. Adelophialides (short phialidic necks arising directly from sides of hyphae
and not delimited by a basal wall) may be present. Conidia are often borne at
first in chains that later coalesce as slimy heads. They are smooth-walled, subhy-
aline to dark grey, obovoid with a weakly apiculate base, and 3.5–4.8 ⫻ 1.8–
2.1 µm. Chlamydospores are not formed.
5. Acremonium {aff. unknown} blochii (Matr.) W. Gams

This species was obtained by expressing pus from intact nodular lesions seen on
extensive areas of skin of two West Bengali water buffaloes (339). The pus was
confirmed as containing fungal elements in direct microscopy, and the heavily
outgrowing A. blochii was identified by the former Commonwealth Mycological
Institute (CMI; now Commonwealth Agricultural Bureau International [CABI]).
The original 1911 case description by Bloch and Vischer (340) claiming
that A. blochii caused gummatous skin ulcers in two relatively severely affected
patients was not substantiated by demonstrating fungal etiology (341); tissue sec-
Ascomycetes 373
tions were negative for invasive fungal elements. The lesions resembled syphilitic
ulcers (341). Bloch and Vischer, however, felt that syphilis had been ruled out
by a negative Wasserman test.
The habitat of this species is unknown; most isolations have been from
medical or veterinary specimens.
Description. Colonies grow 7 to 8.5 mm in 7 days. They are moist, with
appressed to tufted aerial mycelium, which are often in ropy bundles, becoming
powdery with heavy conidiation on certain sporulation media, such as oatmeal
agar. They are usually whitish on both the surface and reverse, whether grown
in light or darkness.
Conidiophores consist mainly of solitary phialides (Fig. 65). Phialides are
spinelike, tapering, and 8–20 (⫺25) ⫻ 1–1.6 (basal width) µm. Conidia are borne
in long chains or slimy heads. They are smooth-walled, hyaline, subglobose with
slightly pointed base, and 2.9–3.3 ⫻ 1.9–2.4 µm. Chlamydospores are not formed.
6. Acremonium {aff. unknown} curvulum W. Gams.

This fungus was reported without etiologic or mycological substantiation from
exogenous endophthalmitis (342). De Hoog et al. (69) pointed out that the similar-
Figure 65 Acremonium blochii CBS 424.93.

374 Summerbell
ity of the well-known opportunist Acremonium recifei makes such a record prob-
lematical. It is not accepted here.
7. Acremonium {aff. unknown} falciforme (Carrión) W. Gams

This fungus uncommonly but regularly causes pale-grain mycetoma in tropical
and subtropical areas worldwide (327, 343–345). Successful treatment of such
a mycetoma by oral itraconazole alone has been reported in an unusual case in-
volving lesions on the right temporal scalp area of a 70-year-old male (346).
Two mycetoma cases involving renal transplant patients have been reported (347,
348), one of which provided sufficient description to allow the identity of the
organism to be confirmed (347). In this detailed report, A. falciforme was isolated
from two arm lesions as well as severely dystrophic fingernails. Direct micros-
copy to confirm establishment of the organism was done in the skin lesions but
not in the nails.
A Kentucky man who had undergone splenectomy and nephrectomy after
a diagnosis of renal cell carcinoma developed a disseminated A. falciforme infec-
tion manifesting as endophthalmitis and diskitis of the lumbar spine (349). No
identification characters were given, but the fungus was identified by mycologist
Norman Goodman. Itraconazole was again an effective therapy. An endophthal-
mitis occurring 9 months after penetrating ocular trauma in a Saudi Arabian man
was successfully treated with repeated surgical aspiration of the fungal mass, oral
ketoconazole, topical natamycin and amphotericin B, subconjunctival micona-
zole, and an injection of amphotericin B into the anterior chamber (350). The
identity of the etiologic agent was confirmed by two unnamed laboratories and
substantiated by a morphologically consistent photograph.
A well-documented case of upper gastrointestinal tract lesions caused by
A. falciforme was reported by Lau et al. (351) in an 11-year-old Hong Kong
girl who had undergone bone marrow transplantation to treat severe combined
immunodeficiency. The infection was successfully treated with amphotericin B
and itraconazole, combined with granulocyte-macrophage colony-stimulating
factor.
The habitat of A. falciforme in nature is unknown.
Description. Colonies are slow-growing, reaching a diameter of 30 to
32.5 mm in 7 days at 25°C. They are typically wet-looking with sparse, granular
to tufted aerial mycelium. They are initially grey-brown but then becoming grey-
violet. The colony reverse becomes violet-purple, most notably on Sabouraud
agar. Conidiation may not occur until after 2 to 3 weeks of growth, and is most
abundant at 25°C, not forming at all below 20°C (334).
Conidiophores are erect, barely distinguishable from aerial hyphae, mostly
unbranched, and often multiseptate, over 50 µm long ⫻ 1.9 to 2.1 µm wide
(Fig. 66). Phialides simply consist of the terminal cells of the conidiophores
Ascomycetes 375
Figure 66 Acremonium falciforme CBS 101427.
described, and are not otherwise differentiated. Conidia are borne in slimy heads.
They are smooth-walled, hyaline, and aseptate or with a single septum (rarely
two- or three-septate). They are usually broadly sausage-shaped to broadly cres-
cent-shaped with a rounded apex and a slightly tapered base terminating in a flat,
broad detachment scar, mostly 7.8–10 ⫻ 2.7–3.3 µm. An isolate from Vanuatu
depicted by McCormack et al. (343) has mostly two-celled conidia with hooked
and bluntly pointed, Fusarium-like apices, in contrast to the more rounded apices
depicted and described by Gams (334). Some similar conidia are also depicted
by De Hoog et al. (69). Chlamydospores are numerous, some intercalary but
mostly terminal on short side branches. They are often in chains. They are
rounded, brownish at maturity, smooth, thick-walled, and 5 to 10 µm long.
This fungus may appear Fusarium-like in culture, particularly in its forma-
tion of two-celled, curved conidia and purple colony colors, but was excluded
from this genus by Gams (334) on the basis of its slow growth rate and the broad
attachment of conidia to the conidiophore.
8. Acremonium {aff. unknown} hyalinulum (Sacc.) W. Gams

A German shepherd dog in South Africa with severe osteomyelitis was found
on autopsy to have disseminated mycosis caused by an Acremonium species
376 Summerbell
(352). The isolate was sent to Acremonium authority W. Gams of CBS, who
commented that it was ‘‘reminiscent’’ of A. hyalinulum (miscopied as hyalinum
by the authors), but differed from the typical isolates by forming conidia in heads
instead of chains, by having shorter phialides, and by growing well at 37°C. It
also produced dark green pigment on Sabouraud agar, something not done by A.
hyalinulum. The authors did not comment on whether polyphialides, a distinctive
feature ordinarily found in A. hyalinulum, were produced.
The organism described clearly requires further investigation and cannot
be ascribed to A. hyalinulum. Regrettably, the isolate no longer exists at CBS.
9. Acremonium {aff. unknown} implicatum (Gilman & Abbott)

W. Gams (Synonyms: Acremonium terricola, Fusidium
terricola, Paecilomyces terricola)
A fungus identified as Fusidium terricola, a name now considered a synonym
of A. implicatum, was co-isolated with Penicillium sp. (or Penicillium spinulo-
sum—see discussion under that species above) from an infected eye and attrib-
uted pathogenicity by Anderson et al. (92). Although a photomicrograph of mate-
rial from the excised corneal button shows fungal filaments, these are broad
filaments compatible with Penicillium but not Acremonium. The identification of
F. terricola is also unsubstantiated. At the time of the study, members of a number
of Acremonium species with catenulate conidia may have been identified under
this name. A. implicatum differs from A. blochii and A. alabamense—known
opportunists producing conidia in chains—by having fusiform (narrowly spindle-
shaped, with two pointed ends) conidia rather than conidia with wider shapes,
such as subglobose, clavate (club-shaped), or pyriform (pear-shaped). Conidia are
pale, not colored like those of A. atrogriseum. Polyphialides are not produced, in
contrast to A. hyalinulum. There are several species closely similar to A. implicatum.
10. Acremonium {aff. Emericellopsis} kiliense Grütz

Common synonyms in older literature, and less commonly used synonyms con-
nected with well-known case reports include Cephalosporium acremonium (pro
parte; name also applied to A. strictum and other species), Cephalosporium in-
festans, Cephalosporium madurae, and Hyalopus bogolepofii sensu auct. (in the
sense used by some authors).
This species has long been known as an agent of pale grain mycetoma,
mainly in tropical areas. Cases are relatively uncommon (327), but may be quite
extensive and severe (345).
Some serious systemic infections have been well documented, including a
disseminated infection in a myeloma patient who had undergone bone marrow
transplantation (353). The infection was seen as septicemia and catheter coloniza-
tion combined with papular skin lesions. The fungus was also repeatedly isolated
Ascomycetes 377
from stool. Mycotic esophagitis causing stenosis was described from an otherwise
healthy 11-year-old boy by Simon et al. (354); the identity of A. kiliense was
well established. Two cases of CAPD-related peritonitis were reported by Lopes
et al. (355) from Brazilian patients. The published descriptions and photo of the
isolates are nonspecific, not ruling out A. strictum.
A case of cranial osteomyelitis subsequent to automobile accident trauma
was attributed to A. kiliense by Brabender et al. (356). The main affected area,
a forehead wound, grew only Staphylococcus aureus on culture; however, A.
kiliense was cultured from three of four subgaleal abscesses remote from the
wound. Several direct microscopic and histopathologic examinations disclosed
no trace of micro-organisms in any of the samples taken. The patient responded
to combined antifungal and antibacterial therapy plus surgery. No diagnostic
characters were mentioned for the isolates, but a commercial reference center
was credited with identification. Despite impeccable investigation of the case
etiology on the part of the attending physicians, this case must still be considered
suggestive rather than definitively demonstrated.
A. kiliense was definitively implicated in a fatal case involving a prosthetic
heart valve vegetation that embolized and gave rise to a brain abscess in an im-
munocompetent patient (357). The isolate’s identification was confirmed by L.
Ajello of CDC and a consistent photograph was published.
A dramatic outbreak of postsurgical endophthalmitis caused by A. kiliense
was traced by CDC investigators to a building’s air system humidifier in an ambu-
latory center for cataract surgery (358, 359). Patients contracting the infection
were operated on early in the day shortly after the air-handling system had been
turned on. One apparently cured case from this outbreak recrudesced later as
keratitis (360). An earlier case of endophthalmitis secondary to cataract surgery
yielded cultures of a fungus identified as Hyalopus bogolepofii (Vuill.) Simões
Barbosa (361). The relatively detailed description appears to be consistent with
A. kiliense, the fungus most commonly called by this now disused name, despite
a crude line drawing that appears to misrepresent the described and photographed
long-ellipsoidal conidia as falcate. A recent case of keratitis progressing to en-
dophthalmitis was attributed to a fungus cited as a ‘‘probable A. kiliense’’ by
Wang et al. (362). No identification characters were given. Canine keratoconjunc-
tivitis caused by A. kiliense was well demonstrated by Mendoza et al. (363). The
causal isolate is in ATCC. A well-demonstrated case of keratitis connected to
contact lens invasion was described by Lund et al. (364), but the fungus depicted
has broadly ovoidal conidia inconsistent with A. kiliense. It may be A. potronii.
Though not depicted, chlamydospores are mentioned in descriptive notes; how-
ever, some A. potronii isolates may have hyphal swellings, as mentioned in the
description below.
An anomalous case of scalp kerion yielding only A. kiliense was described
by Lopes et al. (365). Direct microscopy showed relatively thin (2 to 4 µm)
378 Summerbell
hyphae and no dermatophyte-like arthroconidia or hair invasion. The patient, a

4-year-old Brazilian boy, had no known immunodeficiency or history of trauma.
Unfortunately it is not clear whether the fungus was consistently isolated on two
or more successive culture attempts made from the lesion as is generally recom-
mended for purported nondermatophytic cutaneous infections. The authors stated
that pus and scrapings were collected from the lesion, and that pus grew A. kili-
ense. They then went on to state that A. kiliense was ‘‘the unique microorganism
isolated from the rest of the scrapings on several occasions’’ (emphasis added).
The word rest is emphasized to highlight an ambiguity that may not at first be
obvious. Extensive editorial experience has taught that the expression ‘‘isolated
. . . on several occasions’’ used by persons other than native English speakers
may well simply indicate separate isolation from several inoculum pieces planted
at the same time. In the crucial passage quoted, ‘‘the rest of the scrapings’’ may
be a mistranslation intended to mean ‘‘subsequent samples obtained by scraping
the same lesion,’’ in which case A. kiliense was consistently isolated in temporally
separate examinations and its role in etiology is relatively strongly attested. On
the other hand, ‘‘the rest of the scrapings’’ may mean exactly what it appears to
mean, while ‘‘isolated . . . on several occasions’’ may be an incorrect rendering
of ‘‘isolated from several additional inocula.’’ That would make this case weakly
evidenced, and possibly a misinterpretation of A. kiliense contamination adhering
to a purulent dermatophytosis lesion. It is not unknown for dermatophytes such
as T. rubrum to invade scalp skin without invading hair (366); moreover, sparse
hair colonization may be difficult to detect in material from serous, inflamed
lesions unless fluorescent dyes or fungal stains are utilized. The etiologic attribu-
tion in this case must remain dubious until more explicitly documented later case
reports yield consistent findings.
White piedra-like hair concretions repeatedly yielding only a member of
the genus Acremonium were described by Liao et al. (367). The apparent causal
agent, anachronistically determined as Cephalosporium acremonium, had A.
kiliense-like phialides and cylindrical conidia, and a pinkish colony described as
becoming pale yellow at maturity on both Sabouraud and potato dextrose agar
(PDA) media. Contamination, secondary colonization, or in situ mycoparasitism
of poorly viable, classic white piedra concretions (Trichosporon ovoides) would
be difficult to rule out in such a case without sophisticated techniques such as
immunohistochemistry.
An anomalous subcutaneous hyalohyphomycosis affecting the hip and leg of
an otherwise healthy French stonecutter was ascribed to C. acremonium by Lahour-
cade and Texier (368, 369). The extensively described and illustrated isolate appears
to have been A. kiliense. Characteristic chlamydospores were noted in culture.
Well-confirmed A. kiliense bovine mycotic abortion was demonstrated by
Dion and Dukes (370). The isolate’s identity was confirmed by L. Brady of CMI
(now CABI).
Ascomycetes 379
Description. Colonies grow 12.5 to 16 mm in 7 days. They are flat to

slightly wrinkled with a moist surface, often somewhat ropy or slightly cottony,
and occasionally spiralling across the medium surface. They are dirty white to
pale orange or very pale brown. The reverse is pale brownish, and on Sabouraud
agar characteristically rich ochraceous to grey-brown after 14 to 21 days of
growth.
Conidiophores consist of solitary phialides or short side branches bearing
up to four phialides, sometimes rebranched once or twice, arising from medium
surface or more often from ropy strands consisting of several hyphae fascicled
together (Fig. 67). Phialides are acicular, tapering in outline, often flexuose, and
mostly 25–50 ⫻ 1.5–2 (basal width) µm. Also, submerged in growth medium,
short adelophialides (reduced phialidic necks) up to 6 µm long protrude at right
angles from vegetative hyphae. Conidia are borne in slimy heads. They are
smooth-walled, hyaline, ellipsoidal to cylindrical, and 3.1–5.8 ⫻ 1–1.6 µm. Co-
nidia formed from submerged adelophialides may be curved. Chlamydospores
are usually present on submerged mycelium after more than 1 week growth. They
are globose to ellipsoidal, thick-walled, and 4 to 8 µm in diameter (Fig. 68).
According to Gams (334), chlamydospores form most reliably on oatmeal agar.
Figure 67 Acremonium kiliense, phialides soumis.

380 Summerbell
Figure 68 Acremonium kiliense, chlamydospores.
Other media have not been extensively investigated to determine if these struc-
tures are reliably produced.
11. Acremonium {aff. unknown} potronii W. Gams

This fungus has been well connected with keratitis (371) and has been repeatedly
identified as a suspected but not rigorously proven agent of onychomycosis (334).
A well-documented case of an ulcer beneath the tongue of a cat featured granule-
like masses of mycelium (372). The etiologic agent was identified by an unnamed
mycologist at CBS, but no identification details were given and the culture was
not preserved. This identification, done long before Gams’s critical monograph
of Acremonium (334), cannot be accepted in the absence of documentation. A
recent keratitis case attributed to A. potronii featured a fungus described as having
pale olive-green colonies and spherical conidia (373). A connection to this species
is therefore uncertain. A statement that conidia were ‘‘spherical, smooth-walled
and grouped in verticils,’’ with the last term clearly intended to mean ‘‘sticky
heads’’ in context (phialides were described separately as ‘‘developed laterally’’),
suggests mycological confusion.
The original isolates described as this species were isolated by Potron and
Ascomycetes 381
Noisette (374) from an apparent septicemia of a French winegrower, whose

course of disease began as a cyst at the site of a horsefly bite. After some weeks
in which the patient experienced multiple systemic symptoms, A. potronii was
isolated both from septic arthritis of the right knee and in association with Can-
dida albicans from oral thrush. Concomitant skin lesions were culture-negative.
Consistent thin, nonbudding filaments were seen along with C. albicans elements
in direct smears from the oral lesions; examinations of knee and skin lesion fluid
gave negative results.
The knee fluid was only sparsely culture-positive, and the negative direct
microscopy was noted as not necessarily contradictory. The aggressive infection
responded dramatically to therapy with potassium iodide, the antifungal drug
of choice at the time (1911), then recrudesced after the patient ceased therapy
prematurely, and was finally eliminated after prolonged additional therapy. Al-
though now lost, the isolate was described and illustrated in detail by Vuillemin
(375) and Pollacci and Nannizzi (376), and its connection with the current con-
cept of the species was upheld unequivocally by Gams (334).
In the years just after the description of A. potronii two etiologically well-
attested cases of aggravated tonsillitis were attributed to this fungus (142a, 376a).
In one case (142a) the identification was documented with illustrations as well
as confirmation from G. Pollacci, who had studied the original A. potronii isolate.
It is not clear if the illustrations were from the case strain or from the Vuillemin
reference strain. In the other case (376a), the identification was simply attributed
to mycologist R. Motta, who worked under the supervision of Pollacci. Since
the taxonomy of Acremonium was meagerly known in those times, it is difficult
to be certain that the same species was actually studied in each case.
An Algerian mycetoma attributed by Montpellier and Catanei (377) to an
Acremonium ‘‘presenting the principal characters of A. potronii’’ was caused by
an organism with off-white to pale pinkish, wrinkled colonies producing cylindri-
cal to subglobose but mostly ovoid conidia averaging 5.5 ⫻ 3 µm but sometimes
elongate, 6.5 ⫻ 2.5 µm. Some conidia were formed on adelophialides. Chlamydo-
spores were formed on glucose-free Sabouraud agar. The organism grew more
rapidly than Potron and Noisette’s isolate (actual rates were not given) and sporu-
lated poorly except on sterilized rye grains. This may be A. potronii, but an A.
kiliense isolate in which some atypically shaped conidia were observed cannot
be excluded. Atypically swollen conidia in Acremonium species may be formed
from subsurface phialides or especially when older conidia begin to swell prior
to germination. Montpellier and Catanei based their descriptions on cultures ob-
served over several weeks. Modern students of Acremonium would avoid such
senescent material.
Kinnas (378) illustrated A. potronii in 1965 in connection with a keratitis
case from Greece. The case isolate, however, was not identified to species, and
the illustrations are an uncredited, redrawn pastiche of Vuillemin’s 1910 (375)
382 Summerbell
Figure 69 Acremonium potronii CBS 251.95.
descriptive drawings of A. potronii. Kinnas’s text indicates that they are intended
only to illustrate the genus Acremonium, not A. potronii per se. Some later authors
interpreted this paper as an A. potronii record.
Description. Colonies grow 2 to 5 mm in 7 days. They are typically al-
most smooth to slightly granular, usually pale, but may be pale salmon if exposed
to light during growth. The reverse is concolorous with the surface.
Conidiophores consist mainly of solitary phialides, occasionally arising
from small ropy hyphal bundles (Fig. 69). Phialides are spinelike, tapering, often
gently curving, and mostly (7⫺) 11–27 ⫻ 1–2 (basal width) µm. Conidia are
borne in slimy heads. They are smooth-walled, hyaline, mostly obovate (egg-
shaped, with thick end formed first), and 2.1–4 (⫺5) ⫻ 1.3–2.5 (⫺3) µm.
Chlamydospores are not formed; one nail isolate in CBS evinces irregular hyphal
swellings (334).
12. Acremonium {aff. unknown} recifei (Arêa Leão & Lobo)

W. Gams
A. recifei is most commonly isolated from white-grain eumycetoma in tropical
areas worldwide (379–381). A case of hyalohyphomycosis not organized as my-
Ascomycetes 383
Figure 70 Acremonium recifei CBS 485.77.
cetoma was reported by Zaitz et al. (381); however, this Brazilian case concerned
a hand dorsum injured only 3 months prior to diagnosis, so it may well have
been an infection that would develop into mycetoma if left untreated. Oral itra-
conazole was effective therapy; no debridement was done. A man in the Nether-
lands with an eye injury caused by a fragment of coconut shell developed well-
confirmed A. recifei keratitis (382). A patient with fungemia and respiratory infec-
tion after autologous bone marrow transplantation in a multiple myeloma grew
multiple cultures identified as A. recifei (383). Direct microscopy of respiratory
material was positive for consistent filaments. The fungus was identified by my-
cologist C. de Bièvre, and a consistent description was published. Photographs,
however, showed adelophialides, a structure not mentioned by Gams (334) in
his description of this species, but possibly occurring under some conditions.
The fungus in nature grows on decaying parts of tropical plants and has
been isolated several times from decaying coconut and brazil nut shells (334).
Description. Colonies grow 8.5 to 21 mm in 7 days. They are typically
dusty-looking, thinly felted, or—especially in fresh isolates—sometimes rag-
gedly tufted, but also commonly smooth and wet-looking. They are whitish to
yellow-greenish if kept in darkness, but pale pink if exposed to light during
384 Summerbell
growth. The reverse is the same as the surface color on most media, but ochre-
brown on Sabouraud.
Conidiophores arise directly from the substrate or from bundled aerial my-
celium and consist of solitary phialides or, predominantly, of short side branches
bearing up to 1 to 3 (or occasionally more) phialides (Fig. 70). Phialides are
acicular, and tapering in outline, often gently curving or undulate. They are
mostly 15–55 ⫻ 1.7–2.5 (basal width) µm. Conidia are borne in slimy heads.
They are smooth-walled, hyaline, strongly curving, and sausage- or cashew-
shaped with an apiculate base and sometimes with a thickened apical end, 4–6
(⫺7.5) ⫻ 1.3–2 µm. Chlamydospores may be formed in small quantities in older
cultures, and are thin-walled and 3.5 µm in diameter.
13. Acremonium {aff. unknown} roseogriseum (S.B. Saksena)

W. Gams (Common Synonym: Cephalosporium
roseogriseum S. B. Saksena)
This species was reported in connection with mycotic arthritis of the knee of a
Florida patient who had experienced puncture wounds from spiny palm fronds
in his garden (384). Etiology was well documented, but unfortunately the identi-
fication of the agent as A. roseogriseum was incorrect. The causal fungus was
described as having conidia 20–30 ⫻ 4–8 µm, incompatible with any Acremo-
nium species, and no mention was made of dark conidial pigmentation. The de-
scription of a colony with purple reverse naturally suggests Fusarium or possibly
Cylindrocarpon lichenicola. The isolate was not deposited and its identity cannot
be traced.
Zaias (49) gave Cephalosporium roseogriseum as the name of a common
etiologic agent from superficial white onychomycosis. The description and illus-
trations do not correspond to this species but rather to an amalgam of Acremonium
potronii and A. strictum, the two species later confirmed by Gams (334) from
cultures sent by Zaias to CBS.
A. roseogriseum is a tropical soil fungus with mostly dark grey, clavate to
pyriform conidia (334). It has no pathogenic record in humans or animals.
14. Acremonium {aff. unknown} spinosum (Negroni) W. Gams

This fungus has been well documented as an agent of superficial white onycho-
mycosis by Negroni (385, 386) in conjunction with its original species descrip-
tion. Confirmatory repeat isolations are reported in only one (386) of the two
strongly overlapping papers published on this case.
Description. Colonies grow 7.7 to 8.4 mm in 7 days. They are tough in
texture, moist to slightly scurfy or ropy, and flat, wrinkled, or heaped. They are
whitish, ochraceous, orange, or sordid red-brown on the surface. The reverse is
pale to purple-brown.
Ascomycetes 385
Figure 71 Acremonium spinosum CBS 136.33.
Conidiophores consist mainly of solitary phialides arising from single hy-

phae or hphal bundles (Fig. 71). Sometimes side branches bearing 2 to 3 phialides
are seen. Phialides are spinelike, tapering, often gently curving, and 8–20
(⫺30) ⫻ 1–1.5 (basal width) µm. Conidia are borne in sticky heads or occasion-
ally in chains, mostly with fine warts or spines. They are hyaline, subglobose to
broadly ellipsoidal, and 2.7–3.3 ⫻ 2.2–2.6 µm. Chlamydospores are not formed.
15. Acremonium {aff. Emericellopsis} strictum W. Gams

[Common Synonym in Older Literature: Cephalosporium
acremonium (Also Applied to A. kiliense and Other
Species)]
This species has been well etiologically connected and well identified from two
cases of disseminated mycosis in severely immunocompromised patients, in one
case beginning as a gastrointestinal colonization (303) and in another beginning
as onycholysis (387). A culture from the former case was deposited in ATCC.
In limited infections of immunocompromised patients, a fatal brain abscess in
an immunosuppressed carcinoma patient proved to contain both A. strictum and
Fusarium oxysporum (388), while A. strictum was causally connected with a
pulmonary infection in a 15-year-old chronic granulomatous disease patient
386 Summerbell
(389). The latter case isolate is also in ATCC. Well-demonstrated A. strictum

meningitis in a steroid-treated child with Landry-Guillain-Barré syndrome was
tentatively attributed to introduction via repeated lumbar punctures (390). Soft
tissue infections by A. strictum in the gluteal and femoral regions of an immuno-
competent but multiply traumatized, comatose automobile accident patient may
have contributed to the patient’s death a few days later (388), although the imme-
diate cause of death was a pneumonia that was not microbiologically character-
ized. A. strictum peritonitis in a CAPD patient was recently reported (391).
In the brain and automobile accident cases mentioned above, the identifica-
tion of the organism is not verifiable in the case report, but one or more reference
mycology centers were consulted. In the CAPD case, a partial description is
given, but it lacks sufficient detail to confirm the identification. A recent case of
fungemia attributed to A. strictum in a neutropenic child (392) both describes
and depicts a case isolate with ovoid conidia, which is incompatible with this
species. Induction of aberrant growth by use of an inappropriate medium or con-
ditions in identification studies cannot be ruled out in this case; a photograph from
a slide culture shows vesiculate and distorted forms not found in any Acremonium
species growing in favorable conditions.
A. strictum onychomycosis is reported by Contet-Audonneau et al. (48),
and the pattern of its infection is illustrated in a biopsy study. No identification
characters are given, but the distinctive, not at all dermatophyte-like morphology
of Acremonium filaments in biopsy is here accepted as sufficient evidence of
confirmed infection. As detailed above under A. roseogriseum, several onycho-
mycosis isolates obtained in classic studies by Nardo Zaias were later identified
as A. strictum.
In nature this species is commonly isolated from soils and decaying mush-
rooms and plant parts.
Description. Colonies grow 11 to 17.5 mm in 7 days. They are variable

from isolate to isolate, typically almost smooth to slightly velvety, and less com-
monly felty or cottony. They are sometimes whitish, especially if kept in dark-
ness, but more typically intensely orange or salmon, especially if exposed to light
during growth. The reverse is pale to orange.
Conidiophores consist of solitary phialides or short side branches bearing
up to 1 to 3 or occasionally more phialides (Fig. 72). Side branches are sometimes
rebranched once or twice, with at least the first second-order side branch typically
basitonous (arising from near the base of the main side branch). Phialides are
acicular, tapering in outline, often gently curving or flexuose, and mostly 25–
50 ⫻ 1.5–2 (basal width) µm. Also, submerged in growth medium, short adelo-
phialides (reduced phialidic necks), mostly 1–5 (⫺10) µm long, protrude at
right angles from vegetative hyphae. Conidia are borne in slimy heads. They
are smooth-walled, hyaline, long-ellipsoidal to cylindrical, and 3.3–5.5 (⫺7) ⫻
Ascomycetes 387
Figure 72 Acremonium strictum.
1–2 µm. Conidia formed from submerged adelophialides may be curved. Chlam-
ydospores are not formed.
C. Cylindrocarpon
The genus Cylindrocarpon consists mainly of fungi related to the teleomorph
genus Neonectria, and is thus closely related to some Fusarium species, such
as F. solani. Most human infections, however, are ascribed to Cylindrocarpon
lichenicola, a species without a known teleomorph. The genus as a whole is
primarily associated with plant pathogenesis. There is a smattering of reports
giving genus-level identifications of Cylindrocarpon strains from human (393)
and animal (394, 395) keratitis, including one human case of keratitis progressing
to endophthalmitis (30), but the majority of reported cases are for identified spe-
cies, as noted below.
388 Summerbell
Figure 73 Cylindrocarpon cyanescens CBS 518.82.
1. Identification
The distinction of Cylindrocarpon from Fusarium is mainly on the basis of mac-
roconidial shape; Cylindrocarpon macroconidia lack the distinctive (although
sometimes subtle) foot cell possessed by Fusarium macroconidia, and also have
a rounded apical end, unlike the pointed apices seen in most Fusarium species.
Also, the elongate, cylindrical phialides that are restricted to F. solani and related
members of Fusarium subgenus Martiella (see also F. coeruleum below) in Fu-
sarium are the norm in Cylindrocarpon. Cylindrocarpon destructans may be es-
pecially likely to cause confusion with F. solani (See the discussion under the
former name.) Cylindrocarpon species should be grown for identification on fun-
gal sporulation media such as PDA, oatmeal agar, modified Leonian’s agar, or
pablum cereal agar (396).
2. Cylindrocarpon {aff. unknown} cyanescens (de Vries et al.)

Sigler
This fungus is known from a single case of pale-grain mycetoma originally con-
tracted from a puncture of the foot dorsum in Indonesia (397). It was originally
described as a Phialophora species (398), and was moved out of this notoriously
Ascomycetes 389
heterogeneous genus by Sigler (399) because the culture, chlamydospores, and

phialides suggested Cylindrocarpon, even though no macroconidia were pro-
duced. The hypothesis proposed by the current name requires molecular testing.
Description. Colonies grow very slowly, 1 to 1.5 mm after 7 days at
20°C. They are velvety to heaped [described as formed of ‘‘cauliflowerlike pus-
tules’’ by de Vries et al. (398)] and often consist mainly of chains of chlamydo-
spores. They are a pale cream color at first on the surface and reverse. The reverse
becomes brownish to black, secreting blue pigment into the agar when freshly
isolated. Conidiophores are often extended branches bearing a small number of
phialides as side branches as well as a terminal phialide. Phialides are cylindrical
with tapered ends, often rather short and curved when arising as a side branch.
They are sometimes aseptate, but often with up to three septa, which are 10 to
40 µm long with a small collarette. Conidia are smooth, hyaline, ovoid to ellipsoi-
dal, and 4–7.5 ⫻ 2–3 µm. Chlamydospores are predominant, mostly in chains
or clusters, nearly hyaline at first, then becoming olivaceous brown and partially
encrusted with a thin layer of loosely adhering brown material.
This species does not strongly resemble any other arising in medical mycol-
ogy. It is very slow growing and secretes blue pigment when fresh, and in micros-
copy of cultures is composed mainly of chlamydospores, while producing a small
number of septate phialides giving rise to small, ellipsoidal conidia. Unlike the
Phialophora species it was once included with, it lacks melanized (dark pig-
mented) hyphae or conidiophores, although its chlamydospores tend to be pig-
mented.
3. Cylindrocarpon destructans (Zins.) Scholten Anamorph of

Nectria radicicola Gerlach & L. Nilsson
This species has been well confirmed by Zoutman and Sigler (399) as an agent
of mycetoma contracted as the result of a nail puncture of the foot in Antigua.
Four cases of keratitis in horses from Florida, including two cases con-
firmed with direct microscopy, were reported by Hendrix et al. (400). Identifica-
tion criteria were given, and the identification of the fungus was confirmed by
phytopathological mycologist J. Kimbrough. Despite this, the photographs pre-
sented—a view of a primary isolation plate showing loosely textured fungal colo-
nies and a light micrograph showing a preponderance of uncurved macroconidia
with three evenly spaced septa and an abruptly tapered, truncate base—suggest
that further study of the identity of at least one of the isolated strains may be
warranted. The authors state that they ruled out C. lichenicola because their
strains produced microconidia, unlike that species. In the micrograph (their Fig.
2), however, the few reduced conidia present, referred to as both ‘‘microconidia’’
and ‘‘microconidiospores’’ in the figure legend, are similar to those formed in
low proportion in many Fusarium and Cylindrocarpon species conventionally
390 Summerbell
said not to produce microconidia. Similar 0- or 1-septate conidia are shown in

micrographs of C. lichenicola in papers by Booth et al. (401), Laverde et al.
(402), and Iwen et al. (403), as well as in line drawings in the monographic
study (as C. tonkinense) of Booth (404). Also, the deeply floccose, effuse, frothy-
looking C. lichenicola colonies depicted by Laverde et al. (402) are strikingly
similar to the colonies depicted by Hendrix et al. (400), and contrast strongly
with the compact, felted, and centrally tufted C. destructans colony depicted by
Zoutman and Sigler (399). In nature, C. destructans grows on many types of
plant roots in both cultivated and uncultivated soils.
Description. Colonies grow 7 to 12 mm in 7 days at 25°C. They are
densely felty to floccose, usually with raised centers and somewhat folded mar-
ginal zones. They are pale at the margin and in floccose areas but otherwise
reddish-brown on the surface, sometimes spotted with slimy cream to beige spor-
odochia. The reverse is light brown or more commonly reddish-brown, with a
brownish diffusing pigment. Conidiophores are simple or repeatedly branched,
with 1 to 3 branches at branch points (Fig. 74). Phialides are long and slender,
cylindrical to awl-shaped, and 18–35 ⫻ 2.5–3 µm, with a small collarette. Mac-
roconidia are uncommon in some cultures. They are cylindrical to slightly
Figure 74 Cylindrocarpon destructans CBS 301.93, showing formation of both micro-

conidia and macroconidia.
Ascomycetes 391
curved, with a rounded apex and rounded to slightly truncate base. They are
mostly 1 to 3-septate, sometimes composed of cells of markedly unequal length,
and 20–40 (-50) ⫻ 5–6.5 (-7.5 µm). Microconidia intergrade in size and shape
with macroconidia. They are long-ovoid to long-elliptical and sometimes slightly
curved, with no or one septa. Aseptate conidia are 6–14 ⫻ 3–4.5 µm. Chlamydo-
spores are abundant, terminal or intercalary, solitary, in chains, or in clusters.
They are pale and smooth, becoming brownish and sometimes warted at maturity.
They are more or less rounded and 8 to 16 µm long. The teleomorph is found
only on infected plants and is not described here.
This fungus bears a slight resemblance to F. solani, but grows much more
slowly. Its macroconidia lack the asymmetrical basal foot cell that F. solani mac-
roconidia have. In the C. destructans, microconidia are mostly under 10 µm long
(some larger ones occur), while F. solani has microconidia mostly over 10 µm,
with some smaller ones also seen.
4. Cylindrocarpon {aff. Nectria} lichenicola (C. Massal.)

Hawksw. (Common Synonym: Cylindrocarpon tonkinense
Bugn.)
This species has primarily been reported in well-substantiated cases of keratitis
from Colombia (402), Japan (405), and Argentina (406). There is also an endoph-
thalmitis record from Florida, with etiology attested by growth in multiple cul-
tures and the identification evidenced only by the appearance of the name C.
tonkinense in a table (407). (See also the discussion of Florida horse keratitis
cases attributed to C. destructans above.) Other opportunistic infections are also
rarely seen. A Nebraska leukemia patient experienced a localized hand ulceration
caused by C. lichenicola; since the patient was undergoing high-dose chemother-
apy, partial amputation of the hand was used in addition to amphotericin B to cure
the infection (403). A case of CAPD-related peritonitis is reported by Sharma et
al. (408) in a British patient who had had a prolonged Jamaican vacation. The
etiology in this case was well verified, but the identification of the organism was
not documented or credited. A disseminated C. lichenicola infection in an acute
myeloid leukemia patient was documented by James et al. (409). The infection
was treated successfully with liposomal amphotericin B. The authors speculated
that the patient’s ‘‘past history of athlete’s foot’’ was the source of the infection,
even though the patient had ‘‘no particular skin or nail lesions’’ at the time he
presented for leukemia chemotherapy. This hypothesis seems highly far-fetched.
(See below.) In addition, the question of whether fungal etiology was confirmed
in the previous foot condition is not addressed. The C. lichenicola isolate involved
in dissemination, however, was authoritatively identified by two expert labora-
tories and collected at the second, the International Mycological Institute, in
Egham, UK (now CABI).
392 Summerbell
The notion of a C. lichenicola from athlete’s foot in the James et al. (409)
study was supported by the citation of a case report by Lamey et al. (410), in
which an unidentified Cylindrocarpon was reported from intertrigo involving all
the toe webs of a male Beninese patient examined in France. In this case, a fungus
disclosed by its photograph and description as compatible with C. lichenicola
was isolated repeatedly and consistently over a 2-month period from scrapings
that were positive in direct examination for compatible fungal filaments. No der-
matophyte was isolated in four or more consecutive attempts, all positive for the
Cylindrocarpon. The case appears comparable to intertrigo cases involving Afri-
can patients discussed under Fusarium solani and F. oxysporum below. The rea-
son this emerging pattern of hypocrealean intertrigo in otherwise healthy patients
appears mainly to affect Africans may be at least partially adumbrated by the case
report of Comparot et al. in 1995 (411), discussed below under F. oxysporum, in
which a possible connection to foot washing multiple times per day for religious
reasons is mentioned. (This in itself could not be a sufficient cause, since this
religious practice is carried out worldwide, but may combine with other factors
such as use of contaminated water.) The probability that a case of typical athlete’s
foot seen in a non-African patient could be caused by a hypocrealean fungus
appears to be extremely small. With regard to the Lamey et al. (410) case report,
it should be noted that the conidial measurements given for the fungus seen are
compatible with C. lichenicola in length (20 to 30 µm), but are too narrow in
width (3 to 4 µm). The measurements given, however, conflict with the paper’s
Fig. 3; according to the measurements, conidia should have a length–width
(l/w) ratio of (5-) 6.6–7.5 (-10), whereas the mature conidia shown in the
photo, measured with a ruler, have l/w ratios of around 3.5. A conidium 20 µm
long at this ratio would have a width of 5.7 µm, while one 30 µm long would
be 8.5 µm wide. If the published lengths are correct, therefore, the conidia are
within the width range of C. lichenicola or even slightly too wide, rather than
too narrow. It appears that the conidial measurements given by Lamey et al.
(410) could not possibly have been accurate and therefore do not contradict the
probability that C. lichenicola was involved in this case. Alternatively, the photo
may be completely unrepresentative of the isolate seen; in fact, however, it ap-
pears typical of this most common Cylindrocarpon from human disease.
Description. Colonies grow approximately 41 mm in 7 days at 25°C on

PDA. They are velvety to moderately floccose with minute, upright, heavily co-
nidium-producing strands giving a frothy appearance. They are white initially,
then yellow to pale brown, sometimes becoming red-brown to purplish-red at the
center, especially when the reverse is strongly pigmented. The reverse is buff at
first but is soon dark brown, sometimes with a brown diffusing pigment. Conidio-
phores are long or simple, or with a small number of mostly basitonously arising
(i.e., formed on the lower half of the conidiophore) branches (Fig. 75). Phialides
are long-cylindrical, subulate (awl-shaped; i.e., long and tapered) to filiform
Ascomycetes 393
Figure 75 Cylindrocarpon lichenicola CBS 109048.
(threadlike, sometimes with a degree of irregular sinuous curvature), and 38–

50 ⫻ 3–5 (basal diameter) µm. Macroconidia are formed in sticky heads. They
are smooth-walled, hyaline, cylindrical, long-ellipsoidal, or slightly clavate (i.e.,
with slightly expanded apical end), with a rounded apex and a distinct, protruding,
often slightly laterally displaced truncate basal hilum, usually with three evenly
spaced septa, occasionally to five-septate, and mostly 18–40 ⫻ 5–7 µm. Reduced
forms with 0 or 1 septum but more or less consistent in diameter with typical
macroconidia may be present in low proportions. Chlamydospores are abundant,
especially after 14 days. They are formed terminally, laterally on short branches,
sessile along hyphae, intercalated into macroconidia, in dyads or clumps or in
intercalary chains, and are hyaline to brownish, smooth to finely roughened, glo-
bose, and 6 to 15 µm in diameter.
This very distinctive species can be distinguished from many other Cylin-
drocarpon species by its mostly straight macroconidia with a truncate hilum, and
by its lack of differentiated microconidia. (See comments about reduced conidia
in discussion of C. destructans above.) The same characters distinguish it from
the only slightly similar Fusarium species, F. solani.
394 Summerbell
Figure 76 Fusarium chlamydosporum CBS 615.87 polyphialides.
5. Cylindrocarpon {aff. unknown} vaginae Booth

This species was described by Booth et al. (401) in a publication stated to be
about Cylindrocarpon species associated with keratitis. No details were given,
however, about the isolate obtained from ‘‘the eye of a British farmer following
an injury.’’ This is thus an unsubstantiated record. C. vaginae has predominantly
0 to 1-septate conidia, with a few mainly curved, larger two- or three-septate
conidia to 24 µm long, and is primarily distinguished by producing hyaline
clumps of up to six chlamydospores from globose vesicles held on short lateral
stalks. Colonies are pale with a reverse that gradually becomes grey-brown, and
grow rapidly, covering a 90-mm petri plate of PDA medium in 7 days at 30°C
(25°C measurement not given).
D. Fusarium
The anamorph genus Fusarium contains the asexual states of numerous ascomy-
cetous fungi in the genera Gibberella, Nectria, and Cosmospora, as well as re-
lated purely anamorphic species for which no teleomorph is known. At the pres-
ent moment, a few phylogenetically outlying but morphologically similar
organisms, such as Fusarium dimerum, remain in the genus. Teleomorphs of this
group are not seen in the clinical laboratory and are not described here.
Ascomycetes 395
This loosely related group of Hypocrealean anamorphs was regarded as

rarely pathogenic, except in ocular and nail infections, until the number of neutro-
penic patients increased dramatically in connection with new anticancer chemo-
therapies in the 1980s (302, 412, 413). The widespread use of amphotericin B
therapy or prophylaxis has also contributed to this emergence, since the isolates
of these species are relatively likely to manifest typical Hypocrealean resistance
to this drug (susceptible strains do occur) (412–414). Since this emergence, sev-
eral species have been recognized as particularly significant (412). As in Aspergil-
lus, in Fusarium there are a small number of major opportunistic species and a
much greater number of marginal opportunists. The major agents are Fusarium
oxysporum, F. solani, F. verticillioides (⫽F. moniliforme), and F. proliferatum.
1. General Pattern of Involvement by Major Opportunistic

Fusarium Species
The major opportunistic fusaria show a similar pattern of involvement in dissemi-
nated infection of the neutropenic patient. Generally patients with acute leukemia
are involved, although chronic leukemia, aplastic anemia, and lymphoma-related
cases have also been observed repeatedly (412, 415). Many other strongly immu-
nocompromising conditions yield occasional cases. Cases frequently feature rapid
blood-borne dissemination and the development of local lesions where inoculum
lodges in capillary beds. This is dramatically seen as the appearance of ecthyma
gangrenosa-like, necrotic skin lesions, but also may be seen in organ and bone
lesions, (e.g., in magnetic resonance images) (416). The mediation of this pattern
by copious microconidial production in the host tissue and circulatory system
was proposed by Richardson et al. (302) based on the extensive conidial produc-
tion seen in these species in liquid culture, and was definitively confirmed in a
series of studies by W. Schell and collaborators at Duke University Medical Cen-
ter. In one of these studies, Liu et al. (276) showed microconidia and phialides
in numerous histopathological preparations. Even multicelled Fusarium conidia
could rarely be seen in skin lesions and peritoneal fluid. The proliferation of
conidia in situ facilitates rapid diagnosis in that positive blood cultures are readily
obtained (302, 413, 417).
Common portals of entry for these disseminated infections appear to be
the sinuses, the lungs, and the skin. Emergence from onychomycosis has been
documented several times (415, 418). The common environmental association
between generalist decomposer Fusarium species (a group overlapping strongly
with the opportunistic mammalian pathogens) and water systems (74) appears to
have been responsible for some nosocomial infections (419); patients may acquire
the organism, for example, by inhaling aerosols while showering.
Although localized pulmonary infection is rare in immunocompromised
patients, presumably because infections disseminate too readily, localized skin
396 Summerbell
and soft tissue infections are uncommonly but repeatedly seen. Several related
cases are contrasted by Guarro and Gené (412).
As with most fungi causing disseminated infections in immunosuppressed
patients, opportunistic Fusarium species may also be isolated from highly vulner-
able sites in immunocompetent patients with major barrier breaks, giving rise to
cases of catheter-related peritonitis, endocarditis, keratitis, exogenous endoph-
thalmitis, and trauma-related osteomyelitis and septic arthritis (412, 415). With
regard to ocular infections, there is a noteworthy tendency for F. solani (see
below) to be the predominant organism involved as well as the most aggressive,
but the other major opportunistic fusaria may also cause these infections (229,
393, 420).
Even though disseminated Fusarium infection may not infrequently begin
as sinusitis, members of this genus appear to have little involvement in allergic
fungal sinusitis of otherwise healthy individuals (421). Likewise there appear to
be few or no well-substantiated records from etiologically similar colonizations
of surfaces exposed to air within immunocompetent patients, such as otomycosis,
allergic bronchopulmonary mycosis, and pulmonary fungus ball.
Fusarium species have long been known to cause onychomycosis, particu-
larly of the superficial white variety (49). Here F. oxysporum appears to be the
main agent involved, but other major opportunistic fusaria are also seen in this
role.
One underpublicized but frequently encountered aspect of opportunistic
fusarial biology is the tendency of these fungi to grow saprobically on wound
and ulcer surfaces, as well as on suppurating intertriginous fissures (422, 423).
Such growths are heavily positive for fungal filaments in direct microscopy, but
detailed analysis shows that generally no tissue penetration occurs in immuno-
competent hosts. Exceptions are discussed under F. oxysporum and F. solani
below. Burn patients also may experience superficial Fusarium growth, which
may then progress to life-threatening systemic infection (424, 425).
In mammals other than humans, Fusarium species may cause keratitis
(426), especially in horses (395). Infections of reptiles and amphibians are occa-
sionally encountered (see individual Fusarium species discussions below), and
occasional skin infections, often not well confirmed, have been reported from a
variety of mostly aquatic mammals (426). Infections of arthropods and of bird and
reptile eggs are not discussed in this chapter. The complex topic of mycotoxicoses
caused in humans and animals ingesting food colonized by Fusarium species is
also beyond the scope of this chapter.
2. Unidentified Fusarium in Opportunistic Pathogenesis

Fusarium species require some skill to identify morphologically, and molecular
identification is at least temporarily entangled in the remarkable complexity of
Ascomycetes 397
sibling speciation that has been disclosed within this genus (e.g., the existence
of at least 26 entities within the morphospecies F. solani that would be considered
distinct species using a DNA-based phylogenetic species concept) (427), with
more such entities undoubtedly still uncharacterized. Generic-level identification
is therefore still common in case reports. Since the major opportunistic fusaria
so far have not been shown to have strong clinical differentiation in terms of
their involvement in serious systemic infection or in their responses to therapy,
generic identification is not as problematical as it is in the more heterogeneous
Paecilomyces and Acremonium. The information in the reports that lack species
identification is essentially summarized in Sec. 1 above. Species identification is
still recommended in this genus, however, so that emerging patterns of species-
specific behavior can be revealed, particularly in connection with novel infections
or therapies. F. solani in particular is not especially closely related to the other
major opportunistic fusaria, and is easily distinguished by its elongate microconi-
dial phialides.
3. Identification
For practical purposes in the clinical laboratory, correct genus-level identification
of Fusarium species is the most critically important action to enable correct treat-
ment and infection control. Although antifungal susceptibility testing is indicated
for each Fusarium-like isolate causing a confirmed systemic infection, genus-
level identification as Fusarium suggests that itraconazole and other azole drugs
more directed at Aspergillus or Candida infections may need to be replaced or
supplemented with amphotericin B.
When Fusarium forms characteristic elongate, canoe-shaped macroconidia
rapidly, genus-level identification can be extremely straightforward. The labora-
torian need only be aware that Cylindrocarpon species may have somewhat simi-
lar macroconidia. The macroconidia of this genus, however, differ by being either
straight or sausage-shaped, with rounded apices. The distinctive bootlike pedicel
that is found at the base of most Fusarium macroconidia is not present. The
toelike part of this structure serves in most Fusarium species to stabilize forming
macroconidia against the side of the phialide apex so that they do not break off
due to shear stress while still immature. Only a few species, such as F. incarna-
tum, are adapted to form macroconidia in ways that do not involve such stabiliza-
tion structures. The Fusarium solani complex has perhaps the most Cylindrocar-
pon-like macroconidia, yet these conidia do have a small but perceptible
pedicellate structure. Also, in any comparisons with most similar-looking organ-
isms, the elongate phialides of F. solani are distinctive.
In heavily microconidial Fusarium species such as F. proliferatum, in
which macroconidia may form belatedly or only after special stimulation, the
isolates may need to be distinguished from Acremonium species. With experience
398 Summerbell
this is normally done at a glance. The underlying characters permitting this are
the much faster growth rate of Fusarium species, and in most species, the much
greater amount of floccose aerial mycelium produced. As mentioned previously,
the colonial growth rate of Acremonium species is generally under 35 mm in 7
days at 25°C, often under 20 mm, whereas Fusarium species generally grow over
50 mm in 7 days, and in most cases exceed 60 mm. Many Fusarium species,
including members of subgenera Liseola (e.g., F. proliferatum, F. verticillioides)
most likely to be purely microconidial in early growth, are notably floccose.
Among the medically important fusaria in general, only F. dimerum, which nor-
mally forms copious pointed two-celled macroconidia, is relatively flat, slimy,
and Acremonium-like. (It grows significantly more quickly than an Acremonium
in any case.) Note, however, that heavily bacterially contaminated Fusarium cul-
tures of all kinds may be flat, slimy, and intensely pigmented, probably in part
because mycolytic bacteria have digested most of the aerial hyphae. These bacte-
ria may be antibiotic-resistant pseudomonads, not eliminated simply by culturing
to antibiotic media. Microscopically, Fusarium has hyphae that are generally
relatively broad with diameters mostly over 2.5 µm. Acremonium species have
thin filaments mostly below 2.5 µm in diameter.
Means of rapid molecular identification of Fusarium species have recently
appeared. Hue et al. (428) identified a ribosomal primer pair that selectively am-
plified DNA from Fusarium species with affinities to the teleomorph genera Gib-
berella and Nectria, including F. oxysporum, F. solani, and Fusarium subgenus
Liseola (e.g., F. proliferatum, F. verticillioides), in addition to related nectri-
aceous species such as Fusarium dimerum and Neocosmospora vasinfecta. A
Monographella nivalis (referred to by former name Fusarium nivale) isolate
giving a positive response appears to have been misidentified, since the real M.
nivalis (see section on this fungus below) is much more distantly related to Fu-
sarium species than are many of the isolates that gave negative reactions with
the primers of Hue et al. The sole Acremonium species tested, A. strictum, was
not amplified. Given the taxonomic heterogeneity of Acremonium species and
their dispersion throughout the Hypocreales and several related orders (296), it
seems likely that at least some species of this genus would probably cross-react
with any probes reacting to all Fusarium species. Whether medically important
species would be included is difficult to predict. Fusarium, like Acremonium, is
by no means monophyletic as currently defined. It may be argued that phyloge-
netic clade identification is more predictive than current anamorph genus identi-
fication in any case.
Hue et al. (428) also identified a primer pair apparently specific to the
intersection of the phylogenetically related Fusarium subgenus Elegans and F.
subgenus Liseola clades, as delimited by O’Donnell et al. (429), as well as to
the more distant F. subgenus Martiella (e.g., F. solani). Not enough species or
isolates were tested to advance this result beyond the preliminary stage, however.
Ascomycetes 399
A more specific but clearly more specialized rapid 28S ribosomal sequence
identification technique for six medically important Fusarium species was de-
scribed by Hennequin et al. (430). Since most of the ‘‘species’’ tested are in fact
species complexes that were by no means fully explored in this study, it must
be considered very preliminary, although more widely applicable in principle.
A number of papers have recently appeared on detection and identification
of Fusarium from infected eyes using polymerase chain reaction in combination
with panfungal 18S rDNA (431) or cutinase (432) primers. The studies are again
in a preliminary stage, neither dealing with taxonomic diversity in the species
tested nor with the problem of false positives derived from airborne contamina-
tion. They nonetheless show considerable promise.
Presumptive genus-level recognition of Fusarium elements in histopathol-
ogy, and possible differentiation from Aspergillus elements in some cases by
means of detecting in vivo phialide and conidium formation, is admirably summa-
rized by Liu et al. (276). Still clearer genus-specific histopathology using cross-
adsorbed fluorescent antibody reagents was developed by Kaufman et al. (433)
to distinguish Fusarium from Aspergillus and Pseudallescheria in fixed tissue
specimens. Due to close antigenic similarity, Fusarium filaments could not be
distinguished from those of another hypocrealean fungus tested, Paecilomyces
lilacinus, and even distinction from the Microascalean Pseudallescheria boydii
required the use of two reagents. Microascales are relatively closely related to
Hypocreales phylogenetically (296).
Morphological species identification in Fusarium may be relatively easy
or very difficult, depending on the species involved and on the attention paid to
newly recognized phylogenetic sibling species. The most important character by
far in most medically important species is the shape of the phialides producing
microconidia. It is important to distinguish monophialides, which have only a
single apical opening, from polyphialides, which begin as a monophialide but
rapidly grow extra toothlike extensions that give rise to additional clumps or
chains of microconidia. In some species, such as F. chlamydosporum, that rarely
cause human or animal disease, such proliferating extensions may each give rise
to only a single conidium. The phialides showing this pattern are called ‘‘poly-
blastic phialides,’’ and the resulting conidia, whether micro- or macro- in mor-
phology, are referred to by some authors as ‘‘blastoconidia’’ or ‘‘mesoconidia.’’
(See text below for F. chlamydosporum and F. incarnatum.)
The next most important character in species identification is to observe
whether any microconidia that form do so in sticky heads or chains. Chain forma-
tion is not reliable on most common medical mycology laboratory media, which
are too rich (434). F. verticillioides may form chains on PDA, but in at least
some isolates of this species as well as many isolates of F. proliferatum, the use
of special Fusarium media such as synthetic nutrient agar (435) or carnation leaf
agar (436) may be required. As mentioned below, the present author has had
400 Summerbell
good preliminary results using modified Leonian’s agar (396). The best way to
detect chains is to examine plates dry under the 10⫻ compound microscope lens,
or to use a dissecting microscope at its highest available magnification. The long
and robust chains of F. verticillioides are especially conspicuous. In the absence
of actual chain formation, the species potentially forming these structures all form
radially symmetrical microconidia with a flattened base, suitable for balancing
in chains. The conidia may be club-shaped, pyriform (pear-shaped), or napiform
(turnip-shaped) in all cases with the truncate base just mentioned.
An intuitively appealing but much more difficult character to use in identi-
fication is the shape of macroconidia. This is relatively straightforward in ususual
species such as F. dimerum, but otherwise can be very difficult to apply without
extensive experience. The common medically important species with the most
distinctive macroconidia is F. solani (i.e., from the phylogenetic point of view,
the F. solani species complex). In many isolates, these conidia may be distinc-
tively thick and blunt-ended, ruling out all such species as F. oxysporum with
more sharply pointed macroconidia. There are, however, F. solani isolates with
relatively pointed macroconidia. [See the range of shapes drawn by Gerlach and
Nirenberg (437)]. Phialide shape is far superior for making the distinction among
the less obviously distinguished members of these two species.
Also useful but relatively difficult to use is colony pigmentation. Many
Fusarium species have characteristic colors, at least in uncontaminated isolates
growing on PDA. Many begin as pale colonies, and F. oxysporum, F. solani, F.
verticillioides, and F. proliferatum may remain quite pale in some isolates. As
a multimembered species complex, F. solani is especially variable. The chestnut
red-brown colors that form in some of its subtypes need to be distinguished from
the vinaceous (red-wine-colored) and violaceous (purplish) colors that are charac-
teristic of F. oxysporum and members of F. subgenus Liseola, as well as the
carmine (vivid lipstick red) pigments that form in F. chlamydosporum and a
variety of Fusarium species not known from human infection. It cannot be over-
stressed that these characters are highly variable when different growth media
are used and as is frequently true in the identification of non-Onygenalean molds,
Sabouraud agar is particularly unsuitable. The media and conditions specified in
descriptive literature must be used to obtain interpretable results and make correct
identifications. Note that many Fusarium species undergo a dermatophyte-like
cultural degeneration, and isolates should if possible be identified before such
processes can begin. Persons organizing quality control surveys must ensure that
Fusarium isolates sent out to participants are still in a representative natural con-
dition.
Distinction of most common Fusarium species at the biomedically useful
level in which some closely related species complexes are lumped as single spe-
cies (e.g., F. solani) may be accomplished using the monograph of Nelson et al.
(438). Distinction of common medically important species at this level may be
Ascomycetes 401
aided by exoantigen testing (439). The 20⫹-year-old synoptic key to common

Fusarium species in Compendium of Soil Fungi by Domsch et al. (74) remains
remarkably effective, although the treatment of the ‘‘F. moniliforme’’ complex
in that manual must be compared with more modern concepts, and a few other
names (e.g., that of the former Fusarium semitectum, now F. incarnatum) have
changed. Higher-level morphological identification may be carried out using the
pictorial atlas published by Gerlach and Nirenberg (437). A useful key for many
species is Nirenberg’s (440) key to fusaria on European crop plants. It includes
all the repeatedly seen medically important fungi in temperate areas (the seldom-
seen F. chlamydosporum is absent), as well as most potential clinical contaminant
species in these areas. It also includes a key to the temperate and cosmopolitan
subgenera of Fusarium. The recent molecularly informed revision of morphologi-
cal distinctions in subgenus Liseola by Nirenberg and O’Donnell (441) must be
considered. In general, accurate Fusarium identification at the reference level
requires constant vigilance with regard to new literature.
The current state of the art in molecular identification of most medically
important Fusarium species and species complexes is summarized by O’Donnell
et al. (429) and O’Donnell (427). Considerably more work must be done, how-
ever, to place all the medically important Fusarium types into phylogenetically
defined clusters and species concepts. There is currently a tendency for molecular
studies less ambitious than those of O’Donnell and collaborators to be based on
a naively small representation of the relevant biodiversity in this genus, and cau-
tion must be used in interpretation of such studies.
4. Fusarium {aff. Gibberella} anthophilum (A. Braun) Wollenw.

A disseminated infection in a Japanese acute lymphocytic leukemia patient gave
rise to numerous skin lesions that were separately investigated via one biopsy
and nine surface scrapings (442). Biopsy culture was negative, but cultures from
the scrapings of six lesions gave rise to Fusarium solani, while two lesions grew
F. anthophilum. The latter fungus was depicted and described, and its identity
was stated to have been determined by Fusarium authority P. Nelson (after mis-
identification as F. oxysporum by two less specialized authorities). The authors,
however, explicitly stated that ‘‘septate hyphae with chlamydospores’’ (emphasis
added) were seen in direct microscopy of all scraping samples, while their F.
anthophilum isolate in culture was noted not to produce these structures. Indeed,
the species is not known to do so (437). F. solani may produce abundant chlam-
ydospores. The balance of probabilities in this case suggests that F. solani was
the sole etiologic agent in the skin lesions and F. anthophilum was insignificant.
(See the discussion under F. proliferatum for the distinction of F. anthophilum
from similar-looking, known medically important members of Fusarium subge-
nus Liseola.)
402 Summerbell
5. Fusarium {aff. Cosmospora} aquaeductuum (Radl. &

Rabenh.) Lagerh. Anamorph of Nectria episphaeria
(Tode: Fr.) Fr. (Fusarium Subgenus: Eupionnotes)
This species is indexed as being of medical significance by Guarro and Gené
(443) and de Hoog et al. (69) on the basis of a record of ‘‘Fusarium episphaeria’’
keratitis in a review by Pflugfelder et al. (342). The original authors of the record
[Mandelbaum, Forster et al. (444)], however, probably used this name in the
broad sense of Snyder and Hansen [see Booth (445)] to indicate F. dimerum, as
has commonly been done by several ophthalmological authors, particularly R. K.
Forster, the second author of the Pflugfelder et al. report. (See Ref. 393.) F.
aquaeductuum is not known to date from human or animal disease.
6. Fusarium {aff. Gibberella} chlamydosporum Wollenw. &

Reink. (Fusarium Subgenus: Sporotrichiella)
A non-neutropenic American woman with lymphocytic lymphoma presented
with fever and chills that transpired to be at least partially caused by a persistent
F. chlamydosporum fungemia associated with colonization of the tip of her Brov-
iac catheter (446). Catheter removal and amphotericin B cured the infection. A
neutropenic woman was found to have a limited lesion of the nasal turbinate
caused by this fungus (447). Chlamydospores consistent with the species were
found intermixed with fungal filaments in the lesion and initially raised suspicions
of phaeohyphomycosis because of their dark color and positive Masson–Fontana
staining reaction. The infection was eliminated with surgery and amphotericin
B-lipid complex.
This fungus is a soil fungus, associated with rhizospheres of many plants
as well as with various decaying plant materials, mostly in the tropics and sub-
tropics. Both reported medical cases, however, were in the New York–Washing-
ton area. F. chlamydosporum produces the mycotoxin moniliformin (448).
Description. Colonies grow on PDA 65.5 to 70 mm in 7 days. They are

floccose, and sometimes powdery with conidia. They are whitish at first, then
ochraceous to brownish to bright pink or rose. The reverse begins pinkish and
becomes carmine, wine-red to reddish-brown (437). Conidiophores begin as phi-
alides arising singly on aerial hyphae, soon forming branched clusters, and less
commonly, closely appressed sporodochial clumps. Phialides appear monophia-
lidic at first, but soon often proliferate, especially near the apex, to form polyphia-
lide-like ‘‘polyblastic phialides.’’ These structures at maturity bear multiple,
short, toothlike fertile protuberances on which they give rise to so-called meso-
conidia (412, 449) [terminology not supported by some Fusarium authorities
(415)] or blastoconidia (69, 450). In this species, these conidia are shaped for
the most part like typical Fusarium microconidia and are referred to as such by
Ascomycetes 403
Segal et al. (447). They differ from the conidia ordinarily formed on fusarial
polyphialides (as seen in F. proliferatum, e.g.) not just in their size but also
in their ontogeny; only a single conidium is formed per fertile denticle, not a
multiconidial cluster or chain. Mature polyblastic phialides, then, are mostly ir-
regularly elongate, straight, or curved, with up to 10 protuberant conidiogenous
openings, 8–18 ⫻ 2–3 µm, giving an overall spiny appearance. Sporodochial
phialides producing macroconidia are monophialidic and cylindrical with narrow
apex, 10–16 ⫻ 2–3 µm. Microconidia are strongly predominant, mostly spindle-
shaped and smooth walled. They are commonly one- or two-celled, but some
conidia with up to five cells may be formed on polyblastic phialides. Single-
celled forms are mostly (5-) 9–10 (-14) ⫻ 2–3 (-4) µm, two-celled forms up to
17 µm long. Macroconidia are formed sparsely or not at all, generally only in
sporodochia. They are hyaline and gently crescent-shaped, with a hooked and
sharply pointed apical cell and a small basal pedicel. They are mostly three-
septate but commonly up to five-septate. When three-septate they are (21-) 30–34
(-41) ⫻ (2.5-) 3–4 (-4.5) µm; when five-septate commonly to 38 µm and uncom-
monly to 47 µm. Chlamydospores are abundant, intercalary or terminal, and often
in chains or clusters. They are smooth or strongly roughened and globose or nearly
so, becoming brown and measuring 7 to 17 µm when formed singly.
For distinction from other medically important fusaria, see the discussion
under F. incarnatum below, as well as Guarro and Gené (443). Morphological
distinction from species traditionally clustered with F. chlamydosporum in Fu-
sarium subgenus Sporotrichiella is briefly summarized by Kiehn et al. (446);
molecular distinction has also been possible since the 25S ribosomal sequence
study of Logrieco et al. (451).
7. Fusarium {aff. Nectria} coeruleum (Libert) Sacc. (Common

Synonym: Fusarium solani var. coeruleum; Fusarium
subgenus: Martiella)
This strongly pigmented fungus related to F. solani was indicated as causing
black-grain mycetoma in the ankle of a Thai farmer (452). The identification was
confirmed (as F. solani var. coeruleum) by Fusarium authority C. Booth of CMI
(CABI). Recent molecular studies have cast doubt on this then-credible identifi-
cation, as molecularly confirmed F. coeruleum isolates derive exclusively from
infected potatoes (452a).
This species is primarily known as a pathogen of stored potatoes, but has
been also reported from other plant species and from nematode eggs. The other
reports from substrates other than potatoes are currently considered dubious until
similar reports are molecularly substantiated.
Colonies on PDA grow 47.5 to 52.5 mm in 7 days. They are usually thinly
or nearly appressed-felty. They are locally low-floccose, and beige, yellow-
404 Summerbell
brown, dull purple to bluish, with cream or purple, slimy sporodochial masses.
The reverse is typically strong ink blue-purple, sometimes yellow-brown to dull
violet. Conidiophores consist of single phialides arising laterally on aerial hy-
phae, or small groups of phialides formed on rudimentary branching structures,
or closely appressed sporodochial clumps (Fig. 77). Phialides produce microconi-
dia in aerial mycelium and are strictly monophialidic, long-cylindrical, and 15–
25 ⫻ 2.5–3.5 µm. Microconidia are not formed as a separate conidial morph,
but a few microconidium-like, single-celled, ‘‘subdeveloped’’ (437) conidia may
be present, always in sticky heads, and never in chains. They are hyaline, smooth-
walled, ellipsoidal to obovoidal, or sometimes slightly curved, and (8-) 10–17
(-22) ⫻ 4–5 µm. Macroconidia are overwhelmingly predominant, sometimes in
sporodochia, hyaline, ‘‘only slightly curved’’ (437), mostly three-septate, less
commonly 4 to 5-septate, relatively broad in relation to length, when three-septate
(21-) 28–45 (-50) ⫻ 4–6 µm, with little or no pedicellate character in the basal
cell, and blunt apical cells. Chlamydospores are usually abundant, smooth-walled,
terminal or intercalary, single, in chains or clustered, pale or brownish, sometimes
in macroconidia, and 7 to 10 µm.
This species is distinguished from most F. solani isolates by its heavy ink-
blue colony reverse pigmentation. Occasional F. solani isolates will produce
bluish or dark reverse pigments, but can be distinguished by their abundant and
Figure 77 Fusarium coeruleum (CBS 133.73 macroconidia, phialide, and chlamydo-

spore).
Ascomycetes 405
well-differentiated production of microconidia on PDA or specialized Fusarium

media. Although F. coeruleum has relatively long and thin phialides for a Fu-
sarium species, F. solani isolates may have phialides that are considerably longer
and thinner. F. solani phialides commonly approach 40 µm, while F. coeruleum
phialides are seldom longer than 25 µm.
8. Fusarium {aff. unknown} dimerum Penzig (in Sacc.)

[Common Synonym: Fusarium episphaeria (Tode: Fr.)
Emend Snyder & Hansen Pro Parte (i.e., Lumped with
Other Taxa Under This Name)
Fusarium Subgenus: Eupionnotes. This fungus is uncommonly but regu-
larly isolated from keratitis; cases have been documented by Zapater and collabo-
rators (453, 454) as well as by more recent investigators (455) in Argentina and
by Liesegang and Forster (393) in Florida. (See also the discussion of F. aquae-
ductuum above.) Identifications in the Zapater studies were confirmed at CBS.
Like most fusaria in eye infections, F. dimerum generally responds to pimaricin
(456). F. dimerum has also been confirmed as an agent of endocarditis in
France (457).
Fatal F. dimerum fungemia has been reported in a catheterized Slovak leu-
kemia patient who had experienced prolonged neutropenia (417). Poirot et al.
(458) reported an acute myeloblastic leukemia patient who grew F. dimerum in
several urine cultures just prior to bone marrow regeneration, but became culture-
negative with regeneration and amphotericin B treatment.
In the present author’s experience in Ontario, Canada, this is a common
contaminant producing, saprobic, noninvasive growth on wounds and ulcers. Un-
published cases of CAPD-related peritonitis are under investigation. F. dimerum
is a soil fungus also found on various plant substrates.
Description. Colonies grow on PDA 27.3 to 31.5 mm in 7 days. They
are completely slimy with conidia or produce limited amounts of aerial mycelium.
They are orange on both the surface and reverse.
Conidiophores consisting of phialides arise singly on both aerial and sub-
merged hyphae, or in small or moderately complex branched tufts (Fig. 78). Phi-
alides are monophialidic, mostly short and inflated to wine-bottle-shaped with a
narrowed apical neck. They are occasionally longer and cylindrical, and 6–32 ⫻
2.5–5 µm. Microconidia are not considered to be formed; there are, however, a
small proportion of single-celled conidia mixed with and intergrading in size and
shape with the macroconidia, mostly 7–11 ⫻ 2.0–2.8 µm. Macroconidia are
abundant, hyaline, and crescent-shaped. They are unicellular at first, but mostly
become one-septate at maturity, less commonly two- or three-septate; when one-
septate 7–30 ⫻ 2–4 µm, with pointed apical ends and minutely blunted or pedi-
cellate basal ends. Chlamydospores are uncommon to common, mostly interca-
lary, usually smooth, globose or nearly so, and 6 to 12 µm.
406 Summerbell
Figure 78 Fusarium dimerum.
Isolates from clinical settings often appear to correspond to the subtaxon

F. dimerum var. pusillum (Wollenw.) Wollenw., with a preponderance of unicel-
lular or belatedly one-septate macroconidia mostly in the 7 to 9 µm length range.
This little-recognized (437) subtaxon requires molecular investigation.
9. Fusarium equiseti (Cda.) Sacc. Anamorph of Gibberella

intricans Wollenw.
Expressed blisters from a pustular dermatitis in a dog in 1928 grew Mucor race-
mosus as well as a fungus that was investigated biomedically by Leinati (459)
and described taxonomically by Curzi (460) as Fusarium moronei Curzi. Wollen-
weber and Reinking (461) later synonymized this taxon with Fusarium scirpi
var. caudatum, which in turn was synonymized with F. equiseti by Booth (445).
The animal disease record was indexed by Austwick (426) as ‘‘one of the earliest
records of Fusarium infection.’’ The case report, however, did not include direct
Ascomycetes 407
microscopy of the pustule contents, and the concurrently isolated M. racemosus,

assuming it was correctly identified, is probably nonpathogenic (69). Moreover,
F. equiseti isolates studied so far have a maximum growth temperature of 28°C
(74), and there have been no subsequent valid records of zoopathogenesis by this
fungus. This, then, appears to be a spurious record based on contamination.
10. Fusarium fujikoroi Nirenberg Anamorph of Gibberella

fujikoroi (Sawada) Wollenw. (Fusarium Subgenus: Liseola)
This organism was reported without mycological substantiation under the teleo-
morph name Gibberella fujikoroi from an etiologically well-documented North
Carolina keratitis case by Anderson et al. (92). It seems unlikely that a teleomorph
was actually observed for this heterothallic species, in which laborious mating
with tester isolates must be done to make this observation in culture. Possibly a
member of Fusarium subgenus Liseola was isolated and linked to G. fujikoroi
(used in the broad sense for the related complex of then mostly unnamed species)
via literature descriptions or by an individual with phytopathological experience.
(See the discussion of Aspergillus versicolor and Penicillium spinulosum case
records above for further analysis of the eccentric but by no means wholly dis-
creditable mycology in the Anderson et al. publication.)
F. fujikoroi is a rice pathogen known only from Asia and Australia (437).
In culture it is scarcely distinguishable from F. proliferatum except that very few
polyphialides are formed, and conidial chains are short. Its production of the
mycotoxin and plant hormone gibberellic acid is distinctively high compared to
that of other members of subgenus Liseola (437).
Distinction of F. fujikoroi from numerous newly described and minutely
morphologically distinguished species in the ‘‘Gibberella fujikoroi species com-
plex,’’ most not known from mammalian pathogenesis, is provided by Nirenberg
and O’Donnell (441). Molecular characterization is coordinately given by
O’Donnell et al. (429).
11. Fusarium {aff. Gibberella} incarnatum (Rob.) Sacc.

(Common Synonyms: F. Semitectum, F. pallidoroseum;
Fusarium Subgenus: Arthrosporiella)
A Brazilian man suffered fatal endocarditis after a newly implanted metallic pros-
thetic heart valve became heavily colonized with F. incarnatum (reported as F.
pallidoroseum) (462). The identity of the organism was well documented. An
earlier report of the same organism (as F. semitectum) from an electroshock-
related burn wound in Thailand (463) described ‘‘dark-brown’’ filaments in tissue
stained with hematoxylin and eosin. The discord between this finding and the
general finding that this fungus produces only hyaline hyphae was queried by
McGinnis et al. (462). F. incarnatum may produce short chains of pale to moder-
408 Summerbell
ately brownish chlamydospores, and these may possibly be seen in tissue (see
notes on F. chlamydosporum above), but the repeated and exclusive use of dark
brown to describe the structures seen makes it appear more likely that the burn
was infected by more than one species or that F. incarnatum occurred as a surface
contaminant or colonizer and overgrew an invasive melanized fungus in culture.
Another case reported in the same paper featured electrical burns colonized by
Curvularia lunata, a melanized fungus. Rush-Munro et al. (464) depicted fila-
ments attributed to F. incarnatum in intertriginous toe skin, but gave no support-
ing etiologic or mycological details.
F. incarnatum is a mostly tropical and subtropical soil fungus causing rots
in stored crops with high water content, particularly potatoes and fruits. It pro-
duces moniliformin as well as mycotoxins in the zearalenone class (448).
Description. Colonies grow on PDA 65.5 to 70 mm in 7 days. They are

floccose and sometimes powdery with conidia. They are dirty-white to cinnamon-
brown above, very rarely with small (⬍1 mm) and pale brown sclerotial bodies.
The colony reverse begins pale to pinkish or peach, soon becoming ochraceous
to cinnamon-brown. [Gerlach and Nirenberg (437) stress ‘‘red, violet or bluish
pigment lacking’’.] Conidiophores beginning as phialides arise singly on aerial
hyphae, soon forming branched clusters (Fig. 79). Phialides are mostly monophi-
alidic at first, but soon often proliferate sympodially, especially near the apex,
to give rise to new phialidic extensions. In addition, polyphialidelike ‘‘polyblastic
phialides’’ with multiple, short, toothlike fertile protuberances are formed, giving
rise to so-called mesoconidia (412, 449) or blastoconidia (69). These conidia in
F. incarnatum (unlike those in F. chlamydosporum; see above) are shaped for
the most part like typical Fusarium macroconidia and will be referred to here as
such. They differ from normal macroconidia by lacking the foot cell typically
seen in Fusarium. In comparison with the microconidia ordinarily formed on
fusarial polyphialides (as seen in F. proliferatum, e.g.), they differ not just in
their size but also in their ontogeny; only a single conidium is formed per fertile
denticle, not a multiconidial cluster or chain. Mature polyblastic phialides, then,
are mostly irregular, with two to four toothlike conidiogenous openings, 8–25 ⫻
2.5–5 µm. Microconidia are relatively uncommon, intergrading with macroco-
nidia. They are mostly spindle-shaped or slightly curved, and smooth-walled.
They are usually one-celled, but some two-celled conidia also present. The single-
celled forms are mostly (4-) 7–12 (-17) ⫻ 2–3.5 (-4) µm. Macroconidia are
predominant, mostly scattered in the aerial mycelium. They are hyaline and
nearly straight in the midsection, especially in the inner wall of curvature, with a
hooked and sharply pointed apical cell and a thinly truncate-conical, apedicellate
basal cell. They are mostly three-septate, with an admixture of commonly four
to five- (rarely to seven-) septate conidia; when three-septate (13-) 20–40 (-50)
⫻ (2.5-) 3.5–5 (-6.5) µm. Chlamydospores are sparse, mostly intercalary, and
smooth. They are globose or nearly so, become brown, and are 6 to 12 µm.
Ascomycetes 409
Figure 79 Fusarium incarnatum CBS 132.73 polyphialide.
This species can be distinguished from most other Fusarium species in-
volved in human disease by its macroconidia that arise as solitary blastoconidia
on individual denticles of polyblastic phialides. It also completely lacks the purple
colors of Fusarium subgenera Liseola and Elegans (e.g., F. proliferatum, F. oxy-
sporum) and the red colors of another rarely etiologic species with polyblastic
phialides, F. chlamydosporum. At least in fresh isolates, it forms macroconidia
commonly on PDA, unlike F. chlamydosporum, in which macroconidia are
scarce and strictly associated with sporodochia.
12. Fusarium {aff. unknown} lacertarum Subrahmanyam

(Published in Orthographic Error as Fusarium laceratum;
Fusarium Subgenus: Compared in Its Original Description
with Members of F. subgenus Discolor; Unlikely to Be
a true Fusarium)
This species, which needs further study to determine its correct taxonomic posi-
tion, has only been recorded from parasitized lizards. It has Fusarium-like co-
nidia, but the existing descriptions of its conidiogenesis suggest it may belong
to another genus. The species description by Subrahmanyam (465) was based on
410 Summerbell
an isolate from an infected ‘‘house lizard’’ from Poona, India. The animal was
profusely colonized by white mycelium, which bore copious conidia. This finding
appeared belatedly to confirm the detailed description of a similar fungus by
Blanchard (466) from equally profusely colonized skin cankers on a green lizard
(Lacerta viridis) of Italian provenance. Blanchard compared the fungus to a de-
scription of Selenosporium urticearum Corda, now considered a synonym of Fu-
sarium lateritium [F. lateritium var. mori fide Booth (445)]. Austwick (426) as-
cribed Blanchard’s record to this species. He was evidently unaware that
Blanchard’s organism had subsequently been named Fusarium cuticola (Blanch-
ard) Guégen (467, 468) and had also been recorded in additional lizards, including
a chameleon. In any case, the now long-lost isolate described by Blanchard has
no resemblance to the tree-parasitizing F. lateritium, but is similar to F. lacer-
tarum in several distinctive respects; namely, yellow colony pigmentation, a rapid
growth rate on agar medium, sharply pointed macroconidia mostly under 30 µm
with one to three indistinct (465) or extremely thin (466) septa, and most notably
the production of most conidia on short, lateral protuberances rather than well-
defined phialides, as well as the apparent production of at least some conidia by
direct blastoconidiation from the sides of hyphae (possibly from adelophialides or
short Microdochium-like sympodial polyblastic initials). The description below is
after Subrahmanyam (465), with direct quotes indicated.
Description. Colonies on potato sucrose agar at 28°C grow 70 mm in 3
days, with thinly floccose, white mycelium. At first they are pale on the surface
and reverse, but after 30 days become buff above and pale lemon-yellow below
or near the colony center. Conidiophores are ‘‘short, lateral, branched or un-
branched’’ up to 7 µm long, and disposed laterally along the hyphae. Discrete
‘‘phialides’’ are rarely present, 2.5–4 ⫻ 1–1.5 µm. Conidia are found mostly
with a nearly straight or gently curved midregion and curved and sharply pointed
apical and basal regions. They are hyaline, smooth-walled, zero- to four-septate,
and 6.6–30.8 ⫻ 2.2–3.3 µm, with notably thin or obscure septa and with basal
cell lacking a pedicel. A small proportion of small, clavate to broadly pyriform
no to one-septate conidia are also produced. Chlamydospores are common, termi-
nal or intercalary, solitary, catenulate, or ‘‘in clumps.’’ They are hyaline, smooth
or rarely roughened, and 6.6–18.5 ⫻ 5.5–18.7 µm.
Blanchard (466) depicts mostly two to three-septate conidia but his draw-
ings also include one five-septate conidium. He gives 25 µm as the maximal
conidial length seen.
13. Fusarium {aff. Gibberella} moniliforme Sheldon

(See F. verticillioides Below)
Fusarium Subgenus: Liseola. Discussion of why the earlier epithet, com-
bined as F. verticillioides, is preferred to F. moniliforme is given by Gams (469).
Ascomycetes 411
14. Fusarium {aff. Gibberella} napiforme Marasas, Nelson &

Rabie (Fusarium Subgenus: Liseola (Referred to by Some
Authors as F. Subgenus Dlaminia, an Unacceptably
Artificial, Paraphyletic Group Created to Encompass
Organisms with Affinities to F. Subgenus Liseola but
Possessing Chlamydospores)
This species has been verified from a disseminated infection in a leukemia patient
(470). F. napiforme is mainly known from millet and sorghum from Africa, but
has also been found in warm grassland soils elsewhere (471) (Fig. 80).
Colonies grow on PDA 46 to 82 mm in 7 days. They are floccose and
whitish above, with pale to deep purple reverse. Conidiophores consisting of
single phialides arise laterally on aerial hyphae, or small groups of phialides
formed on rudimentary branching structures, or closely appressed sporodochial
clumps. Phialides are monophialidic, subulate (tapered, awl shaped), and 14–31
⫻ 1.5–3 µm. Microconidia are formed in short chains or sticky heads. They
are hyaline, smooth-walled, and always with truncate bases but otherwise very
variable, with a proportion of conidia obovoid to fusoid, no to one-(less com-
monly to three-) septate and 6–23 ⫻ 1.5–4.5 µm. Others are lemon-shaped or
the characteristic napiform (turnip or rutabaga-shaped), no to one-septate, and
9–15 ⫻ 6.5–10.5 µm.
Macroconidia are produced from scattered monophialides or from sporo-
Figure 80 Fusarium napiforme CBS 673.94, phialides and microconidia.

412 Summerbell
Figure 81 Fusarium napiforme CBS 673.94, chlamydospores.
dochia (in the latter case forming orange conidial masses). They are hyaline,
falcate, typically with the inner wall nearly straight and the outer wall more dis-
tinctly curved, mostly five-septate with a lower proportion three- or four-septate
and 29–90 ⫻ 3.5–5.0 µm, with pedicellate basal cells. Chlamydospores (Fig. 81)
are common. They are smooth-walled, hyaline to pale brown, and 5–12 ⫻ 4–8 µm.
This fungus differs from the more common F. verticillioides by producing
well-developed chlamydospores, as well as by producing a proportion of inflated,
napiform, or limoniform microconidia. F. proliferatum may produce inflated mi-
croconidia but never produces chlamydospores. Several other related and similar
chlamydospore-producing species, most notably F. oxysporum, never produce
microconidia in chains, and may have microconidia with different shapes (e.g.,
allantoid in F. oxysporum).
15. Fusarium nygamai Burgess & Trimboli Anamorph of

Gibberella nygamai Klaasen & Nelson [Fusarium
Subgenus: Liseola (Referred to by Some Authors as
F. Subgenus Dlaminia, an Unacceptably Artificial,
Paraphyletic Group Created to Encompass Organisms
with Affinities to F. Subgenus Liseola but Possessing
Chlamydospores)]
This species caused disseminated infection in a granulocytopenic lymphoma pa-
tient who had recently traveled to Egypt (472). The fungus in nature appears to
Ascomycetes 413
be pantropical in distribution, and is associated with plant roots, particularly of

sorghum, maize, and beans, as well as with sorghum grain heads (473).
Description. Colonies grow on PDA 58 to 82 mm in 7 days. They are
floccose to powdery, whitish above at first, later dull violet with a greyish-orange
or dark violet central conidial mass with pale to deep purple reverse. Conidio-
phores consisting of single phialides arise laterally on aerial hyphae, or small
groups of phialides are formed on rudimentary branching structures or closely
appressed sporodochial clumps (Fig. 82). Phialides are usually monophialidic
and subulate (tapered, awl shaped) and 13–40 ⫻ 1–3 µm, with polyphialides
also uncommonly present. Microconidia form in sticky heads or—especially on
specialized media such as carnation leaf agar or synthetic nutrient agar—in a
mixture of heads and short (usually 10 or fewer conidia) chains. They are hyaline,
smooth-walled, obovoid to fusoid, no- to one-septate, and 5–27 ⫻ 2–3 µm, with
truncate bases. Macroconidia produced mainly from sporodochia, forming
greyish-orange, slimy masses, appear under the microscope as hyaline, nearly
straight to falcate, mostly three-septate with a lower proportion one-, two-, or
four-septate. They are 25–54 ⫻ 2–5 µm, with pedicellate basal cells. Chlamydo-
spores are usually common but uncommon in some isolates. They are often in
chains or clumps, smooth-walled, and hyaline to pale yellow-brown (Fig. 83).
This fungus is distinguished from the superficially similar F. oxysporum
by the sometimes difficult to detect production of conidia in chains, as well as
Figure 82 Fusarium nygamai CBS 675.94, phialides and microconidia in heads and
chains.
414 Summerbell
Figure 83 Fusarium nygamai CBS 675.94, chlamydospore.
the production of predominantly catenulate (in chains) or clumped chlamydo-

spores, as opposed to the single or paired chlamydospores generally seen in F.
oxysporum. Detecting radially symmetrical microconidia with conspicuous trun-
cate bases is often an indication that a fungus other than F. oxysporum should
be considered. F. verticillioides and F. proliferatum are normally distinguished
by their lack of chlamydospores (but see comment about thickened cells in de-
scription of F. verticillioides below). F. napiforme differs by its high proportion
of swollen, napiform microconidia.
16. Fusarium {aff. Gibberella} oxysporum Schlecht. (Fusarium

subgenus: Elegans)
F. oxysporum shows the ‘‘general pattern of involvement by major opportunistic
Fusarium species’’ described in the introductory section for Fusarium above.
Case literature for this well-established opportunist has been reviewed previously
(see same section) and will not be reviewed in detail here. De Hoog et al. (69)
have recently given a species-specific synopsis. In general, this fungus is a major
agent of disseminated and cutaneous infection in the immunocompromised—
especially the neutropenic—patient. It also causes infections related to major
barrier breaks, such as CAPD peritonitis (474). In a rare case of AIDS-related
Fusarium infection, a port-a-cath implanted for cytomegalovirus therapy was the
Ascomycetes 415
locus of entry of a disseminated F. oxysporum infection in an HIV patient (475).

The infection responded to liposomal amphotericin B. An apparently nonimmuno-
compromised patient who had adult respiratory distress syndrome and was receiv-
ing extracorporal membrane oxygenation died of a disseminated F. oxysporum
infection that was first detected as a positive bronchial lavage specimen (476).
There are some distinctive features about the pathogenic record of F. oxy-
sporum. In keratitis, it is frequently seen in some surveys (420), but is generally
significantly less common than F. solani (393, 456). It is correspondingly less
common than F. solani in exogenous endophthalmitis (412). It may be less viru-
lent and therefore less likely to spread spontaneously from keratitis to the pos-
terior chamber and the vitreous cavity (342). Rosa et al. (30) reported that F.
oxysporum became more common than F. solani in Florida keratitis and endoph-
thalmitis in the period from 1982 to 1992, and also caused most cases associated
with the use of soft contact lenses. The large numbers of cases reported for F.
oxysporum statistically overwhelm the etiologic confirmation shortcomings of
this paper as discussed above under Aspergillus glaucus (e.g., potentially as few
as one-third of the cases confirmed by direct microscopy). No identification crite-
ria were given for the species, but the distinction among the major Fusarium
species may have been considered routine after many years of investigation in
Florida. Chodosh et al. (477) recently reported a keratitis ascribed to F. oxy-
sporum, apparently contracted during routine ophthalmic examination, respond-
ing to tobramycin in vivo while the fungal isolate also showed high susceptibility
in vitro. Natamycin therapy was begun as soon as a fungus was grown, but the eye
had already substantially improved with antibacterials. Corneal scrapings showed
no fungal elements, making it quite possible that the fungal isolation was purely
fortuitous; however, no bacteria were obtained in culture. The identification was
again undocumented. Ocular infection by F. oxysporum has also been recorded
in snakes (426).
F. oxysporum is by far the most commonly reported Fusarium from ony-
chomycosis, and shares a distinctive niche with a few other fungi, most notably
Trichophyton mentagrophytes and members of Acremonium subgenus Acremo-
nium, in the causation of superficial white onychomycosis (49, 50, 464). Another
distinctive regularity of F. oxysporum onychomycosis is that the nails of the hand
are often affected, as in the first well-substantiated case reported by Ritchie and
Pinkerton (478). Regular occurrence of fingernail infections, including superfi-
cial, distal–subungual, and paronychial onychomycoses, was documented by
Gianni et al. (479). An unusual type of nail infection, proximal subungual ony-
chomycosis, was well documented from both finger- and toenails in cases in
which paronychia was also seen (480, 481). Four pedal and three digital onycho-
mycoses, as well as two cases of pedal intertrigo, were studied by Romano et
al. (482). All cases were meticulously confirmed as Fusarium infections. Species
identification criteria were given in a later paper (483). In the intertrigo cases,
416 Summerbell
in response to earlier demonstrations that Fusarium species could grow saprobi-

cally on wounds, ulcers, and other moist lesions of the foot, including tinea pedis
lesions, without penetrating tissue (422, 423), the authors made biopsy slides that
unequivocally demonstrated fungal tissue penetration. Also, they repeated culture
and direct microscopy four times at 20-day intervals to probabilistically exclude
cryptic dermatophytosis.
Very rarely, F. oxysporum may cause an extensive subcutaneous infection
in an immunocompetent patient (e.g., a case in which it invaded an arterial leg
ulcer with invasion of nearby blood vessels). In this case it failed to respond to
amphotericin B and local debridement, and the leg was ultimately amputated
(484). Some tissue penetration in less severe ulcer cases was seen by English
(485). Landau et al. (486) described a severe foot ulcer that had extended over
a 1-year period in a 69-year-old man and that had a heavy superficial growth of
F. oxysporum with copious filaments in direct smears but no detectable tissue
penetration in biopsies. Despite the lack of tissue penetration, treatment with
ketoconazole appeared to cause rapid resolution of the lesion. Interestingly, F.
oxysporum is almost completely resistant to this drug in vitro (414). Extensive,
purely cutaneous infections also occur in immunocompromised patients whose
immunodeficiencies differ from those seen in the leukemia and aplastic anemia
patients most likely to experience Fusarium infection. A 16-year-old Sri Lankan
girl, thought likely to have an uncharacterized immunodeficiency, suffered a life-
long syndrome characterized by superficially chromoblastomycosis-like areas of
subcutaneous Fusarium infection on her arms (487). The tissue was heavily in-
vested with hyphae and showed microabscess formation and a chronic granulo-
matous reaction. The identification of F. oxysporum was confirmed by L. Ajello
of CDC.
F. oxysporum has a widespread distribution in soils and has many geneti-
cally differentiated forms causing wilt, damping off, and other diseases in various
plants. Thrane (448) states that it produces moniliform and a variety of other
secondary metabolites, although many isolates are not discernibly toxigenic.
Description. Colonies on PDA grow 65.5 to 70 mm in 7 days. They are

felty to floccose, not infrequently bald and wet with bacterial contamination in
primary isolates. They are whitish above or with a purple tinge, deep vinaceous
to violet in heavily bacterially affected isolates, sometimes spotted with orange
sporodochia, and very rarely (in isolates seen clinically) with small (⬍1 mm), cauli-
flowerlike, pale brown to bluish sclerotial bodies. The colony reverse is usually pale
vinaceous to deep violet. Conidiophores arise singly on both aerial and submerged
hyphae, in small branched tufts, or uncommonly, in sporodochial clumps (Fig. 84).
Phialides are monophialidic, essentially flask-shaped, cylindrical or wine-bottle-
shaped with a narrowed apical neck, but often somewhat inflated near the base and
sometimes gently curved. They are rather short, 8–14 ⫻ 2.5–3.0 µm (10 to 25
µm long in sporodochia), and sometimes reduced to short, adelophialidic fertile
Ascomycetes 417
Figure 84 Fusarium oxysporum. From Laboratory Handbook of Dermatophytes, by J.

Kane et al., 1997, Star Publishing Company, Belmont, CA (see Ref. 89). Used with per-
mission.
openings on the sides of hyphae in submerged mycelium. Microconidia are in sticky

heads, never chains. They vary from ellipsoidal to cylindrical, to slightly curved
to allantoid (sausage-shaped) to reniform (kidney-shaped). They are hyaline and
smooth-walled. They are usually one-celled but some two-celled conidia are also
present. Single-celled forms are mostly 5–9 (-13) ⫻ 2.4–3.5 µm. Macroconidia
are commonly formed, in sporodochia or in ordinary aerial mycelium. They are
hyaline, nearly straight in the midsection or more often moderately curved, mostly
three-septate, less commonly 4- to 5 (-7)-septate; when three-septate (18-) 27–42
(-54) ⫻ 3–5 µm. They have pedicellate basal cells and pointed, often somewhat
hooked apical cells. Chlamydospores are commonly formed, often delayed until
after 7 days, and terminal or intercalary. They are usually smooth, globose or nearly
so, and 7 to 11 µm.
This variable species can easily be confused with opportunistic members
of Fusarium subgenus Liseola, especially F. nygamai, F. napiforme, and imma-
ture F. proliferatum, as well as a host of species not known from human or
animal infection. The distinction should not be attempted with Sabouraud agar.
418 Summerbell
Specialized Fusarium media such as synthetic nutrient agar and carnation leaf
agar are optimal. PDA is less so but serviceable in cases of confirmed infection.
Modified Leonian’s agar seems to work well for the present author but has not
been subjected to formal comparison. The chlamydospores of F. oxysporum,
when suitably promptly formed, can distinguish it from immature F. prolifera-
tum. Examination of aerial mycelium under the dissecting microscope usually
reveals at least some conidial chains in F. proliferatum, at least on specialized
Fusarium media and Leonian’s. F. proliferatum isolates may sometimes form
polyphialides rapidly and be easily distinguishable. Some isolates, however, have
only sparse polyphialides until after approximately 10 days. The clavate shape
of most F. proliferatum microconidia strongly suggests this species even in iso-
lates in which polyphialides and chains are not immediately seen, and further
investigation is necessary in all F. oxysporum-like strains in which clavate micro-
conidia with truncate bases predominate. This symmetrical shape with a flat base
suggests adaptation to formation of the carefully balanced conidial chains that
are typical in many species of the subgenus Liseola. The kidney- to sausage-
shaped microconidia commonly seen in F. oxysporum would not be physically
capable of forming chains; however, some Liseola members may form a few
curving conidia from submerged mycelium. (See the additional discussion under
F. nygamai for distinction from that species, which so far is only known from
tropical areas. For distinction from F. solani, see that species.)
17. Fusarium proliferatum (Mats.) Nirenberg ex Gerlach &

Nirenberg, Anamorph of an Unnamed Gibberella Species
[Mating Population D of the G. fujikoroi complex (488),
Fusarium Subgenus: Liseola]
The involvement of this fungus in human disease is primarily known from three
disseminated infections in leukemia patients (489–491), two of which were suc-
cessfully cured (490, 491). Based on several unpublished cases seen by the pres-
ent author in Ontario, where this fungus was a predominant cause of opportunistic
mycosis in connection with neutropenia, it probably shares the ‘‘general pattern
of involvement by major opportunistic Fusarium species’’ described above in
the introductory section for Fusarium. Isolates with sparse or delayed formation
of polyphialides could easily be misidentified as F. oxysporum, and some of the
medical history of F. proliferatum may be concealed under this name.
In nature, F. proliferatum has been frequently isolated from corn (Zea
mays), sorghum, wheat, figs, diverse other plant substrates, various insects, and
soil (429). It produces moniliformin as a secondary metabolite (448), as well as
fumonisins (492).
thinly floccose to raggedly tufted. They are whitish above, sometimes becoming
dull violet, with pale to deep vinaceous reverse. Conidiophores consisting of
Ascomycetes 419
Figure 85 Fusarium proliferatum polyphialide and monophialide.
phialides arise singly on aerial hyphae, in small branched tufts, or in more elabo-
rate antlerlike branching structures (Fig. 85). Uncommonly, sporodochial clumps
may appear, especially in cultures over 14 days old. Phialides are monophialidic
and candle-shaped at first but after some days producing spinelike lateral prolifer-
ations, usually from just beneath the apex, giving rise to asymmetrically forking
phialides, occasionally producing further lateral proliferations, giving an overall
antlerlike appearance, 11–32 ⫻ 2.3–3.5 (-4.2) µm. Microconidia are in sticky
heads or long chains, with the latter most abundantly formed on specialized media
such as carnation leaf agar or synthetic nutrient agar (Fig. 86). They are hyaline,
smooth-walled, variable in shape, mostly clavate with a flattened base but inter-
mixed with inflated, pyriform to globose-apiculate forms, usually aseptate, and
mostly 7–9 (-11 when inflated) ⫻ 2.2–3.2 (-7.7 or occasionally as wide as 12.5
when inflated) µm. Macroconidia are often formed only after extended culture
or not at all, sometimes in sporodochia, hyaline, nearly straight to falcate, mostly
420 Summerbell
Figure 86 Fusarium proliferatum conidia in chains as seen in tape mount. (Swollen

conidiophore base seen is not typical.)
three- or five-septate, (19-) 30–58 (-79) ⫻ 2.6–5 µm, with pedicellate basal cells.
Chlamydospores are absent.
Distinction of F. proliferatum from F. oxysporum can be achieved by show-
ing polyphialides, at least some conidial chains, mainly clavate microconidia with
a truncate base (i.e., blackjack- or cosh-shaped), often mixed with some pyriform
conidia, and an absence of chlamydospores. (For additional details, see the de-
scription of F. oxysporum.)
F. verticillioides is readily distinguished by its lack of polyphialides. F.
anthophilum produces polyphialides but differs from F. proliferatum by produc-
ing only sticky heads, never chains, of mostly broadly pyriform microconidia,
especially in early growth on media with relatively low nutrient content. Later mi-
croconidia may be almost cylindrical to allantoid (sausage-shaped) (437). There
are a number of Fusarium species specific to common household plant sub-
Ascomycetes 421
strates [e.g., F. lactis Pirotta & Riboni from figs and F. phyllophilum Nirenb.
& O’Donn. (⫽F. proliferatum var. minus Nirenb.) from potted Sansevieria
(‘‘good luck plant’’) and Dracaena] that strongly resemble the more ecologically
catholic F. proliferatum. Causation of opportunistic infection by such fungi is
not recorded. With patience and special media, these species can be distinguished
morphologically using the key provided by Nirenberg and O’Donnell (441); oth-
erwise, they may be identified by β-tubulin gene sequencing as noted by O’Don-
nell et al. (429). As an example of the types of morphological distinctions made,
here are Nirenberg and O’Donnell’s summaries of salient characters distinguish-
ing F. proliferatum and F. lactis: F. proliferatum ‘‘produces clavate conidia
mainly in long, linear, crowded chains from mono- and polyphialides in the dark’’
on synthetic nutrient agar, while F. lactis ‘‘produces obovoid conidia in zig-
zaglike, short (⬍15 conidia) to medium (15–30 conidia), crowded chains, mainly
on polyphialides.’’ F. phyllophilum, formerly called F. proliferatum var. minus,
differs from F. proliferatum by producing ‘‘short (⬍15 conidia) linear conidial
chains formed abundantly only in the dark.’’ The conidial chains of F. prolifera-
tum in these conditions on synthetic nutrient agar comprise more than 30 conidia.
18. Fusarium sacchari (Butler) W. Gams Anamorph of an

Unnamed Gibberella Species [Mating Population B of the
G. fujikoroi Complex (488); Fusarium Subgenus: Liseola]
A keratitis was ascribed to this fungus without etiologic or mycological substanti-
ation by Zapater (456). It is unlikely that this experienced investigator of mycotic
keratitis was incorrect about the etiology, but since F. sacchari is in a complex
of species that are particularly difficult to identify correctly, mycological substan-
tiation is necessary for the record to be accepted.
Guarro et al. (493) reported a case of fungemia caused by F. sacchari in a
nonneutropenic Brazilian renal transplant patient. Both etiology and identification
were well documented. A small dose of amphotericin B was sufficient to cure
the infection, which had no obvious portal of entry in the uncatheterized patient.
F. sacchari in nature is commonly isolated from a variety of tropical plants,
usually sugar cane and banana, but also such other diverse plants as Cattleya
orchids and sorghum (429). It produces moniliformin as a secondary metabolite.
floccose with abundant hyphal strands. They are whitish above, sometimes be-
coming dark vinaceous, with yellowish or ochre to deep vinaceous reverse. Co-
nidiophores consisting initially of phialides arise singly or in small, branched
groups along aerial hyphal strands, soon forming more elaborate antlerlike
branching structures (Fig. 87). Phialides are monophialidic and candle-shaped at
first, but rapidly produce spinelike lateral proliferations, usually from just beneath
the apex or just above the base, giving rise to asymmetrically bifurcate or trifur-
422 Summerbell
Figure 87 Fusarium sacchari CBS 223.76 polyphialide.
cate phialides and often giving rise to additional proliferations giving an overall
antlerlike appearance, 17–30 ⫻ 2.5–3.8 µm. Microconidia are in sticky heads,
never in chains. They are hyaline, smooth-walled, ellipsoidal, ovoid, or allantoid
(curved, sausage-shaped), mostly aseptate but occasionally two-celled, and
mostly (4-) 7–11 (-18) ⫻ 2–3 µm. Macroconidia are often formed only after
near-ultraviolet light stimulation or not at all. They are hyaline, nearly straight to
falcate, mostly three-septate, and (24-) 33–43 (-52) ⫻ 3.3–3.5 µm, with slightly
beaked, sharply pointed apical cells and pedicellate basal cells. Chlamydospores
are absent. (For distinction from other taxa, see discussion under F. subglutinans.)
19. Fusarium solani (Mart.) Sacc. Anamorph of Nectria

haematococca Berk. & Br. (Fusarium Subgenus: Martiella)
F. solani shows the ‘‘general pattern of involvement by major opportunistic Fu-
sarium species’’ described in the introductory section for Fusarium above. Case
Ascomycetes 423
literature for this well-established opportunist has been reviewed previously (see
same section) and will not be reviewed in detail here. De Hoog et al. (69) have
recently given a species-specific synopsis.
Along with F. oxysporum, this fungus is one of the most common fusarial
agents of disseminated and cutaneous infection in the immunocompromised—
especially the neutropenic—patient.
One very distinctive feature of F. solani’s biology is its high importance
worldwide as an agent of keratitis and as a major cause of human blindness in
tropical areas. Most surveys show the incidence of F. solani keratitis to be far
more frequent than keratitis caused by other fusaria; for example, in Miami,
Florida [11 identified F. solani: one F. oxysporum (229); 76 F. solani: six other
identified Fusarium isolates (393)], in Enugu, Nigeria [12 F. solani: no other
fusaria (230)], and in Buenos Aires, Argentina [20 F. solani: nine other identified
fusaria (456)]. Affected persons often live in rural areas (456). Trauma involving
vegetative matter is the normal predisposing factor. Use of soft contact lenses
may also predispose significantly, but can be controlled by good hygiene and use
of overnight storage fluids containing polyhexamide biguanide, which inhibits
F. solani (494). A dramatic contact lens-related case recently reported from an
immunocompetent patient featured keratitis that initially required penetrating ker-
atoplasty, then progressed to endophthalmitis successfully treated with amphoter-
icin B lipid complex (495). Filaments were not seen in corneal scrapings but
were seen in in vivo confocal microscopy of the cornea and confirmed later in
an anterior chamber tap. Recently an epidemiological study by Mselle (496) in
Dar es Salaam, Tanzania, showed that HIV infection is now a powerful predispos-
ing factor in Africa, with 81.2% of studied fungal keratitis patients and only
33% of nonfungal keratitis patients being HIV⫹. F. solani is attested (without
identification characters) to have caused 75% of the total fungal keratitis seen.
In leukemia patients, endogenous endophthalmitis caused by F. solani may de-
velop as a sole disease manifestation or as part of a dissemination (497). There
are a small number of cases in which persons with no or slight predisposing
factors acquired F. solani endophthalmitis. The origin and portal of entry of inoc-
ulum in such cases is uncertain (497). It appears likely that the F. solani isolates
involved in oculomycosis will ultimately be ascribed to only a limited number of
the biological entities in this species complex. (See taxonomic discussion below
description.) Jones et al. (229) showed that 16 isolates of this species from infected
eyes in Miami, San Francisco, and Singapore were morphologically similar, grew
well at 37°C, and survived at 40°C. Four comparison isolates of plant-pathogenic
F. solani grew poorly at 37°C. Their survival success at 40°C is not recorded except
in a general comment that most plant-pathogenic fusaria of the various species
tested did not survive. English (485) similarly found an F. solani isolate from kerati-
tis significantly more thermotolerant than those from leg ulcers or plants.
Some additional connections between F. solani and HIV-related syndromes
have been noted. A female AIDS patient with late-stage non-Hodgkins malignant
424 Summerbell
lymphoma was well demonstrated to have a soft palatal ulceration caused by F.

solani (498). The identity of the isolate was determined by C. de Bièvre and C.
Hennequin of Institut Pasteur.
F. solani is sufficiently virulent to rarely cause mycetoma and other subcu-
taneous infection (possibly prodromal mycetoma) subsequent to dermal trauma
in otherwise healthy patients. A mycetoma from South America was reported by
Luque et al. (499), while an erythematous, indurated lesion with copious fungal
filaments developed 14 days after surgical removal of a stingray barb from the
hand of a diver (500). According to Austwick (426), the U.K. National Collection
of Pathogenic Fungi held two F. solani isolates from confirmed mycetoma as of
1984, as well as one F. oxysporum isolate. Three other relatively recent case
reports involving F. solani mycetoma are reviewed by Guarro and Gené (412).
A case of intertrigo in an otherwise healthy male, a very recent immigrant
from Senegal to Italy, was well linked by Romano et al. (483) to F. solani by
repeated antifungal culture and biopsy examination. Similar cases of intertrigo
linked by the same authors to F. oxysporum, discussed above, were also in Afri-
can patients. A nearly identical, well-confirmed case reported from France (411)
concerned a patient whose travel history was not discussed, but who was de-
scribed as a ‘‘practicing Muslim . . . who washes his feet 5 times per day (prior to)
his prayers.’’ (See further discussion of this matter above under Cylindrocarpon
lichenicola.) Both the Italian and the French cases failed to respond to terbinafine
therapy; the isolate in the latter case showed resistance in vitro to all other topical
and systemic drugs considered.
In veterinary medicine, F. solani is particularly noted for causing cutaneous
infection in turtles, particularly in young turtles and their eggshells (501). The
genetic type causing one such infection was markedly different from a selection
of culture collection isolates from various sources, as gauged by random ampli-
fied polymorphic DNA (RAPD) analysis (502). Given the diversity of F. solani
as outlined below, however, this is perhaps to be expected. Outbreaks of infection
caused by F. solani have been observed in Australian crocodile farms (503, 504).
A dramatic macular skin infection, authoritatively linked to F. solani, was de-
scribed in sea lions (Zalophus californianus) and grey seals (Halichoerus grypus)
held in chlorinated freshwater pools (505). The infections responded poorly to
various antifungals (all retrospectively well known to be ineffective against F.
solani in vitro) and were best controlled by manipulating environmental condi-
tions. The identifications in this study were credited to Fusarium authority C.
Booth of CMI (CABI). A report by Jacobson (506) of necrotizing skin lesions
caused by F. solani in a caged Burmese python is etiologically well demonstrated
but does not substantiate the fungal identification.
In nature, F. solani is well known from various soils and plant associations.
Like F. oxysporum, it has numerous forms associated with root or other dis-
eases of various plants. It is also well known from ponds, rivers, sewage facilities,
Ascomycetes 425
and water pipes (74). Its mycotoxins include fusaric acid and naphthoquinones
(448).
Description. Colonies on PDA grow 64 to 70 mm in 7 days. They are
usually thinly or nearly appressed-felty. They are locally low-floccose, whitish-
cream to buff, pale brownish, pale red-brown, or in some isolates pale blue-green
above, sometimes spotted with creamy or dusky blue-green, slimy sporodochial
masses, with pale, tea- or tea-with-milk-brown, red-brown, or very uncommonly
(in clinical isolates) blue-green to ink-blue reverse. Conidiophores consisting of
single phialides arise laterally on aerial hyphae, or small groups of phialides
formed on rudimentary branching structures, or closely appressed sporodochial
clumps. Phialides when producing microconidia in aerial mycelium are strictly
monophialidic, very characteristically filiform (elongated and slender), and 15–
40 ⫻ 2–3 µm (Fig. 88). Those producing macroconidia are shorter and more in-
flated, subcylindric to flask-shaped, and 10–25 ⫻ 3–4.5 µm. Microconidia are al-
Figure 88 Fusarium solani microconidial phialide.

426 Summerbell
Figure 89 Fusarium solani macroconidia, one with a chlamydospore attached.
ways in sticky heads, never in chains. They are hyaline, smooth-walled, ellipsoidal
to nearly cylindrical, and sometimes slightly curved. They are usually aseptate,
less commonly one- or two-septate. When aseptate they are (5-) 8–13 (-17)
⫻ 3–4 (-5) µm.
Macroconidia are usually common, sometimes in sporodochia. They are
hyaline, gently curved, mostly three-septate, less commonly four-to five-septate,
characteristically broad in relation to length (Fig. 89). When three-septate they
are (22-) 27–50 (-58) ⫻ 4–6 (-7) µm, with scarcely pedicellate, thick basal cells
and relatively broadly pointed to tapered but nearly round-ended apical cells.
Chlamydospores are usually abundant. They are smooth or more often rough-
walled, terminal or intercalary, single or clustered, pale or brownish, sometimes
protruding from sides of macroconidia, and 6 to 11 µm.
Pale to tea-colored forms within this complex taxon are common from in-
ternal infection. A form with vivid red-brown reverse, sometimes overshadowed
with bluish (but with typical elongated phialides and abundant microconidia, un-
like the deeply blue-pigmented potato-rot and soil fungus Fusarium coeruleum,
Ascomycetes 427
discussed above) may also be seen from such infections, but is particularly com-
mon from onychomycosis. Such a form producing red to brown colony colors
and blue-greenish sporodochia on oatmeal agar, as well as yellow to greenish
colonies on more osmotically active (2–5% glucose-supplemented) media, was
described in detail (in Chinese) from a keratitis isolate by Ming and Yu (507).
The isolate grew at 37°C. Ming and Yu’s proposal of a new taxonomic forma
(a rank below variety) based on this study was nomenclaturally invalid; a Latin
diagnosis was lacking, and no type material was designated.
Recent molecular studies of plant-derived and saprobic F. solani isolates
have disclosed that this complex consists of at least 26 entities that would be
considered distinct species using a phylogenetic species concept (427). This con-
firmed numerous earlier mating studies, reviewed by O’Donnell (427), that
showed numerous intersterile heterothallic mating groups within F. solani, all of
which would be considered separate species under the older biological species
concept. In addition, there are several homothallic entities, and some possibly
clonal entities. The medically important isolates have not yet been placed in terms
of their relation to elements of this complex. The variation seen in culture sug-
gests that several of these sibling species are involved in pathogenesis.
The F. solani complex as a whole is readily distinguished from the some-
times similar F. oxysporum by the stark contrast between its long, thin phialides
and the stubby, inflated phialides of the latter. Although macroconidia in F. solani
are generally distinctly thicker and blunter than those of F. oxysporum, there is a
degree of overlap in certain isolates, so this character can be a screening character
revealing obvious F. solani isolates as such, but it cannot be used for consistent
mutual exclusion of the two species. F. solani seldom has the violaceous purplish
colors frequently seen in F. oxysporum; the red-brown and dusky blue-green to
ink-blue colors occasionally seen in the former species are all readily distin-
guished with experience.
20. Fusarium subglutinans (Wollenw. & Reink.) Nelson et al.

Anamorph of Gibberella fujikoroi (Sawada) Wollenw. var.
subglutinans Edwards [Synonyms: Fusarium sacchari var.
Subglutinans, F. moniliforme, var. subglutinans Pro Parte
(i.e., the Latter Name Was Applied to a Broad Concept
Including This and Other Species) Fusarium Subgenus:
Liseola]
An isolate from well-verified mycotic keratitis in northern Italy (508) was identi-
fied prior to March 1975 by medical mycology authority R. Vanbreuseghem as
Fusarium moniliforme Sheldon var. subglutinans Wollenw. & Reink., consistent
with contemporary taxonomic concepts. On the basis of this record, F. subgluti-
nans has been indexed in reviews of medically important fungi (69, 443). The
428 Summerbell
complex group of isolates referred to as F. moniliforme var. subglutinans, how-

ever, was split into two separate varietal concepts and placed into F. sacchari
along with the type variety by Nirenberg in 1976 (435). These varieties have
since been elevated to species status as F. subglutinans, F. bulbicola, and F.
sacchari ss. str. (⫽ in the strict sense), based on molecular investigations (429).
Moreover, another closely related medically important species, Fusarium prolif-
eratum, may appear very morphologically similar to F. subglutinans unless char-
acters related to microconidial chain formation are emphasized. Only in the mid-
1970s, however, did these characters begin to be more broadly publicized and
made more accessible by the introduction of better media such as carnation leaf
agar (434). F. proliferatum was only distinguished as a separate species from the
long-standing Wollenweber and Reinking concept of F. moniliforme by Niren-
berg in 1976 (435) [apart from being described in 1971 as a Cephalosporium
species by Matsushima (509), who did not see macroconidia in his material],
and was therefore not well conceptualized in 1975. An example of early 1970s
confusion about these fungi was given by Gerlach and Nirenberg (437), who
pointed that the microphoto alleged to depict F. moniliforme var. subglutinans
in the authoritative Fusarium monograph by Booth (445) showed a species that
‘‘produces its microconidia in chains, because the microconidia in the picture
are clavate with a flattened base.’’ In other words, the concept of F. moniliforme
var. subglutinans was mistakenly illustrated in the most modern monograph avail-
able in 1975 by a species that was similar to F. proliferatum in its conidiogenesis.
There is thus considerable doubt about which currently recognized species the
isolate examined by Vanbreuseghem belonged to, and this isolate, RV 32739,
should be re-examined if it remains available.
F. subglutinans is very similar to F. sacchari (see above) in colony charac-
ters and conidial morphology. It differs in that a high proportion of its microco-
nidia are septate, showing an intergradation between microconidia and macro-
conidia, and in that macroconidia tend to form readily in cultures on PDA. F.
sacchari, on the other hand, has mainly single-celled microconidia and forms
macroconidia only rarely after induction with near-ultraviolet light. The conidio-
phores in F. subglutinans are often erect, arising directly from the substrate as
small treelike formations. In F. sacchari, however, the conidiophores mostly arise
from the aerial mycelium and are therefore referred to as ‘‘prostrate’’ by Niren-
berg and O’Donnell (441). F. subglutinans is a cosmopolitan decomposer,
whereas F. sacchari is a tropical organism usually associated with sugar cane
and banana plants. Both species have slightly to distinctly curved microconidia
with more or less rounded bases, and are unable to form microconidia in chains.
The club-shaped microconidia of F. proliferatum, however, with their flattened
bases, form chains readily whenever suitable nutrient and moisture conditions
permit. F. verticillioides (⫽F. moniliforme) differs strongly from all the species
Ascomycetes 429
mentioned so far by not forming polyphialides. Molecular distinction of all these

species is summarized by O’Donnell et al. (429), and a morphological key is
provided by Nirenberg and O’Donnell (441).
21. Fusarium verticillioides (Sacc.) Nirenberg Anamorph of

Gibberella moniliformis Wineland (Common Synonym:
Fusarium moniliforme Sheldon; Fusarium Subgenus:
Liseola)
F. verticillioides, frequently referred to as F. moniliforme in the literature, shows
the ‘‘general pattern of involvement by major opportunistic Fusarium species’’
described in the introductory section for Fusarium above. Case literature for this
well-established opportunist has been reviewed previously (see the same section)
and will not be reviewed in detail here. De Hoog et al. (69) have recently given
a species-specific synopsis. Although not as frequently reported as F. solani, this
fungus vies with F. oxysporum as the second most common fusarial agents of
disseminated and cutaneous infection in neutropenic and other severely immuno-
compromised patients (412). A prototypical case is that published by Young et
al. (510) regarding disseminated infection in a granulocytopenic lymphoma pa-
tient. Skin disrupted by varicella zoster infection was thought to be the portal of
entry. In some areas, F. verticillioides can become the predominant Fusarium
species infecting the compromised patient (511).
Like F. solani, F. verticillioides may occasionally cause subcutaneous
infection in apparently immunocompetent individuals. Collins and Rinaldi
(512) described a case of a man with an inflamed hand pustule that grew this
species. Although no fungal filaments were seen in the expressed pus on Gram
staining, destaining and restaining with periodic-acid-Schiff (PAS) stain allowed
confirmation of fungal filaments. The patient was an avid gardener but recalled
no injury. A well-developed F. verticillioides mycetoma in an Italian miner was
described by Ajello et al. (513). The case had originally been reported as being
caused by Acremonium sp., since only microconidia were then seen (514). The
case isolate is now in most major fungal culture collections. On the other hand,
an infected hyperkeratotic heel lesion attributed by Pereiro et al. (515) to F. verti-
cillioides in a prostatic adenocarcinoma patient appears to be better ascribed to
an Acremonium sp. The authors state that they made the identification based on
formation of small phialoconidia in chains from a floccose, off-white colony.
No purple coloration was noted. A photograph depicts solitary, thin, sometimes
flexuose phialides rather than the rigid-looking primary phialides of F. vert-
icillioides, which are not infrequently borne on branched conidiophores. The
authors’ description of the conidia as ‘‘8–9 ⫻ 2–3 µm’’; that is, as elongated
conidia with a length–width ratio of not less than 2.5: 1 [the average for F. vert-
430 Summerbell
icillioides is 2.7 :1, based on the measurements of Gerlach and Nirenberg

(437)] appears to be contradicted by their unscaled but otherwise excellent
photo showing short, obovate conidia with a length–width ratio of not more than
1.5 :1.
F. verticillioides is occasionally involved in ocular infections, particularly
keratitis, although the incidence of such infection is much less than the incidence
of F. solani infections (393, 456, 516). The strong ecological affinity of this
organism for maize corn may be significant in etiology; for example, Zapater
(456) recounts a keratitis that began after a moth flying from a fungally colonized
bin of chicken-feed corn struck a woman in the eye without causing immediate
discernible injury. One case of apparent F. verticillioides keratitis (direct micros-
copy was not performed on ocular material) was successfully treated with cyclo-
piroxolamine after disk susceptibility testing suggested it was resistant to the
other agents available for testing (pimaricin was not tried, but resistance was
shown to two other polyenes) (516).
In veterinary mycology, F. verticillioides has been reported to cause fatal
pneumonia in a captive American alligator (517). The isolate was atypical, but
its identity was later confirmed by sequencing (429).
In nature, F. verticillioides is frequently associated with corn (Zea mays),
but also with various other plants (grains, pines, cabbage family) and also insects
(429). It is the best-known producer of fumonisins, mycotoxins implicated in the
etiology of the fatal brain degeneration known as equine leukoencephalomalacia
in horses eating contaminated feed. A variety of other secondary metabolites such
as fusaric acid and moniliformin are also produced (448).

floccose to powdery. They are whitish above, sometimes becoming dull violet
with pale to deep vinaceous reverse. Conidiophores consisting of single phialides
arise laterally on aerial hyphae, or small groups of phialides formed on rudimen-
tary branching structures, or closely appressed sporodochial clumps (Fig. 90).
Phialides are strictly monophialidic and subulate (tapered, awl shaped), and 11–
32 ⫻ 2–3.5 (-4.5) µm. Microconidia have a strong tendency to form in long
chains, especially on specialized media such as carnation leaf agar or synthetic
nutrient agar. They are sometimes also in sticky heads, especially on rich media.
They are hyaline, smooth-walled, clavate with a flattened base, usually aseptate,
rarely one- or two-septate, and 7–10 (-19) ⫻ 2.5–3.2 (-4.2) µm. Macroconidia
are often formed only after extended culture or not at all, sometimes in sporo-
dochia. They are hyaline, nearly straight to falcate, mostly three- or five-septate,
and (18-) 30–58 (-73) ⫻ 2–4.3 µm, with pedicellate basal cells. Chlamydospores
are conventionally said to be absent; however, according to Gerlach and Niren-
berg (437) ‘‘inflated cells with thickened walls occur.’’
Ascomycetes 431
Figure 90 Fusarium verticillioides, monophialide with conidia in a chain.
E. Microdochium
1. Microdochium nivalis (Fries) Samuels & Hallett Anamorph
of Monographella nivalis (Schaffnit) E. Müller (Synonym:
Fusarium nivale Ces. ex Sacc.)
This is one of numerous problematic fungi that have several records from human
disease despite lacking the ability to grow at body temperature.
An allegedly significant isolation of this fungus, recorded as F. nivale, was
included without specific etiologic or mycological documentation in an omnibus
report on fungi causing keratitis in south Florida (393). In general, this study
accepted only fungi that grew from specimens on multiple media or those that
grew on a single medium and also correlated with positive direct microscopy.
Mycology was performed at the Mayo Clinic. Since this fungus has a maximum
growth temperature of 28°C (74, 518), this record must be questioned. It may
432 Summerbell
possibly reflect either misidentification or a contamination problem. Inclusion of

some contamination events into such lists is facilitated by the ‘‘positive culture
on ⬎1 media’’ acceptance criterion. Such a criterion is ethically necessary in the
clinic, since it is conservatively presumptive of fungal infection in ambivalent
cases requiring rapid treatment, but it is mycologically dubious, since nonpatho-
genic airborne conidia often settle on surfaces in coherent clumps or chains or
in conidia-laden aqueous aerosols from sources such as tap water or splashed
outdoor leaf surfaces. Dispersal of such aggregations on the ocular surface prior
to sampling would lead to clinically insignificant, single-species growth on multi-
ple media. Another less likely source of low-temperature fungi in keratitis sam-
ples is retention of inoculum as dormant material on residual debris at the site
of trauma. Finally, serous exudates on bodily surfaces may be colonized by sap-
robes, especially fusaria (422, 423, 485); a possible ocular instance appears im-
mediately below.
A seemingly very carefully confirmed case of keratitis attributed to M. ni-
valis var. major, a variant of M. nivalis with mostly three-septate, relatively thick
macroconidia, was presented by Perz et al. (519) (Fig. 91). A patient under un-
specified treatment for kidney stones but otherwise healthy and with no history of
ocular trauma, spontaneously developed a fungal keratitis, confirmed by biopsy.
Figure 91 Microdochium nivalis var. major CBS 105.90.

Ascomycetes 433
Scrapings grew M. nivalis, and a compatible description was published, along

with nonspecific but nondiscrepant photographs. K. Mańka, a forest phytopathol-
ogist experienced with hypocrealean soil fungi, was a coinvestigator. According
to Gams and Müller (518), M. nivalis var. major is ecologically indistinguishable
from the type variety; it would therefore not be predicted to grow at body temper-
ature, but appears not to have been tested. A typical isolate, CBS 106.90, was
tested ad hoc by me for purposes of this chapter, and was found to have a maximal
growth temperature of approximately 27°C. Perz et al. noted that conjunctival
sacs and ocular secretions of their patient grew M. nivalis in both the affected
and unaffected eyes, as well as Geotrichum candidum. It is conceivable that this
surface colonization overgrew inoculum of an unelucidated true agent of the kera-
titis; otherwise, an atypical heat-tolerant variant of M. nivalis var. major or a
fungus very similar to it must cause occasional keratitis. The culture described
had a brown reverse on PDA, and formed mostly (1-) three-septate apedicellate
macroconidia with very few aseptate conidia and no chlamydospores, a combina-
tion of characters ruling out most common Fusarium and Cylindrocarpon species
involved in keratitis.
Microdochium oryzae (Hashioka & Yokogi) Samuels & Hallett (anamorph
of Monographella albescens [von Thümen] Parkinson et al.), the agent of rice
leaf scald disease, is morphologically similar to M. nivalis but grows up to 36°C
(518). This fungus, only isolated from infected rice plants so far, may be consid-
ered if M. nivalis-like fungi are seen in areas of suitable climate. It has conidia
with a somewhat swollen basal cell.

sparsely cobwebby or floccose to felty. They are whitish above, becoming pale
pinkish to peach, orange, or amber, with these colors often visible on the surface
but most prominent on the reverse. Conidiophores consisting of annellides arise
laterally on aerial hyphae or in small groups on rudimentary branching structures,
and also commonly on closely appressed sporodochial clumps. Annellides have
a nearly cylindrical to bulbous, barrel- to pear-shaped base and an elongating,
thin percurrently proliferating apical tubule, overall 6–15 ⫻ 2.2–4.0 (basal mea-
surement) µm. Microconidia are not considered to be formed; there is, however,
a small proportion of single-celled conidia mixed with and intergrading in size
and shape with the macroconidia, mostly 8–12 ⫻ 2.0–2.8 µm. Macroconidia are
crescent-shaped, sometimes with a relatively straight basal half, hyaline, smooth-
walled, mostly one-septate, fairly commonly two- or three-septate, and exception-
ally up to seven-septate. When one-septate they are (9-) 13–18 (-23) ⫻ 2.2–3.0
(-4.5) µm; when three-septate they are up to 36 µm long, with a wedge-shaped,
obtuse base and a sharply pointed apex. In M. nivalis var. major conidia are
mostly three-septate, ranging from one- to 7-septate, and are 19–30 (-37) ⫻ 3.5–
4.5 (-6) µm. Chlamydospores are absent.
434 Summerbell
The production of annellides by this fungus and its connection to the tel-
eomorph genus Monographella in the family Amphisphaeriaceae show that any
resemblance between the former F. nivale and the genus Fusarium is coinciden-
tal. Nonetheless, the resemblance of the conidia to small Fusarium conidia is
high. The maximum growth temperature, 28°C, can easily be used to distinguish
typical isolates of this fungus from mammalian opportunists.
F. Neocosmospora
1. Neocosmospora vasinfecta E. F. Smith [Common Synonym:
Fusarium vasinfectum (an Inappropriate Combination of an
Anamorph Genus Name with a Species Concept Including
the Teleomorph)]
A case of allergic bronchopulmonary mycosis in a patient with asthmalike symp-
toms and hemoptysis was attributed to N. vasinfecta (as Fusarium vasinfectum)
by Backman et al. (520). The fungus was never cultured. A panel of mold allergy
tests indicated this fungus as the only one to which the patient reacted; further
tests showed positive precipitins against N. vasinfecta antigens. N. vasinfecta,
however, happened to be the only Hypocrealean fungus included in the panel;
thus the reaction seen could have been a cross-reaction against an indefinite num-
ber of fungi from this group or its relatives. [See Kaufman et al. (433).] The
possibility of a coincidentally cross-reacting antigen from an entirely different
group of organisms also cannot be ruled out. Diagnoses of allergic bronchopul-
monary mycosis caused by fungi other than Aspergillus fumigatus clearly need to
be established by repeated isolation of positive cultures, correlated with specific
immunological responses. The criteria designed to make the troublesome cultur-
ing step unnecessary for classic allergic bronchopulmonary aspergillosis do not
apply to other molds. The case record is rejected.
N. vasinfecta was, however, authoritatively associated with a localized my-
cotic cyst of soft tissue in the leg of a renal transplant and dialysis patient (521,
522). No trauma had occurred at the infection site, suggesting a circulatory source
for the inoculum. Surgery and a small dose of ketoconazole (truncated prema-
turely because of a cyclosporin toxicity side effect) eliminated the infection. A
French parachutist who suffered an open ankle dislocation in Senegal contracted
an N. vasinfecta osteoarthritis, ultimately requiring amputation (523). The fungus
was not seen in several histopathologic studies but grew repeatedly from the
wound and from the progressing osteitis despite extensive cleaning measures and
treatment with amphotericin B.
The fungus in nature is common in tropical and subtropical soils. Molecular
phylogeny studies show that it is closely related to the species in the Fusarium
solani complex (427).
Ascomycetes 435
Description. Colonies are fast-growing—40 mm in diameter after 7 days

at 25°C. They are thinly floccose, white to buff or pinkish, soon becoming stip-
pled with production of white (immature) to orange-brown or red-brown (mature)
ascomata. Conidiophores are acremonium-like, consisting of short to long lateral
branches, sometimes with one or rarely more branch points (Fig. 92). Phialides
are subulate (awl-shaped, long, and tapered) to cylindrical, 30–100 ⫻ 1–2 µm,
and bearing conidia in sticky heads. Conidia are hyaline, smooth, aseptate or
less often one-septate, long-ellipsoidal, cylindrical, or fusoid (Fig. 93). They are
sometimes slightly curved, and are 5–13 ⫻ 2–3.5 µm. Ascomata are bright or-
ange to orange-brown or red-brown overall. They are nearly spherical, 200 to
500 µm in diameter, with a short, protruding apical neck bearing an ostiole (open-
ing), and with thick walls that become yellow at maturity and are composed of
textura angularis (composed of polygonal cells as seen in face view) tissue. Asci
are cylindrical and 80–100 ⫻ 11–15 µm, containing eight linearly arranged asco-
spores. Ascospores are ellipsoidal to subglobose and orange-brown, with thick,
heavily roughened walls. They are 10–16 ⫻ 7.5–12 µm, and lack a germ pore.
Chlamydospores are present. Growth occurs at 37°C in vitro (Fig. 94).
Figure 92 Neocosmospora vasinfecta conidiophores CBS 554.94 conidiophore.

436 Summerbell
Figure 93 Neocosmospora vasinfecta CBS 554.94 zero- and one-septate conidia.
Figure 94 Neocosmospora vasinfecta ascospores (large, rough structures) and conidia.

Ascomycetes 437
G. Trichoderma
Trichoderma species, like Chaetomium species, form a large group of difficult
to distinguish, often thermotolerant common saprobes, which, for reasons that
remain unclear, are much less frequently involved in opportunistic pathogenesis
than other common thermotolerant saprobes. In recent years, however, a small
number of legitimate cases of Trichoderma infection have been recorded in hu-
mans, and at least one species, T. longibrachiatum, may reasonably be called an
emerging opportunist of the severely immunocompromised patient.
Trichoderma species as classically defined by Rifai (524) and Bissett (525)
were known aggregates of several closely related, all but indistinguishable sibling
species, some of which were associated with distinguishable Hypocrea teleo-
morphs on natural substrata. With the advent of molecular methodologies, the
species aggregates that had been used in identification schemes were seen as
inadequately precise to specify ecologically distinct biological entities. A strong
impetus for this judgment came from the appearance of a genetically distinct
organism generally fitting the aggregate description of Trichoderma harzianum,
but causing an economically important disease of cultivated mushroom beds that
other genetically distinct T. harzianum groups did not cause (526, 527). This
mushroom pathogen is in the process of being described as a distinct species
subsequent to delineation by sequencing. (Morphological characters have also
been found to distinguish it.) The ‘‘gold standard’’ for species identification in
Trichoderma has therefore been removed to the molecular level, which is most
accurately predictive of natural associations and interactions. Molecular confir-
mation of species identification has been suggested as necessary for precision
in case reports wherever possible (528). At the same time, however, molecular
clarifications of species relationships have allowed more precise morphological
characters to be delineated, so the most up-to-date works such as the keys and
descriptions published by Gams and Bissett (529) may for careful pheneticists
facilitate purely morphological identification at a level comparable to that made
possible by molecular methodologies.
1. Identification
The procedures for morphological identification of Trichoderma species are
unique. The organism is best grown on malt extract, oatmeal, or cornmeal agar
(529). Within approximately 5 to 7 days in most isolates, well-developed sporo-
dochial pustules will be seen. These pustules may be found in four stages: (1)
immature, with branching incomplete and phialides sparse or nil, white to yellow
in species with green conidia; (2) submature, with branching complete and newly
formed conidial heads present, yellow to pale green in species with green conidia;
(3) mature, with phialides mostly intact and large numbers of mature conidia
present, green, dark green, intense yellow-green, or hoary blue-green in species
438 Summerbell
with green conidia; and (4) overmature, with phialides collapsed and masses of
conidia present, deep yellow-green to deep green in green-conidial species.
Trichoderma species can only be analyzed in stage 2. Stage 3, which for any
given sporodochium occurs approximately 24 hr after stage 2, tends to feature
large numbers of conidia that make discernment of structure in the still well-
formed conidiophores impossible. Stage 4, the typical 7-day stage that most per-
sons unfamiliar with Trichoderma would attempt to analyze, is almost completely
devoid of information useful for species identification, except in submature spor-
odochia that may still remain near the colony margin. Trichoderma colonies for
identification should therefore be inoculated so that they may be observed be-
tween their fourth and seventh days of growth. Structures form well at a wide
range of room temperatures in light, as well as at 25°C. Recent species identifica-
tion keys by Gams and Bissett (529), however, establish a standard of 20°C for
growth rate measurements—a standard that unfortunately will seldom be conve-
nient in the clinical laboratory. A recalibration based on 25°C growth is not cur-
rently available.
Before sporodochia are mounted for microscopy they are best examined
under the dissecting microscope or under the 10⫻ compound microscope lens
to discern any sterile setae that are produced, as well as the general branching
structure. Sporodochia of some species bear a characteristic down composed of
long, sinuous setae that arise from the ends of the branches. A few species [e.g.,
Trichoderma (formerly Gliocladium) virens] have convergent, Penicillium-like
conidial structures, and their conidia may coalesce into large, sticky masses that
will be very conspicuous at low magnification. Heavily bacterially affected colo-
nies of other Trichoderma species may mimic this gloeoid appearance, so it is
advisable to ascertain that one is working with a pure culture if such a manifesta-
tion is seen in gross overview.
For making microscopic slide mounts, analysts must be aware that Tricho-
derma sporodochia are highly water-repellent, and simply inserting them into
ordinary mounting media generally results only in the trapping of an intractable
air bubble that prevents meaningful microscopy. Any tearing or heavy squashing
of the material destroys the branching structure that must be observed. The best
technique to use is to carefully pick submature (see above) sporodochia off the
growth medium, being careful to cut from under the base so that the entire struc-
ture is intact. Place onto a slide and add a drop of 95% ethanol to defat the
hydrophobic components, making sure that the sporodochium appears to be wet-
ted. Before the ethanol can dry, add the mounting medium (lactic acid is optimal;
cotton blue may be added) and gently allow a coverslip to angle over the top of
the sporodochium, pressing it as little as possible to make a useable slide. If a
key using conidial measurements is being employed [e.g., that of Gams and Bis-
sett (529)], the same growth medium should be used as was used by the authors
of the key.
Ascomycetes 439
The main character that careful mounting should allow detection of is the
branching pattern of structures within the sporodochia. Some species, such as
Trichoderma koningii, have verticillate structures, in which branch points tend
to give rise to approximately three branches arranged radiately, like spokes in a
wheel. Where only two branches diverge from a branch point in such structures,
they tend to diverge either as opposite pairs, or as two ‘‘spokes’’ diverging at
approximately 120° from each other with the third radially symmetrical spoke
missing. As this verticillate structure iterates through major branches, smaller
branches, and finally the clusters of phialides themselves, a very symmetrical
bushy structure, sometimes referred to as ‘‘pyramidal,’’ is generated. This type
of structure, typical of Trichoderma species in sections Pachybasium and Trich-
oderma, is distinguished from structures featuring long main branches that do
not rebranch extensively, as seen in the section Longibrachiatum. The secondary
branches that do arise from the main branches in this section often arise singly,
and in many species the finest tips of the conidiophores often feature a zone in
which a number of phialides are attached singly and spaced irregularly apart,
along an otherwise nearly unbranched elongated section of the conidiophore.
Another morphological character that has been more emphasized since
molecular methods clarified species distinctions in this group is the shape of
phialides. In section Pachybasium, they are typically short and stout, whereas in
section Longibrachiatum, they are elongate, lageniform (wine-bottle-shaped) or
nearly cylindrical. An excellent key to the species, along with descriptions and
illustrations, is provided by Gams and Bissett (529). Many Trichoderma species
form similar chlamydospores, and this character is seldom used in species de-
scriptions.
A large number of molecular identification techniques have been investi-
gated for Trichoderma species (530). Since relatively few of the species in this
genus are known or suspected to have clinical significance so far, some of these
methodologies deal primarily with species and groups not known to be biomedi-
cally relevant. Recently Kuhls et al. (530) have shown that the widely used M13
and (GACA) 4 primers for moderately repetitive loci can readily be used in PCR
to generate fingerprints that allow species identification of clinical Trichoderma
isolates. Ribosomal internal transcribed spacer (ITS) sequencing may then be
used to confirm identifications, especially for isolates giving atypical banding
patterns. Trichoderma longibrachiatum isolates from confirmed human disease,
however, were shown to have very similar or identical banding patterns, while
saprobic isolates of the same species were more heterogeneous. This would tend
to increase the ease of confirming the identity of clinically significant isolates by
PCR fingerprinting alone.
The majority of credible case reports involving Trichoderma spp. have pro-
posed a species identification for the causal agent. An exception is an etiologically
credible report of infection attributed to Trichoderma sp. in two ball pythons
440 Summerbell
(Python regius) kept as cagemates (506). The generic identification itself is un-
substantiated, and a long-outdated generic name (Oospora) is used for one of
the other fungi identified in the same paper, suggesting use of a questionably
credible laboratory. Nonetheless, an incorrect generic identification of Tricho-
derma seems unlikely.
2. Trichoderma citrinoviride Bissett Anamorph of Hypocrea

schweinitzii (Fr.:Fr.) Sacc. (Trichoderma Section:
Longibrachiatum)
An isolate of T. citrinoviride was among the human-pathogenic isolates included
in a recent study on identification of clinical Trichoderma isolates with molecular
methods (530). The isolate was said to have been from ‘‘hemocultures’’ of a
neutropenic lymphoma patient. No other case information was given, and assur-
ance that the isolate was not a contaminant lies primarily in the s at the end of
the word hemocultures. T. citrinoviride is a common organism on temperate soil
and wood, so far mostly known from northern North America and Europe (531).
whitish at first, with effuse or loosely tufted bright green to yellow-green conid-
iation developing mostly at the colony margin. The reverse is usually yellowish-
green. Conidiophores have long primary branches and with secondary branches
that are short, often rebranched once or twice (Fig. 95). Phialides are variously
arranged, in verticils of three or in pairs (or solitary), with a strong tendency to
Figure 95 Trichoderma citrinoviride conidiophore structure.

Ascomycetes 441
Figure 96 Trichoderma citrinoviride conidia.
appear singly at irregular intervals along the main branch near the branch apex.
They are mostly distinctly constricted at the base, especially when laterally dis-
posed, lageniform (wine-bottle-shaped), or when somewhat shorter, ampulliform
(ampoule-shaped, with a slightly extended apex and a bulge just above the basal
constriction). They are 3.5–6.6 ⫻ 2.0–3.2 µm, with terminal phialides (at major
branch ends) longer to 12 µm. Conidia are green, smooth, ellipsoidal, and 2.2–
3.7 ⫻ 1.5–2.1 µm. T. citrinoviride grows at 40°C (Fig. 96).
This species is morphologically distinguished from T. longibrachiatum by
its conidiophores bearing side branches that are often rebranched once or twice.
Its phialides are mostly distinctly constricted at the base, and its conidia are
smaller than 4.0 ⫻ 2.5 µm. It can be readily distinguished in PCR using M13 and
(GACA) 4 primers (530). (For morphological distinction from other Trichoderma
species, see the discussion under T. harzianum.)
3. Trichoderma {aff. Hypocrea} harzianum Rifai (Trichoderma

Section: Trichoderma)
A renal transplant patient in Spain died of autopsy-confirmed, disseminated T.
harzianum infection, particularly affecting the brain and lungs (532). The case
442 Summerbell
isolate was not molecularly confirmed, but was carefully morphologically charac-
terized and deposited in CBS (CBS 102174). Gams and Bissett (529) state that
this species has a maximum growth temperature of 36°C. Clearly, culturing of
an isolate from multiple human brain lesions is discordant with this and suggests
the matter requires further study. As Richter et al. (528) stated, ‘‘Growth at ele-
vated temperatures is one of the well known virulence factors of neurotropic
fungi.’’ A fatal peritonitis caused by a member of the T. harzianum aggregate
has also been reported (533). Identification of the organism, anomalously referred
to as a ‘‘yeast strain’’ in the case report, was not substantiated in the text, but
was credited to an unspecified staff member of Institut Pasteur. Unfortunately,
however, the isolate was not included in the molecular re-evaluations published
3 years later concerning medically important Trichoderma isolates collected by
that institution (530).
whitish at first, with profuse yellow-green to dark green conidiation developing
effusely or in pustules fringed by white mycelium. Conidiophores regularly re-
branch in verticils or pairs, forming a ‘‘pyramidal structure’’ (see Identification
section on page 437) (Fig. 97). Phialides are usually in verticils of three or four,
Figure 97 Trichoderma harzianum conidiophores.

Ascomycetes 443
less commonly in pairs and seldom solitary. They are mostly distinctly constricted
at the base, especially when laterally disposed, ampulliform (ampoule-shaped,
with a slightly extended apex and a bulge just above the basal constriction), or
when somewhat longer and especially when terminal, lageniform (wine-bottle-
shaped). Terminal phialides are occasionally curved or flexuous (e.g., with a gen-
tle S-curve). Most phialides are 3.5–7.5 ⫻ 2.5–3.8 µm, with terminal phialides
(at major branch ends) longer, to 10 µm. Conidia are nearly hyaline to pale green
in transmitted light. They are smooth, obovoid (egg-shaped with broad end pro-
duced first) to subglobose, and (2.5-) 2.7–3.5 ⫻ 2.1–2.6 (-3) µm. Maximum
growth temperature is 36°C (Fig. 98).
Trichoderma viride and similar species also producing broad conidia are
distinguished from T. harzianum by the rough ornamentation of their conidial
walls. T. koningii and similar species share the highly verticillate ‘‘pyramidal’’
conidiophore structure of T. harzianum but are distinguished by their narrower,
ellipsoidal, not broadly ovoid or subglobose conidia. T. longibrachiatum, T. citri-
noviride, and other members of section Longibrachiatum are distinguished by
their mostly ellipsoidal conidia, and most importantly by their seldom rebranched,
largely nonverticillate conidiophore structure, featuring conidiophore branch
Figure 98 Trichoderma harzianum conidia.

444 Summerbell
ends with irregular rows of singly or asymmetrically paired phialides and few
or no verticils. Many members of section Longibrachiatum are thermotolerant,
growing well at 40°C (531). T. atroviride and T. inhamatum, two species not
recorded from human and animal disease, are more similar to T. harzianum. The
former has dark green conidia in transmitted light, in contrast to the paler conidia
of T. harzianum, and also has slightly larger conidia, 2.6–3.8 (-4.2) ⫻ 2.2–3.4
(-3.8) µm. The latter has relatively strictly pustular conidiation with little effuse
development of conidia, and has shorter, broadly flask-shaped phialides that are
4–5 (-7) ⫻ 2.3–3 µm. T. inhamatum is also similar to T. harzianum molecularly,
but can be distinguished by a two-nucleotide difference in its ribosomal ITS1
sequence (534). The T. harzianum subgroups causing mushroom crop loss, soon
to be described as separate from T. harzianum, can be distinguished from T.
harzianum ss. str. by failure to grow at 36°C (North American Th2 group) or
by conidia that decolorize (‘‘bleach out’’) when placed in plain lactophenol (Brit-
ish Th4 group) (527). The distinction can be made more definitively by molecular
means (534).
Trichoderma species with distinct long, undulate sterile spines extending
from the ends of the conidiophore branches and unusually thick main conidio-
phore branches over 10 µm belong to Trichoderma section Pachybasium, a group
not reported from human and animal disease and, in the present author’s experi-
ence, seldom seen in the clinical laboratory in temperate regions even as contami-
nants. Immature branch ends in underdeveloped pustules of other Trichoderma
sections should not be confused with these well-differentiated sterile spines.
4. Trichoderma koningii Oudemans Anamorph of Hypocrea

koningii Lieckfeldt et al. (Trichoderma Section:
Trichoderma)
This species has been reported to cause infection in dialysis patients by Ragnaud
et al. (535) and Campos-Herrero et al. (536). Molecular methods have allowed
the disassembly of the complex of species once included in the T. koningii aggre-
gate, and a T. koningii sensu stricto has been delimited based on re-collecting
from the same locality and materials as Oudemans originally collected from prior
to 1902. The ‘‘real’’ T. koningii is thus a species that has a maximum growth
temperature of approximately 33°C (537) and is unlikely to play any role in
diseases of warm-blooded animals. Its morphological distinction from the medi-
cally important species is noted in the discussion under T. harzianum above.
In the Ragnaud et al. (535) dialysis case, identification is credited to C. de
Bièvre of Institut Pasteur, but the isolate appears not to have survived at that
institution to be included in the later molecular reassessment by Kuhls et al. (530).
The authors include a technical description that eventually reveals itself as a
textbook-derived generic-level description of Trichoderma grown in pure culture
Ascomycetes 445
(e.g., it states ‘‘conidia subglobose to oblong, smooth or echinulate, with color

hyaline or most often green’’). No clue remains as to the specific characters of
the case isolate.
In the Campos-Herrera et al. case (536), an excellent microphotograph
shows three long terminal conidiophore branches with phialides borne singly
or in offset pairs, completely lacking verticillate structure. This, along with the
ellipsoidal-oblong conidia shown, allows the causal agent to be definitively recog-
nized as a member of Trichoderma section Longibrachiatum, although the species
is not determinable. As such a fungus could not be mistaken for T. koningii by
anyone following Rifai’s 1969 monograph (524) of Trichoderma or works based
on it [e.g., Domsch et al. (74)], this shows that the concept of T. koningii used
in this 1996 paper followed pre-1969 concepts dividing all green Trichoderma
spp. into T. viride (or T. lignorum) for round-conidial species and T. koningii for
ellipsoidal-conidial species.
An isolate from a liver transplant patient reported in a meeting abstract
(538) as T. koningii was later reidentified as T. longibrachiatum (539). The very
thorough compilation by de Hoog et al. (69) indexes both reports separately under
the names given.
5. Trichoderma {aff. Hypocrea} longibrachiatum Rifai

(Trichoderma Section: Longibrachiatum)
This fungus has now repeatedly been confirmed as an opportunist of the severely
immunocompromised patient. Fatal disseminated infections in bone marrow
transplant patients have been well confirmed on two occasions (528, 540). On
one of those occasions the organism was originally identified as T. pseudokoningii
(540), but this judgment was later amended after molecular study (530). Brain
lesions occurred in both cases. (Compare the similar case mentioned under T.
harzianum above for a possible emerging pattern in Trichoderma infections.) An
isolated brain abscess in a neutropenic leukemia patient was cured by resection
and prolonged therapy with ketoconazole and itraconazole (541). Susceptibility
testing had shown the isolate to be resistant to the amphotericin B that had been
used in initial therapy. An invasive sinusitis caused by T. longibrachiatum in a
liver and small bowel transplant recipient was successfully treated with debride-
ment, amphotericin B, and subsequent oral itraconazole (539). A neutropenic
child with severe aplastic anemia had an infection of skin around an intravenous
line site; T. longibrachiatum from the lesion was molecularly confirmed by
Trichoderma authority G. Samuels and was successfully treated with amphoteri-
cin B lipid complex prior to bone marrow transplantation (542). In keeping with
another emerging pattern in Trichoderma infections, a fatal peritonitis was caused
by T. longibrachiatum in a CAPD patient (543). The isolate in this case was
identified by hypocrealean authority W. Gams of CBS.
446 Summerbell

whitish at first, with conidiation first occurring in strands, then coalescing into
crusts that are dark green. The reverse is pale to greenish-yellow.
Conidiophores consist of long primary branches and relatively few second-
ary branches with little rebranching (Fig. 99). Phialides are often solitary, some-
times paired or in verticils of three, with a strong tendency to appear singly or
in offset (not precisely opposite) pairs at irregular intervals along the main branch
near the branch apex. They are lageniform (wine-bottle-shaped), usually with
little or no basal constriction, and are 5.3–11.6 ⫻ 2.0–3.2 µm, with terminal
phialides (at major branch ends) longer to 14 µm. Conidia are pale to medium
green. They are smooth, obovoid, ellipsoidal or narrowly ellipsoidal, and 3.4–
6.6 ⫻ 2.3–3.5 µm. T. longibrachiatum grows well at 42°C.
For distinction from T. citrinoviride, see that species. For distinction from
other Trichoderma groups, see discussion under T. harzianum. Richter et al. (528)
reported that potato flake agar elicited strongly yellow diffusing pigment from
their isolate of T. longibrachiatum, but other species were not compared. T. longi-
brachiatum and T. citrinoviride may both produce such pigments; Gams and
Bissett (529) note the production of ‘‘bright greenish-yellow pigments’’ as a
character of the section Longibrachiatum as a whole and also note about other
Trichoderma groups that ‘‘dull yellowish pigments are common in many species
Figure 99 Trichoderma longibrachiatum conidiophore structure showing several soli-

tary phialides and conidia.
Ascomycetes 447
but are not very distinctive’’ (i.e., not very useful in distinguishing species). T.
harzianum, in Trichoderma section Trichoderma, is one species in which such
‘‘dull yellow’’ pigments are often present. Another species in that group, T. aure-
oviride Rifai, is named for its conspicuous production of brownish-yellow reverse
pigments forming yellow crystals in the agar. The ability of T. longibrachiatum
to grow above 40°C may assist in separating it from many other Trichoderma
types, but temperature ranges are by no means well defined so far for many
species and variants. (See discussion of T. harzianum above.)
6. Trichoderma pseudokoningii Rifai Anamorph of an

Unnamed Hypocrea Species (Trichoderma Section:
Longibrachiatum)
This species was reported by Gautheret et al. (540) as an agent of a fatal infection
in a bone marrow transplant recipient. The isolate, however, was later reidentified
as T. longibrachiatum using molecular methods (530). Studies by Turner et al.
(544) and Kuhls et al. (545) have suggested that the genuine T. pseudokoningii
is very rare outside Australia and New Zealand. The name was long used as an
aggregate designator, as suggested by Rifai (524), and was mainly applied to
isolates that molecular studies now reveal to be T. longibrachiatum or T. citrinovi-
ride. Another case listed by de Hoog et al. (69) for this species based on a meeting
abstract by Degeilh et al. (546) concerns an isolate later identified as T. longi-
brachiatum (541).
7. Trichoderma viride Pers. Anamorph of Hypocrea rufa

(Pers.: Fr.) Fr. (Trichoderma Section: Trichoderma)
As with T. koningii, until recently the name T. viride was applied to a highly
phylogenetically diverse group of similar-looking Trichoderma isolates, essen-
tially all those with rounded, roughened conidia. Prior to 1969 the name was
applied to an even greater range of Trichoderma species with rounded conidia.
It was in the context of these former aggregate concepts that T. viride was noted
by Escudero et al. (547) as forming an aspergilloma-like pulmonary fungus ball,
by Loeppky et al. (548) as causing CAPD-related peritonitis in an amyloidosis
patient, and by Jacobs et al. (549) as causing perihepatic hematoma in a liver
transplant patient. More recently, a T. viride sensu stricto was defined based on
isolates definitely derived from the H. rufa teleomorph (550), and this species
has a maximal growth temperature below 35°C. Species from this complex with
higher growth maxima are beginning to be described [e.g., T. asperellum (551)],
but no isolates have been preserved from the T. viride disease reports to allow
corrected identification of the pathogenic types involved.
The Escudero et al. (547) report on Trichoderma pulmonary colonization
describes conidia of the causal agent only as ‘‘small and rounded . . . forming
heads typical of the genus Trichoderma,’’ without mentioning the characteristic
448 Summerbell
roughening of T. viride aggr. sensu Rifai. The discussion reveals that species
identification was arrived at without reference to Rifai’s (524) monograph pub-
lished 6 years earlier, and the significance of the distinction between smooth-
and rough-walled, rounded Trichoderma conidia was unknown to the authors.
The isolate may have been T. harzianum or another species. The identification
given in the peritonitis report of Loeppky et al. (548) is completely unsubstanti-
ated. The report by Jacobs et al. (549) of T. viride infection in a liver transplant
recipient includes a capsule description that in retrospect rules out identification
as T. viride. The report involved an organism with a conidia reported as ‘‘slightly
rough.’’ Based on the measurements given, their l/w ratio was approximately
1.75, corresponding well with the ‘‘ovoid’’ shape described by Jacobs et al. The
isolate produced chlamydospores and grew at 45°C. A photograph showed a long
branch bearing conspicuous single phialides. T. viride has distinctly rough, round
conidia with a median l/w ratio of 1.1 (551). Its temperature tolerance is men-
tioned above. Only a minority of isolates produce chlamydospores within usual
incubation periods. The newly described T. asperellum does have finely rough-
ened conidia, of which some may be ovoid, but most are subglobose to globose,
and the median l/w is 1.2. Its branching has symmetrical, verticillate ‘‘pyrami-
dal’’ structure; structures with series of single phialides would not be common.
Its maximum growth temperature is not recorded, but photos published by Sam-
uels et al. (551) clearly show that growth is reduced by over 50% between 30°C
and 35°C, a finding that seldom if ever corresponds with thermotolerance. Jacobs
et al.’s isolate, therefore, does not fit either described Trichoderma species with
uniformly rough conidia or taxa such as Trichoderma saturnisporum or T. gha-
nense with irregular conidial roughening. On the other hand, only the mention
of fine roughening prevents this isolate from fitting a description of T. longibrach-
iatum. If the authors mistook cytoplasmic granulation or attached debris on
the conidial wall for fine conidial roughening, as persons inexperienced with
Trichoderma commonly do, T. longibrachiatum would be the most likely identi-
fication of their isolate, with a small chance of T. harzianum.
In general, the T. viride aggregate is similar in most respects to T. harzia-
num (see discussion of that species), but differs by having conidia with distinctly
roughened walls. Isolates from medical and veterinary cases should be molecu-
larly characterized or deposited in major collections, at least until the taxonomy
of this group has become stabilized in molecularly informed species concepts.
H. Tubercularia
1. Tubercularia vulgaris Tode: Fr. Anamorph of Nectria
cinnabarina (Tode: Fr.) Fr.
This distinctive species forms large, stout, heavily conidial synnemata on the
natural substrate (newly killed and weakened branches of various trees and
Ascomycetes 449
shrubs), but only occasionally does so in culture. Indeed, it is seldom identified

or described from cultures. The use of this name to indicate a putative etiologic
agent in a tabulation of Florida endophthalmitis cases (407) is therefore unex-
plained. Cases were evidenced only by multiple culture on one occasion. (See
discussion under Microdochium nivalis above.) The record may well be correct,
but cannot be accepted.
I. Verticillium
1. Verticillium {aff.? Glomerella} nigrescens Pethybr.
(Synonym: Cephalosporium serrae Maffei)
The ex-type isolate of the now-synonymized name C. serrae was obtained from
well-demonstrated keratitis by G. M. Serra, and was described by Maffei (552),
who also recapitulated relevant clinical details. Direct microscopy was positive
for fungal filaments. The isolate is in CBS and other collections, but is degener-
ated, no longer producing the characteristic dark chlamydospores figured by
Maffei.
A severe case of ‘‘C. serrae’’ keratitis reported by Gingrich (420) had
neither etiologic nor mycological substantiation. The same C. serrae case was
summarized earlier by Gingrich in recorded discussion notes following a seem-
ingly unrelated paper on the experimental destruction of rabbit corneas by en-
zymes from an unidentified ‘‘Cephalosporium’’ (553). Explaining the identifica-
tion of his isolate, Gingrich stated: ‘‘Cephalosporium serrae [is] distinct from
all other Cephalosporium species in that [its] conidiophores are branched.’’ This
statement bore no relation to scientific reality at that (1960) or any earlier time,
making this record uninterpretable. Connection with a microconidial Fusarium
may be speculated, since the case isolate was highly resistant to amphotericin B
in vitro. Gingrich (420) later attributed the findings of the rabbit cornea study to
C. serrae. Apart from its identification as a ‘‘Cephalosporium’’ isolate from kera-
titis, however, the fungus used in the experiments was not identified as to species
or provenance. Gingrich’s (420) statement that ‘‘C. serrae’’ is strongly enzymati-
cally destructive toward the eyes thus appears to be unfounded, or if his own
case isolate was the unidentified strain used in the experiments, at least not to
apply correctly to V. nigrescens.
An isolate from mycetoma in Venezuela identified as C. serrae by de Al-
bornoz (554) was more recently reidentified by W. Gams as representative of a
newly described species, Phaeoacremonium inflatipes W. Gams, Crous et Wingf.
(555). The isolate is in various culture collections: CBS 651.85, ATCC 32628,
and the University of Alberta Microfungus Collection and Herbarium (UAMH)
4034. Contrary to a statement by Guarro et al. (327) in their review of infections
caused by Acremonium and species formerly considered Cephalosporium spp.,
the authentic V. nigrescens is not known from mycetoma. V. nigrescens is a soil
450 Summerbell
fungus of areas of temperate climate. It is especially associated with roots of the

family Solanaceae (potato, tomato, pepper, eggplant) (74).
Description. Colonies grow 23 to 28 cm in 7 days on PDA at 25°C. They
are white and velvety, soon becoming greyish. The reverse is pale at first, then
grey to black as chlamydospores form. It is sometimes also orange-brown at the
center. Conidiophores are mostly erect, forming one, two, or uncommonly more
successively elevated branch points giving rise to 1–2 (-3) phialides. Sporulation
is also seen within the agar on short phialides (Fig. 100). Aerial phialides are
subulate (awl-shaped) to candle-shaped, 20–35 (-50) ⫻ 1.5–3 µm. Conidia are
ellipsoidal or short cylindrical, usually aseptate, rarely one-septate, and 4–8.5 ⫻
1.5–2.5 µm. Chlamydospores are produced after 10 days, dark, singly or in
chains, terminal or intercalary, usually distinctly swollen to near globose, and 5
to 10 µm.
The normal maximal temperature for growth of this fungus is 34°C. The
isolate ex-type of C. serrae appears not to have been tested. V. cinerescens is
one of the Verticillium species shown by molecular phylogeny to have affinities
with the order Phyllachorales, and is remotely related to the teleomorph genera
Glomerella and Plectosphaerella. It can be distinguished from Acremonium spe-
cies by the combination of its erect conidiophores and dark chlamydospores.
Figure 100 Verticillium nigrescens CBS 109724 conidiophores.

Ascomycetes 451
J. Volutella
1. Volutella {aff. ?Pseudonectria} cinerescens (Cesati) Sacc.
This fungus was isolated in New York in 1958 by Foster et al. (556) from three
cases of postsurgical endophthalmitis, one of which was thoroughly documented.
Theodore (557) later illuminated the source of this outbreak by stating that the
same organism was isolated from ‘‘the cocaine used during the surgery,’’ possibly
explaining the paucity of similar outbreaks in recent times. Identification was cred-
ited to G. A. de Vries of CBS, and consistent photos and partial descriptions were
given. Curiously, no culture has been retained at CBS. A fourth confirmed case was
reported under the generic name only by Theodore et al. (557). A keratitis case
from Florida was ascribed without details to a member of the genus Volutella by
Liesegang and Forster (393). The CBS collection contains an isolate of V. cineres-
cens, CBS 832.71, listed as isolated in 1971 from ‘‘keratomycosis’’ by R. C. Zapater
in Argentina. No case report definitely documenting this isolation has so far been
traced by the present author. Zapater (558) does, however, refer in a summary table
to a postsurgical ocular infection caused by V. cinerescens.
Description. Colonies grow moderately rapidly. They are pale above and
below, with profuse formation of cushionlike sporodochial tufts bearing conidia
in slimy masses. Conidiophores are sometimes produced as short single side
branches from aerial or submerged hyphae, but are generally also produced in
tangled, bushlike sporodochial complexes over 50 µm wide (Fig. 101). They are
loosely interwoven, multiply rebranched in a monopodial (maple- or fig-tree-like)
fashion, bundling together more tightly at the base of the sporodochium to form
a compacted stipelike region. Phialides are candle-shaped to elongate-cylindrical,
often with a slightly swollen basal region, especially in nonsporodochial phia-
lides, and 8.5–20 ⫻ 1–2 µm. Short adelophialides (phialidelike, fertile short side
branches not delimited from the subtending hypha by a septum) are also present.
The conidia are ellipsoidal, cylindrical, or somewhat curved, smooth, hyaline,
and 2–4 ⫻ 0.8–1.2 µm. Long, thick-walled, erect setae (spines) with rounded
or pointed tips are also commonly formed from substrate mycelium.
The nonsporodochial phialides and conidia of this species are somewhat
reminiscent of those seen in the otherwise quite different Acremonium kiliense.
Growth on PDA or another non-Sabouraud fungal sporulation medium should
be performed to elicit sporodochia in potential Volutella isolates.
IV. OPHIOSTOMATALES
This order is generally well known for plant pathogenicity; for example, Ophios-
toma ulmi and its offshoot O. novo-ulmi are the agents of Dutch elm disease.
452 Summerbell
Figure 101 Volutella cinerescens CBS 832.71 conidioma.
It also contains one notorious animal-pathogenic anamorph species, Sporothrix

schenckii, the dimorphic fungus that is the agent of sporotrichosis. Many ana-
morphs in this group, whether or not they are associated with a teleomorph, are
currently placed in the genus Sporothrix, and some species with no pathogenic
record (e.g., Sporothrix inflata) are commonly seen (although not commonly
identified) as contaminants from skin and nails. This therefore is a group of fungi
that are reasonably frequently seen in the clinical laboratory.
A. Identification
Most Ophiostomatalean anamorphs can be identified at least to the level of spe-
cies complex by morphological studies conducted primarily with slide cultures.
Many species produce a compact sympodially proliferating conidiogenous cell
bearing an apical ‘‘rosette’’ of conidia, as S. schenckii does. Most of these species
Ascomycetes 453
differ from S. schenckii by producing secondary conidia on the still attached

conidia of the primary rosette, significantly longer conidia, well-organized synne-
mata, or sympodial conidiogenous cells with an inflated apex. [See de Hoog (559)
for very useful keys and descriptions, but note that the synonymy of S. schenckii
and Ophiostoma stenoceras mooted in that work was later revealed to be incor-
rect.] S. schenckii is also tested for conversion to a budding yeast phase at 35°
and 37°C. (Different isolates tend to convert better at one of these temperatures
than the other, so using both temperatures speeds the finalization of the test.) It
is not the only species in the group that produces a yeast phase at these tempera-
tures, but it converts more thoroughly than other species and forms a high propor-
tion of distinctive, elongated ‘‘cigar-shaped’’ buds, often in pairs (forming so-
called rabbit ears). S. schenckii is also unique among described Ophiostomatalean
anamorphs in producing copious dark ‘‘secondary conidia’’ that in fresh isolates
arise densely as single dark subglobose or rarely triangular cells on short stalks
from the substrate mycelium. Their density is often so great that they are said
to form ‘‘sleeves’’ along the hyphae (560). The augmentation in numbers of
these dark conidia rapidly turns colonies dark brown to black. These conidia are
important in helping to distinguish S. schenckii from poorly known, possibly
undescribed Sporothrix spp. sharing its environmental habitats (e.g., peat moss)
as well as the very similar early growth stages of the Sporothrix state of O.
stenoceras. The mimics lack secondary conidia. Molecular methods can also
make the distinction between pathogenic and nonpathogenic types (561).
According to Dixon et al. (560), some mimics of S. schenckii occur that are
nonpathogenic in animal models and possess dark secondary conidia, differing
phenotypically from pathogenic S. schenckii in vitro only in their ability to grow
at 37°C. These authors therefore recommended a test for 37° growth as a refer-
ence test for all S. schenckii-like isolates converting to a yeast phase and forming
dark secondary conidia. Definitive studies, however, have shown that some
proven etiologically significant S. schenckii isolates, especially from fixed cutane-
ous sporotrichosis, grow minimally or apparently not at all at 37°C, despite grow-
ing well at 35° [see review by Kwon-Chung and Bennett (68)]; this is also the
present author’s experience. The small number of apparently nonpathogenic iso-
lates forming dark secondary conidia isolated from peat moss by Dixon et al.
(560) are the only such isolates known; therefore, pending clarification of the
nature of these isolates, all isolates obtained from suspected sporotrichosis and
matching S. schenckii in all characters except the ability to grow at 37°C in vitro
(while still growing at 35°C) should be reported as S. schenckii. Molecular con-
firmation (152, 561) may be done if there is significant uncertainty about the
identification. A recommended molecular analysis is mitochondrial DNA HaeIII
restriction fragment analysis, which allows recognition of geographic variants of
S. schenckii (562) based on studies of a large number of isolates from around
the world.
454 Summerbell
B. Sporothrix
1. Sporothrix {aff. Ophiostoma} schenckii Hektoen & Perkins
(Common Synonym in Older Literature: Sporotrichum
schenckii)
This species, the agent of sporotrichosis, is mainly known as an agent of subcuta-
neous infection subsequent to traumatic inoculation. Most primary infections de-
velop into acute lymphocutaneous infections by the spread of the organism into
regional lymph nodes (68). The usual source of inoculum is plant material, partic-
ularly of a grassy or strawlike nature (563); a large outbreak in 1988 in North
America was associated with poorly stored peat moss (560). In Central and South
America, a nonprogressive form of the disease, fixed cutaneous sporotrichosis,
is seen especially in persons who are repeatedly exposed to inoculum of S. schen-
ckii, and who thus manifest an immune response that tends to prevent lymphocu-
taneous spread. Inoculation of S. schenckii into sites other than skin can cause
other local infections (e.g., keratitis, endophthalmitis, invasive otitis, and mycotic
arthritis) (69). In immunocompromised patients, more severe infections may be
seen; chronic alcoholic debilitation has been noted as a relatively frequent predis-
posing factor for rare pulmonary cases of sporotrichosis (68). England and Hoch-
holzer (564) report that the combination of alcoholism and chronic obstructive
pulmonary disease is seen in most patients with pulmonary sporotrichosis; the
majority also are male and middle-aged. Sporotrichosis in general is much more
common in males than females (68), as is often the case with thermodimorphic
mycoses. Rare idiopathic cases of pulmonary and other extracutaneous sporotri-
chosis may occur in individuals with no known predisposing factors (68). Dis-
seminated sporotrichosis is occasionally seen in patients with AIDS (565–567),
but on the other hand, very few cases of sporotrichosis have been seen in connec-
tion with neoplasms (568), and a Medline search of ‘‘sporot* AND neutropeni*’’
elicits no records. The disease may affect a wide range of animal species and is
particularly common in cats.
Description. Colonies are flat and slow-growing—4 to 15 mm in 7 days on

Sabouraud peptone glucose agar at 25°C. They are whitish then partly deep brown
to black, sometimes near black from the start. The reverse is concolorous with the
surface. Conidiogenous cells arise more or less at right angles to surface hyphae.
They are sometimes terminal, cylindrical, and 10 to 40 ⫻ 1 to 2 µm at maturity,
with a slightly swollen, compact sympodial rhachis of toothlike denticles at the
apex. There are also often sparsely scattered denticles along the stalk. Primary
conidia are formed in sympodial rosettes on conidiophore apices. They are ob-
ovoidal (egg-shaped, attached at the thin end), and 3–6 ⫻ 1.5–3 µm (Fig. 102).
The secondary conidia are deep brown, often subglobose, and approxi-
mately 3 µm in diameter (Fig. 103). They are sometimes triangular, are attached
Ascomycetes 455
Figure 102 Sporothrix schenckii primary sympodial conidia.
Figure 103 Sporothrix schenckii secondary sessile conidia.

456 Summerbell
Figure 104 Sporothrix schenckii yeast phase at 37°C in vitro.
individually on denticles on the sides of the hyphae, especially substrate (in the
agar) hyphae, and are often densely packed and surrounding the hyphae in sleeve-
like formation in fresh isolates.
Converted phase on brain–heart infusion agar overlaid with a few drops
of 10% yeast extract solution and incubated in parallel cultures at 35° and 37°C
(as explained above) gives rise to white, glossy masses of yeast cells, 3–10 ⫻
1–3 µm, with long obclavate (‘‘cigar-shaped’’) buds (Fig. 104). Sympodially
formed pairs of such buds (rabbit ears) are typically seen.
S. schenckii has a distinct variant, S. schenckii var. luriei, generally seen
in southern Africa or India, which is very similar to the type variety in most
cultural aspects but differs in its host phase, producing unusually large, irregular
yeast-like cells as well as septate cells resembling fission cells, rather like hyaline
versions of the fission cells of chromoblastomycosis fungi. A recent case investi-
gation by Padhye et al. (569) reported large numbers of incompletely separated
cells in an ‘‘eyeglass’’ configuration as characteristic of S. schenckii var. luriei
infection. The variety has a distinct mitochondrial DNA type (570) and differs
from the type variety in not assimilating creatine, creatinine, or guanidinoacetic
acid (571). A recent Indian case that did not yield a culture was confirmed by
immunohistochemistry specific for S. schenckii.
Ascomycetes 457
C. Ophiostoma
1. Ophiostoma stenoceras (Robak) Melin & Nannf.
This fungus, closely related to S. schenckii, was reported by Summerbell et al.
(572) as associated with long-term paronychia in a 65-year-old woman who had
acquired the infection while laboring in forestry and agricultural work. The lesion
was direct-microscopic-positive for filaments but not for yeast elements. Unfortu-
nately for the case report (but not for the patient), the infection was rapidly cured
with nonspecific iodine therapy while the laboratory report was pending, and no
repeat isolation for confirmation of etiology could be performed. Ophiostomatalean
fungi are among the few pathogenic fungi highly susceptible to iodine, and the cure
tended to correlate the proposed etiology; however, additional and better confirmed
cases are needed before O. stenoceras can be formally listed as a pathogen.
O. stenoceras forms colonies and conidiogenous structures that are similar
to S. schenckii colonies within their first 10 to 14 days of growth, except for the
following features: (1) no dark secondary conidia are formed; (2) conidia may
appear more elongate and clavate than typical S. schenckii primary conidia [ac-
cording to de Hoog (559) the difference between the two species is not statisti-
cally significant, but restudy of this matter using fresh isolates would be ideal];
(3) dark brownish to black spots not associated with secondary conidia may de-
Figure 105 Ophiostoma stenoceras ascomata (immersed in cloud of spores).

458 Summerbell
velop on the colony surface; and (4) attempts to convert the organism to the yeast
phase give a partial conversion, with most elements still recognizably hyphae or
mycelial conidia in microscopic examination even though the colony has a pasty
appearance. After 14 days on modified Leonian’s agar, ascomata form (Fig. 105).
They are black, with a bulbous base 80 to 180 µm, and with an elongate (up to
2 mm), thin neck bearing a crownlike ring of short, sharp, spinelike setae 18 to
35 (-55) µm, at the ostiole, reflexed outwardly to support an extruded, slimy mass
of pale, smooth-walled, bean- or orange-segment-shaped ascospores (2.0-) 2.5–
4.5 ⫻ 1.0–1.5 µm (Figs. 106 and 107). Unlike S. schenckii, O. stenoceras de-
grades starch in vitro (572).
Figure 106 Ophiostoma stenoceras ascoma apex.

Ascomycetes 459
Figure 107 Ophiostoma stenoceras ascospore.
V. DOTHIDEALES, PRO PARTE (OTHER MEMBERS OF

THIS GROUP WILL APPEAR IN OTHER CHAPTERS)
A. Neotestudina
1. i. Neotestudina rosatii Ségr. & Destombes (Synonyms:
Zopfia rosatii, Pseudophaeotrichum sudanense,
Pseudodelitschia coriandri )
This ascomycetous fungus is a well-characterized agent of mycetoma (573). Like
many such fungi, it is currently identified by the characters of its ascomata rather
than from general cultural characters (Fig. 108 and 109).
Figure 108 Neotestudina rosatii ascospores.

460 Summerbell
Figure 109 Neotestudina rosatii: (a) cortical, subcortical, and ascogenous tissues; (b)
surface view of the peridium; (c) asci; (d) ascospores.
Ascomata are globose, black cleistothecia, 100 to 200 µm in diameter. The

peridium (ascomatal wall) is smooth, multilayered, with a pseudoparenchymatous
(cellular-looking) outer layer. It is divided into plates composed of radially ar-
ranged, thick-walled cells 4 to 10 µm wide, which are block-shaped near the
centers of the plates and become elongate and somewhat curved near the margins.
Asci form laterally on ascogenous hyphae. They are bitunicate and broadly ellip-
soidal to subglobose, containing eight ascospores. Ascospores are two-celled,
rhomboidal (i.e., with abruptly curved, nearly pointed ends and with an abrupt
change of cell wall angle at the central septum), with thick, smooth walls. They
are brown, 9–12.5 ⫻ 4.5–8 µm, with germ pores at both ends. No anamorph is
present. Growth occurs to 40°C.
Ascomycetes 461
B. Piedraia
1. Piedraia hortae (Brumpt) da Fonseca & Arêa Leão
This is one of the few truly contagious fungal parasites of humans. It also occurs
on other primates. Black concretions are attached to the shaft of scalp hairs,
and ascomata form within these concretions. Identification is therefore by direct
microscopic analysis of the concretions; the fungus may be grown in pure culture,
but is hard to isolate, slow-growing, and generally nonsporulating. The disease,
piedra, may be acquired by contact with soil and is cultivated as a sign of beauty
in some cultures (11). A synopsis of recent references on the biology of P. hortae
is provided by de Hoog et al. (69). Extensive overviews are given by Rippon
(11) and Kwon-Chung and Bennett (68).
Ascomata are ascostromata, and are compact, black, very variable in size,
up to 1 mm long by 0.33 mm wide, and irregularly lumpy with formation of
fertile locules (Fig. 110). Stromatal material is composed of vertical rows of
thick-walled, dark, polygonal cells that are 2–10 ⫻ 2.5–6.5 µm. Fertile locules
are 30 to 60 µm in inner diameter, opening to the surface by means of an incon-
spicuous ostiolar pore. Asci are bitunicate, ellipsoidal, and evanescent, with two
to eight hyaline. They are elongate, sinuously curved, and spindle shaped (i.e.,
with acutely angled, rounded ends). The ascospores are 30–45 ⫻ 5.5–10 µm,
and bear whip-like extensions at both ends. A similar species, P. quintanilhae van
Uden et al., differs by lacking the whip-like ascospore appendages, and occurs in
wild populations of chimpanzees (574).
VI. PLEOSPORALES, PRO PARTE (OTHER MEMBERS OF

THIS GROUP WILL APPEAR IN OTHER CHAPTERS)
A. Leptosphaeria
1. Leptosphaeria senegalensis Ségretain et al.
This species was described based on isolations from mycetoma in Senegal and
Mauritania (575) (Figs. 111, 112, and 113).
Description. Colonies grow slowly, according to de Hoog et al. (69), but
show ‘‘great variability in growth rate’’ and grow more quickly at 37° than at
30°C, according to De Vries (3). They are velvety to wooly, pale at first, then
becoming dark brownish-grey with a nearly black reverse. Brownish soluble pig-
ment may be formed. Ascomata form after 30 to 60 days on oatmeal agar. They
are black, 100 to 300 µm in diameter, and covered with short, flexuous, dark
hyphae. They are subglobose, lacking an ostiole, with a thick peridium composed
of several layers of isodiametric cells, and the inner ones are less dark than the
462 Summerbell
Figure 110 Piedraia hortae: (a) border of subcuticular zone of a mature ascostroma;
(b) young ascotroma; (c) mature ascus; and (d) ascospores.
outer, cortical layer. Asci are bitunicate, clavate, and 80–100 ⫻ 17–22 µm. They
are eight-spored, arising from a stroma, and intermingled with thin, occasionally
branched paraphyses. The ascospores are hyaline to pale brown, ellipsoidal, and
23–30 ⫻ 8–10 µm, usually with five linearly arranged cells, less commonly six,
distinctly constricted at the septa. Each ascospore is embedded in a thick, hyaline
to slightly colored slimy sheath.
This species is distinguished from L. tompkinsii below by producing mostly
five-celled ascospores with rounded ends rather than mostly seven-celled asco-
spores with pointed ends. The ascospores of L. senegalensis have a broad gel
sheath described by El-Ani (576) as ‘‘turbinate [top-shaped] greatly enlarged
near the spore apex,’’ while sheaths of L. tompkinsii are ‘‘fusoid [spindle-shaped]
Ascomycetes 463
Figure 111 Leptosphaeria senegalensis ascoma peridium with ostiole.
like the spore.’’ Further details of the distinction are given by El-Ani (576).
Ségretain et al. (575) and de Vries (3) note rare ascospores with up to eight
cells in L. senegalensis, but this appears to be questioned by El-Ani (576), who
studied 823 spores of four L. senegalensis isolates and saw 810 four-celled spores,
13 five-celled, and none with more cells. Because there may have been some
early confusion between the two species, I have cited El-Ani’s measurements for
L. senegalensis microscopic structures above in preference to those from other
sources.
2. Leptosphaeria tompkinsii El-Ani

This species was described based on an isolate from mycetoma in Mauritania
(576) (Fig. 114).
Description. Colonies grow slowly, according to de Hoog et al. (69), but
show ‘‘great variability in growth rate’’ and grow more quickly at 37°C than at
30°C, according to de Vries (3). They are velvety to woolly, pale at first, then
becoming dark brownish-grey with a nearly black reverse. Brownish soluble pig-
ment may be formed. Ascomata form after 30 to 60 days on oatmeal agar. They
are black, 214 to 535 µm in diameter, and covered with short, flexuous, dark
464 Summerbell
Figure 112 Leptosphaeria senegalensis asci containing ascospores.
Figure 113 Leptosphaeria senegalensis, Segretain et al. (IP 614): (a) mature ascus; (b)
ascospores.
Ascomycetes 465
Figure 114 Leptosphaeria tompkinsii El-Ani (IP 1151.76): (a) mature asci; (b) asco-
spores.
hyphae. They are subglobose to compressed, lacking an ostiole, with a thick

peridium composed of several layers of isodiametric cells, the inner ones less
dark than the outer, cortical layer. The upper portion of the peridium is often
thinner than the lower portion. Asci are bitunicate, clavate, and 80–115 ⫻ 20–
25 µm. They are eight-spored, arising from a stroma, intermingled with thin,
rarely branched paraphyses. The ascospores are hyaline, fusoid (spindle-shaped;
i.e., with ends tapering to a relatively acute conical point), and 32–45 ⫻ 8.8–11
µm, with (5-) 7 to 8 (-9) linearly arranged cells, slightly constricted at the septa,
with the second or third cell from the apical end often somewhat inflated com-
466 Summerbell
pared to the others. Each ascospore is embedded in a thin, hyaline, slimy sheath.
Distinction from L. senegalensis is given above under that species.
VII. PSEUDEUROTIACEAE SS. STR. SENSU SUH AND

BLACKWELL (1)
A. Pseudeurotium
1. Pseudeurotium ovale Stolk
English et al. (577) investigated an onychomycosis case in which P. ovale was
shown to be established in at least one and likely at least five of an English
patient’s 10 dystrophic nails. In a follow-up study of 200 nail fragments, 20 from
each of the patient’s nails, a single colony of the dermatophyte Trichophyton
rubrum was obtained from one nail, while the same nail and four others, all
positive for fungal elements in direct microscopy, yielded P. ovale. The same
species was also isolated from another patient as a likely incidental contaminant
from a typical Acremonium sp. (recorded as Cephalosporium sp.) superficial
white onychomycosis. In the former case, the etiologic status of P. ovale was
not entirely clarified, as English et al. pointed out. In the later terminology of
Summerbell (578), it may have been an etiologically irrelevant but established
‘‘secondary colonizer’’ of material predigested by the dermatophyte, or it may
have been an etiologically significant ‘‘successional invader,’’ which gained ac-
cess to the nail as a result of dermatophyte disturbance but did not require the
continuing presence of T. rubrum to persist.
Description. Colonies grow 25 to 30 mm in 7 days. They are cottony to

somewhat granular, pale at first, then brownish-grey after ascomata have devel-
oped. The reverse is white, greenish, or brownish. Ascomata are cleistothecia
(i.e., rounded structures lacking an ostiole or differentiated opening), mostly 90 to
180 µm in diameter (Fig. 115). They are dark, bald, with a rather tough peridium
composed of polygonal cells. Asci are ellipsoidal to spherical, and 7–9 ⫻ 6.5–
8 µm. Ascospores are ellipsoidal to ovoidal, 4–6 ⫻ 2.5–4 µm, smooth-walled,
and hyaline to pale olive-brown. Conidiophores are hyaline, vaguely Sporothrix-
like, but lacking toothlike denticles at conidial attachment points. They consist of
minimal stub-like side branches not delimited by a crosswall or more developed
structures, with the most differentiated being delimited, slightly swollen, smooth-
walled, single-celled lateral branches up to 20 µm long with tapering tips bearing
at maturity an apical rosettelike cluster of several sympodially produced conidia
attached by flat bases. Conidia are ellipsoidal to slightly irregular, smooth, pale,
and mostly 4–6 ⫻ 2.5–3.5 µm. The related Pseudeurotium zonatum has spheri-
cal, not ellipsoidal ascospores.
Ascomycetes 467
Figure 115 Pseudeurotium ovale CBS 247.89 ascoma breaking open to reveal asco-
spores.
ACKNOWLEDGMENTS
This chapter is dedicated to Dr. K-J. (June) Kwon-Chung, who was associated
with an impressive number of the best-quality case reports seen in its preparation.
I would like to thank Katsuhiko Ando for Japanese translation, Walter
Gams for German translation and Latin advice, Marjan Erich-Vermaas for pho-
tography, Rob Samson for managerial intervention, and Arien van Iperen for
preparation of cultures. Gerard de Vries is thanked for drawings, and the Instruc-
tional Media Centre, Ontario Ministry of Health, for inking them.
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8
Yeasts
Blastomycetes and Endomycetes
Kevin C. Hazen
University of Virginia Health System, Charlottesville, Virginia, U.S.A.
Susan A. Howell
St. John’s Institute of Dermatology, King’s College of London,
London, England
I. INTRODUCTION
The Blastomycetes and Endomycetes contain a large number of medically impor-

tant yeast species, most notably species within the genus Candida, which is re-
ferred to as a form genus because the genus itself is a repository for yeast species
having certain characteristics and lacking sexual reproduction. The lack of sexual-
ity limits taxonomists in their phylogenetic attempts to relate the various species
when using traditional identification criteria. Molecular methods, however, have
provided a new and exciting means to establish the phylogenetic relationships
of not only the blastomycetous yeasts but also the endomycetous yeasts. In this
chapter, we attempt to review the results of traditional and molecular studies of
yeast taxonomy and relate those results to the clinical mycology laboratory. It is
evident that yeast taxonomy will continue to evolve as more refined techniques
are developed.
II. TAXONOMY AND CLASSIFICATION
The class Blastomycetes belongs in the division Deuteromycota, and the class
Endomycetes are yeasts within the division Ascomycota (Table 1). Each order
has two families of medically important organisms (Table 1).
499
500
Table 1 Taxonomy and Classification of Yeasts Within the Blastomycetes and Endomycetes
Division Class Order Family Description
Ascomycona Produce asci.

Endomycetes Asci not contained in ascocarp (naked asci).
Saccharomycetales Form asci directly from zygote or zygote’s diploid
progeny.
Dipodascaceae Anamorphs with arthroconidia.
Saccharomycetaceae Anamorphs with budding cells.
Deuteromycota Lack meiotic spores.
Blastomycetes Vegetative cells are yeasts. May produce pseudo-
mycelium. When present, true mycelium is
poorly developed.
Sporobolomycetales Produce ballistospores.
Sporobolomycetaceae Reproduction by fission or budding. Produce ballis-
tospores on sterigmata arising from vegetative
cells.
Cryptococcales Nonsexual yeasts that do not produce sterigmata.
Cryptococcaceae Budding cells always present. May produce pseudo-
mycelia, mycelium, and arthroconidia. Cells may
be hyaline or pigmented but rarely brown or
black.
Hazen and Howell
Yeasts 501
Yeast taxonomy is under constant revision. Recent advances in molecular

methods for phylogenetic analysis have particularly caused significant revisions
of taxonomic assignments. These advances have helped to demonstrate ascomy-
cetous and basidiomycetous affinities for certain imperfect (nonsexual) yeasts,
such as Candida albicans. In the absence of a sexual structure, however, these
organisms remain in the fungi imperfecti (Deuteromycota)—a pseudotaxonomic
repository for organisms with no known sexual state.
This entire division, Deuteromycota, is sometimes referred to as a ‘‘form
division’’ to indicate that the term division is used arbitrarily, and unlike the
true divisions, implies no taxonomic (or evolutionary) significance. When sexual
reproduction is observed along with the attendant sexual reproductive structures
(e.g., asci), the form-genus species is reassigned to the appropriate taxonomically
legitimate division and renamed with an epithet consistent with the division. Such
renaming has in practice led to confusion in scientific and clinical discussions
about yeast infections, but adherence to this classification principle is important
because it provides insights into the relationship of the organism to similar or-
ganisms. The demonstration that some species of Candida belong to the asco-
mycetous yeasts and others to the basidiomycetous yeasts illustrates the pseudo-
taxonomic value of the form-order term Cryptococcales. Knowing the correct
taxonomic assignment provides information useful for identifying these organ-
isms isolated from patient specimens.
As noted above, a number of traditional mating and recent molecular analy-
ses have led to the reassignment of various blastomycetous yeasts to either the
endomycetes or one of the basidiomycetous yeast genera. This chapter will not
describe the basidiomycetous yeasts but will focus only on the strictly asexual
yeasts and the endomycetous yeasts. For blastomycetes reassigned to endo-
mycetous genera (Table 2) we will primarily use the more common (albeit taxo-
nomically invalid) blastomycetous species binomial because these organisms are
typically seen only in the anamorphic form. Blastomycetous yeasts with basidio-
mycetous affinities (Table 2) will be described only relative to their blastomyce-
tous features.
III. TRADITIONAL METHODS OF IDENTIFICATION

AND CLASSIFICATION
Several characteristics of fungi can be used to separate different classes. The

definitive characteristics for studying ascomycetous fungi are sexual propagules
and fruiting structures. Other characteristics used to differentiate the ascomyce-
tous yeasts include vegetative cell morphology, cell wall polysaccharides, co-
enzyme Q families (e.g., coenzyme Q 10 is present only in basidiomycetous fungi),
502 Hazen and Howell
Table 2 Classification of the Medically Important Yeast Genera Within the

Blastomycetes and Endomycetes
Class Order Teleomorph genus-species Anamorph
Endomycetes Saccharomycetales Arxiozyma telluris Candida pintolopesii

Citeromyces matritensis Candida globosa
Clavispora lusitaniae Candida lusitaniae
Clavispora capitatus Blastoschizomyces capitatus
Debaryomyces hansenii Candida famata
Galactomyces geotrichum Geotrichum candidum
Hansenula anomala Candida pelliculosa
Issatchenkia orientalis Candida krusei
Kluyveromyces lactis Candida sphaerica
Kluyveromyces marxianus Candida kefyr
Metschnikowia pulcherrima Candida pulcherrima
Pichia guilliermondii Candida guilliermondii
Pichia fermentans Candida lambica
Pichia jadinii Candida utilis
Pichia membranaefaciens Candida valida
Pichia norvegensis Candida norvegensis
Saccharomyces cerevisiae
Saccharomyces exiguus Candida holmii
Stephanoascus ciferrii Candida ciferrii
Yarrowia lipolytica Candida lipolytica
Blastomycetes Sporobolomycetales Sporobolomyces
Cryptococcales Blastoschizomyces
Candida
Cryptococcus
Malassezia
Rhodotorula
Trichosporon
morphology of septa (e.g., the dolipore-parenthesome septum of most basidio-

mycetes versus the ascomycetous simple pore septum with Woronin body), sen-
sitivity to killer toxins, membrane fatty acid components, ascus and ascospore
features, temperature tolerance, urease production, fermentation, nitrate assimila-
tion, and intranuclear mitosis. In contrast, the imperfect yeasts have features that
are either ascomycetous or basidiomycetous but lack sexual structures (asci and
basidia), therefore key characteristics to help identify the asexual blastomycetes
include most of those listed above for ascomycetous fungi plus diazonium B blue
reaction, ballistospore formation, and production of carotenoids (1–3). Molecular
methods have significantly enhanced the ability to classify imperfect yeasts. The
recent development of proteomics may also provide useful classification criteria
to separate the yeasts.
Yeasts 503
The definitive methods of determining classification of an unknown yeast

are not readily available to the clinical laboratory. Clinical laboratories rely on
a limited set of characteristics that provide a most likely approximation of an
organism’s taxonomic position. Morphology can provide some information (e.g.,
in the case of the genus Kloeckera or species Metschnikowia lunata), but other
tests, such as assimilation and fermentation characteristics and exoenzyme pro-
duction, may be used. When possible, the clinical laboratory may be able to
induce mating reactions and obtain the arrangement and morphology of the resul-
tant meiotic progeny. Mating reactions are relatively easy to induce if the yeast
isolate is homothallic. When the organism is heterothallic, it may be necessary
to try several different species along with both mating types before the right
mating cognate is obtained to allow identification. Ascospore formation of most
hemiascomycetous yeasts can be induced by growing them on dilute V-8 juice
agar, but some medically important genera may require specific media, such as
ascospore agar. The incubation temperature is typically 25°C, but Metschnikowia
species may require temperatures between 12–15°C, and the optimum tempera-
ture for sporulation of Debaryomyces species is below 20°C.
The essential characteristics for separating ascomycetous yeasts are the sex-
ual spores and accompanying structures. (See the Appendix.) The color, number,
size, morphology, ornamentation, and arrangement of the ascospores within the
ascus and the characteristics of the asci provide definitive information to ascertain
the classification of an unknown yeast. In the absence of sexual reproduction,
clinical laboratories will use morphology and assimilation/fermentation patterns
as the key criteria to determine an organism’s taxonomic position. Particularly
useful tests for clinical laboratories include urease production (generally ascomy-
cetous yeasts are negative and basidiomycetous yeasts are positive), inositol as-
similation (generally ascomycetous yeasts are negative and basidiomycetous
yeasts are positive), and nitrate assimilation. Commercial test systems (e.g., the
API 20C and the IDS RapID) provide good accuracy for common yeasts but may
not be particularly useful for unusual yeasts. In addition, these tests can some-
times provide species identifications that are at best questionable. Similarly, C.
inconspicua may be misidentified as C. krusei. Karyotyping, in this case, deter-
mined the correct identity (4).
The most useful assimilation/fermentation tests are the auxanographic
methods modeled after the work of Wickerham and Burton (5). Taxonomic trea-
tises provide tables of the reactions with the various substrates based on the Wick-
erham method. Commercial assimilation systems do not show 100% agreement
with the Wickerham method, particularly with the more unusual clinical isolates.
A clinical laboratory using assimilation results from commercial kits must be
aware that some results will not correlate with the Wickerham and Burton
method.
The implications from such studies are significant, given the growing phar-
maceutical interest in developing new agents for treatment of mycoses. The
agents generally show narrower spectra of species efficacy than older agents, such
as amphotericin B. Antibiograms of Candida species have shown that species
identification is important before the choice of appropriate antifungal agent for
a given clinical situation can be made.
One of the common problems faced by clinical laboratories—which rely
on phenotypic tables of biochemical/physiological characteristics for yeast iden-
tification—is the consistency between the methods employed to generate the
characteristics defined on the table and the methods used in the clinical labora-
tory. For example, urease production is essentially a characteristic of the basidio-
mycetous yeasts. When the presence of urease in taxonomically different yeasts
is evaluated, however, positive tests are obtained for many yeast species if the
test medium is not sufficiently buffered. This factor of buffering may account
for the variable urease results reported among isolates of C. krusei. The conditions
for urease production thus must be identical to those used to generate the table
of phenotypic characteristics.
A similar situation occurs with cycloheximide (CHX) sensitivity testing.
Cycloheximide sensitivity is a useful characteristic for separating closely related
species. Clinical laboratories typically use media containing CHX at concentra-
tions of 400 to 500 µg/ml, and may presume that such media are suitable for
testing sensitivity to CHX. At least two major compendia of yeast species (1, 3),
however, noted that CHX concentrations in media may vary from 100 to 1000
µg/ml and that species can fall into groups based on their level of sensitivity.
This point was also determined by Whiffen (6), who originally reported the use
of CHX for species discrimination. Some isolates may develop tolerance to lower
concentrations, making the reliability of species discrimination based on sensitiv-
ity to low concentrations of CHX unreliable. Which specific concentration is used
to assess CHX sensitivity for inclusion in a table of yeast phenotypic characteris-
tics is often not stated, but clinical laboratories should be aware that it is likely
the concentration is not the one found in standard clinical fungal growth media.
The incubation temperature at which the CHX sensitivity test is performed may
also influence the final result.
Nitrate assimilation is also a useful characteristic that can help to identify
the species of an organism and help demonstrate taxonomic associations. The
presence of nitrate reductase (as detected by a rapid swab test) does not imply
that an organism can assimilate nitrate. The test thus must be consistent with the
methods used to generate the phenotypic table.
‘‘Definitive’’ taxonomic and classification evaluation of an isolate with
subsequent placement into a species is not always possible with assimilation and
fermentation tests. Additional tests are always necessary if the organism is under
Yeasts 505
consideration as the type species of a new taxonomic epitaph. In this situation,

molecular methods provide precise information.
IV. NEWER METHODS OF IDENTIFICATION AND

CLASSIFICATION
In recent years there has been an explosion in the variety of molecular methods
developed to examine taxonomic relationships. Two of the original DNA-based
methodologies used the mol % G⫹C ratio and DNA :DNA hybridization to deter-
mine the species identity. Isolates of the same species would be expected to have
similar mol % G⫹C and DNA: DNA reassociation values. The values indicate
if the organisms belong to the same species or genus or are unrelated, but do
not provide information of taxonomic relationships. Phylogenetic relationships
between organisms are measured by using nucleotide sequence divergence. Se-
quences of conserved genes have been studied, but the most popular targets for
these analyses have been the rRNA genes.
Alternative methods that do not require sequencing of nucleic acids have
been developed for identification purposes rather than strictly for taxonomy, and
are more suited to the clinical and research environments. The comparison of
the sizes and numbers of chromosomes by karyotyping with pulsed-field gel elec-
trophoresis has been used for both epidemiology and for examining the genetic
organization of similar species. DNA probes have been produced that were spe-
cies-specific or generated species-specific profiles. Restriction enzyme digests
of DNA have been used to produce profiles characteristic of species or strains.
Fingerprinting techniques that use the polymerase chain reaction have been devel-
oped to demonstrate intraspecies differences for epidemiology and interspecies
differences for identification of yeasts.
V. EFFECT OF MOLECULAR ANALYSIS ON

CLASSIFICATION AND IDENTIFICATION
The morphological and physiological tests for traditional species identification

of yeasts can be unreliable or difficult to interpret, especially if the organism is
metabolically unreactive. Furthermore, the characteristics obtained by these tests
provide little evidence of the evolution of species. In such cases, various cell
components may be examined for taxonomic significance and usefulness in as-
sessing the relatedness of species and genera. Only the methods using nucleic
acids will be discussed here, although many of the cited papers contain references
to alternative phenotypic, physiological, or chemotaxonomic analyses, applicable

to the study of blastomycetous and endomycetous yeasts.
A. Phylogeny Deduced from rRNA Sequence Data

The impact of molecular methods on the phenotypically defined classification of
yeasts was reviewed by Kurtzman (7). Based on the partial sequences of small
and large subunit (SSU and LSU, respectively) rRNA the ascomycetous yeasts
were found to be monophyletic, and the fission yeasts (Schizosaccharomyces)
were separated from budding and filamentous yeasts. Phylogenetic trees con-
structed from the rRNA sequences of the D2 region of the LSU demonstrated
the basidiomycetous yeasts to be divided into two clades broadly reflecting the
type of hyphal septum, the presence or absence of teliospores in the sexual state,
and the occurrence of cellular xylose. Clade A contained species of Rhodotorula
and Sporobolomyces, among others, which have simple septal pores and lack
xylose. Clade B contained species, including Malassezia, Trichosporon, and
Cryptococcus, which have dolipore septa and xylose. Kurtzman (8) also used
nucleotide divergence of the LSU and SSU rRNAs to demonstrate that genera
as diverse as Saccharomyces, Debaryomyces, Metschnikowia, and Galactomyces
belong to a single order, the Endomycetales (now designated Saccharomycetales).
The evolutionary relationships of species within genera have been exam-
ined by sequencing data. Metschnikowia species possess a unique characteristic
among the ascomycetous yeasts, a large deletion in the LSU rRNA 25S-635-
initiated region (9). Analysis of the sequence divergence within this genus
suggests that the aquatic species M. australis, M. bicuspidata, M. krissii, and
M. zobellii form a separate group from the terrestrial species M. hawaiiensis, M.
lunata, M. pulcherrima, and M. reukaufii. Two of the terrestrial species—M.
hawaiiensis and M. lunata—contained even larger deletions in the LSU region
and were well separated from the other members of the group. The authors com-
ment on the surprisingly large sequence divergence for a group of organisms
with relative phenotypic homogeneity, and suggest that either the genus is very
old or underwent rapid evolution, given the parasitic associations of this genus
and the need to adapt to specific niches.
In contrast, isolates identified as Galactomyces geotrichum were heteroge-
neous, yielding inconclusive data from mating reactions with test strains belong-
ing to G. geotrichum sensu stricto and G. citri-aurantii, and exhibited variable
levels of DNA similarity (10). Examination of 57 isolates representing G. geotri-
chum, G. citri-aurantii, and G. reessii identified six groups on the basis of mol
% G⫹C and DNA: DNA reassociation values; isolates of G. geotrichum formed
four of these groups. The only isolates recovered from human sources were all
placed in G. geotrichum group A along with many other environmental isolates.
The authors suggest that the reasons for such heterogeneity in this species could
Yeasts 507
be that the groups are at an early stage of species differentiation or that sexual
reproduction was induced by the organisms themselves.
Phylogenetic studies of some groups of organisms demonstrate relation-
ships not apparent from traditional classification. Ando et al. (11) used partial
sequences of 18S and 26S rRNA to examine phylogenetic relationships of species
of the genus Kluyveromyces, and compared the data with a strain of Saccharo-
myces cerevisiae. The genus was heterogeneous, with several species closely
related to S. cerevisiae. Phenotypically Saccharomyces and Kluyveromyces are
similar in some aspects (e.g., the major ubiquinone is Q-6, and nitrate is not used
as the only source of nitrogen), but differ in both the number of ascospores per
ascus and the morphology of the ascopores. James et al. (12) examined the 18S
rRNA gene sequence of species of Saccharomyces, Kluyveromyces, and Zygosac-
charomyces, and demonstrated Saccharomyces to be heterogeneous with some
species of different genera more closely associated with each other than with
members of their own genus. Some close associations were confirmed by se-
quence analysis, such as for the four species of the Saccharomyces sensu stricto
complex (S. bayanus, S. cerevisiae, S. paradoxus, and S. pastorianus) and S.
exiguus with its anamorph Candida holmii, and these groupings also shared close
phenotype description. Phylogeny could therefore be useful in determining the
phenotype characteristics that have greatest importance in taxonomic classifica-
tion.
Another example where the comparison of phylogenetic and phenotypic
classification needed to be carefully considered concerned the differentiation of
Issatchenkia and Pichia species.
Comparison of the partial sequences of 18S and 26S rRNA of 10 Issatchen-
kia species indicated Issatchenkia orientalis to be more closely related to Pichia
membranaefaciens than to P. anomola or the other Issatchenkia species, and the
genus was described as phylogenetically divergent (13). Pichia species were dem-
onstrated to be heterogeneous when a phylogenetic tree of 204 ascomycetous
yeasts had 20 species of Pichia dispersed throughout (14). An important criterion
of yeast classification is morphology of ascospores. Pichia species produce
smooth hat-shaped ascospores, whereas Issatchenkia produce roughened, round
ascospores. The conflict between morphology and sequencing analysis indicates
that the criteria defining these two genera should be re-examined.
The evolutionary relationships in SSU rRNA sequences between pathogens
of the genus Candida and related species were examined, and organisms were
found to cluster according to the existence of a known teleomorph and disease-
causing capability (15). C. albicans, C. tropicalis, C. parapsilosis, and C. viswa-
nathii form a closely related subgroup, all of which can cause disease in humans
and have no determined teleomorph species. The next branches on the phyloge-
netic tree contained C. guilliermondii and then C. lusitaniae, both disease-causing
agents but with known teleomorphs, Pichia guilliermondii and Clavispora lusi-
taniae, respectively. The evolutionary distance then increased in order with Can-
dida glabrata, Hansenula polymorpha, Candida kefyr, Kluyveromyces marxianus
var. lactis, Saccharomyces cerevisiae, Candida krusei, and distantly Yarrowia
lipolytica. Of these latter species only C. glabrata does not have an identified
teleomorph and is closely associated with S. cerevisiae. Similar groupings of
these species were produced in the survey of nucleotide divergence in the LSU
rDNA gene of 204 ascomycetous yeasts (14). One clade contained Lodderomyces
elongisporus, Candida parapsilosis, C. tropicalis, C. maltosa, C. viswanathii
(and its synonym C. lodderae), C. albicans, and C. dubliniensis. C. glabrata was
again more closely associated with S. cerevisiae. Pathogenic species of yeasts
were not confined to a particular clade, however, as C. guilliermondii, C. zeyla-
noides, and Clavispora lusitaniae were placed in other distinct branches of the
phylogenetic tree.
B. Species Identification and Relationships Demonstrated

by Other Molecular Methods
Yeasts that are germ-tube- and chlamydoconidium-positive have been studied
using a wide spectrum of molecular methods to determine if the yeasts should
be considered. C. albicans, C. stellatoidea, or C. dubliniensis. The karyotypes
and genetic organization of C. albicans and C. stellatoidea type I were shown
to be different, although C. stellatoidea type II was indistinguishable from C.
albicans. The results agreed with the description type II as a sucrose-negative
variant of C. albicans (16, 17). The literature in the early 1990s, however, con-
tained many references to atypical C. albicans, yeasts that differed in their assimi-
lation profiles and produced distinctive patterns with the C. albicans-specific
probe 27A (18–20).
In 1995 Sullivan et al. (21) proposed these atypical C. albicans yeasts as
a separate species, C. dubliniensis, on the basis of genetic comparisons. Isolates
of C. albicans and C. stellatoidea type II were indistinguishable from each other
by fingerprinting with oligonucleotide probes, RAPD patterns, karyotype, and
sequence of the V3 LSU rRNA region; C. stellatoidea type I could be distin-
guished only by karyotype. Isolates of C. dubliniensis, however, were distinguish-
able by these methods, but most importantly by the sequencing data. The C.
dubliniensis strains differed from C. albicans by 14 nucleotides and from C.
stellatoidea by 13 positions. C. albicans and C. stellatoidea differed only at one
position, however. This information suggests that C. albicans and C. stellatoidea
should not be in separate species and that C. dubliniensis should be considered
separate with a sequence divergence of more than 2%. Investigations examining
different loci have reached similar conclusions. Sequencing the ACT1 gene dem-
onstrated C. dubliniensis to be a unique taxon in Candida (22), while amplifica-
tion of ITS regions, or the V3 region of the 23S rRNA, followed by restriction
Yeasts 509
enzyme analysis (REA) showed C. albicans and C. stellatoidea to be indistin-

guishable and C. dubliniensis to be different (23).
Similar techniques have been used to assess relationships between other
yeast species. Karyotyping by pulsed-field gel electrophoresis and hybridization
to restriction enzyme digests with species-specific probes supported the finding
that C. tropicalis was conspecific with C. paratropicalis and that C. krusei was
conspecific with Issatchenkia orientalis (24).
The results of nucleotide sequences do not, however, always agree with
the results from alternative methods. DNA base composition and sequence simi-
larity of strains of Candida utilis and Hansenula jadinii showed these two species
to be indistinguishable (25). The electrophoretic karyotypes of 13 C. utilis isolates
and one H. jadinii isolate, however, demonstrated a large degree of chromosome
length polymorphism, although the mtDNA restriction digest patterns were all
similar (26). The electrophoretic karyotype is known to be variable for some
species, such as C. albicans and C. glabrata, where it has been used as an epide-
miological tool (27, 28). For other organisms the karyotype appears to be remark-
ably consistent within species. Different karyotypes were shown to exist among
the Malassezia species, and some corresponded to the different cell types of
Malassezia furfur (28, 29). Currently the genus Malassezia is divided into seven
species, based on differences in phenotype and genotype. Each species has a
characteristic karyotype, and is molecularly defined by differences in the DNA
base composition, reassociation values, and LSU rRNA sequence data (30–32).
Clearly taxonomic deductions based on karyotyping are highly dependent on the
organism and the frequency of genetic reorganization characteristic for isolates
of that species.
A more rapid method of species identification involves PCR amplification
of sections of the rRNA genes followed by restriction enzyme digestion of the
PCR products to give characteristic electrophoretic patterns. The yeasts C. albi-
cans, C. tropicalis, C. krusei, C. kefyr, C. lusitaniae, C. guilliermondii, C. gla-
brata, and S. cerevisiae were identified by digestion of the 18S rDNA gene with
six restriction enzymes. The method failed to distinguish between C. parapsilosis
and C. viswanathii and between Cryptococcus neoformans and Trichosporon
beigelii (33), however. Isolates of C. krusei, C. inconspicua, and C. norvegensis,
all species that are fluconazole-resistant and occasionally fail to be distinguished
by assimilation tests incorporated in the API 32C strips, were distinguished by
amplification of the internal transcribed spacer (ITS) region and digestion with
the enzyme Hhal (34). Species of the Saccharomyces sensu stricto complex
analyzed by PCR–REA differentiated S. cerevisiae and S. paradoxus from each
other and from S. bayanus and S. pastorianus, which were indistinguishable (35).
Messner and Prillinger (36) used digests of the 18S rDNA with internal tran-
scribed spacer regions and the 25S rDNA to identify 10 type strains of recognized
Saccharomyces species. The results were reproducible and found to be in accor-
dance with sequencing analyses of the same locus. The reproducibility of the
method makes it suitable for isolate identification once a database of restriction
patterns of reference stains is obtained. This contrasts with the results generated
by random amplification of polymorphic DNA (RAPD), where the DNA finger-
print can be affected by many factors.
RAPD is increasingly being applied to species identification. Candida
paratropicalis was shown to be a sucrose-negative variant of C. tropicalis, as
isolates of both species produced nearly identical patterns with a panel of 10-
mer primers (37). Candida guilliermondii and C. fermentati are very similar
yeasts, but can be distinguished phenotypically by the ability of the latter to
ferment galactose at 30°C after 21 days. Rapid identification of these species by
RAPD demonstrated that C. fermentati was not unusual among clinical specimens
(38). Isolates from culture collections representing nine species were compared
by combining the patterns generated by using five 10-mer primers (39). The re-
sults demonstrated C. intermedia and Issatchenkia orientalis to be homogeneous
species, while C. catenulata, Debaryomyces hansenii, C. sake, C. rugosa, and
Arxiozyma telluris were heterogeneous. RAPD patterns also distinguished be-
tween the three subgroups of C. parapsilosis. Arxiozyma telluris was the assigned
teleomorph species of C. pintolopesii; however, the RAPD pattern did not resem-
ble either of the anamorph varieties of C. pintolopesii. The authors caution that
micro-organisms assigned to the same species may not necessarily be closely
related.
Doubt was cast on the validity of the holomorph pairing of C. pintolopesii
and A. telluris by Meyer et al. (40), who found 16 other holomorph pairs to
produce highly similar patterns following amplification with the minisatellite
M13 sequence. Biochemical and physiological tests revealed that there were
marked differences between C. (Torulopsis) pintolopesii and A. (Saccharomyces)
telluris. Although they shared similar mol % G⫹C ratios of 31.8–34.9% C. pinto-
lopesii was respiration-deficient while A. telluris was respiration-competent, and
they differed in their fatty acid and cytochrome composition (41). It is therefore
possible that these yeasts are members of the same taxon but are not holomorphic
pairs.
Others, examining a wider range of species, have found RAPD to be a
suitable tool for recognizing yeast species (42, 43), and that isolates from ana-
morph–teleomorph pairs gave almost identical patterns (42). Molecular methods
are constantly demonstrating the existence of subgroups within species, however.
Candida parapsilosis has been shown to contain three distinct subgroups by mol
% G⫹C content, DNA: DNA reassociation, and RFLP patterns, although group
II isolates were not encountered among a panel of clinical strains (44). Candida
haemulonii has been shown to contain two distinct groups when examined by
isoenzyme analysis and DNA: DNA reassociation, which is in some agreement
with physiological tests (45). For RAPD to be used as a tool for species identifi-
Yeasts 511
cation of medically important yeasts, a suitable panel of reference strains would

therefore be required along with standardized primers and PCR conditions.
C. Molecular Techniques for Epidemiology

The epidemiology of infection requires the gathering of large amounts of circum-
stantial evidence in order to demonstrate a most likely scenario. For example,
following increased recovery of C. glabrata from patients in an intensive care
unit over a 10-week period, the information on the arrival date, location of the
patients in the unit, and dates of isolation of the yeast all suggested that person-
to-person transmission was likely. Molecular typing of the isolates, however,
demonstrated that five of the seven patients affected had distinguishable strains
and that the rise was unlikely to have been due to cross-infection (46). Only the
direct comparison of the yeasts in question could show that infection control
procedures had not broken down.
It is important to use the most appropriate method for each organism under
investigation (Table 3). Karyotyping by pulsed field gel electrophoresis (PFGE)
has been successful when used for C. glabrata, but was less discriminatory for
C. albicans, C. parapsilosis, and C. lusitaniae. Restriction enzyme analysis has
been moderately successful in distinguishing among isolates of C. albicans, C.
tropicalis, and S. cerevisiae, but its sensitivity and interpretation is subject to the
enzyme used. Another contributing factor is the frequency of occurrence of types;
certain types may be more commonly found than others. Clemons et al. (27)
compared large numbers of C. albicans by PFGE and REA. Restriction enzyme
analysis detected 71 types from 112 geographically distinct isolates, of which 32
isolates were assigned to one group IA2, while PFGE detected 18 types and
placed 57 isolates into a single type. Similarly, a study using PFGE (CHEF) to
examine the epidemiology of oral candidiasis in HIV-infected patients found one
type of C. albicans was recovered at some stage from 49 of the 66 patients who
were studied (47). Ideally, therefore, investigations using molecular typing for
any yeast species should include a suitable panel of epidemiologically distinct
isolates and reference strains to establish the discriminatory ability of the method
for that organism. This is particularly important in comparisons of small numbers
of isolates. For example, an investigation of an outbreak of sternal wound infec-
tions due to C. tropicalis involved six patients and one nurse. Restriction enzyme
analysis showed them all to be of the same type but distinct from nine epidemio-
logically unrelated isolates that were each distinguishable (48). For analyses by
REA appropriate restriction enzymes should be selected to maximize the discrim-
ination between isolates. The restriction enzyme HinfI has been demonstrated to
provide reproducible and discriminatory results for species identification and to
be suitable for strain typing of some species but not all (49). Eleven isolates of
C. dubliniensis and 49 isolates of C. parapsilosis produced only two types,
Table 3 Examples of Epidemiologic Studies and Results in Recent Nosocomial

Outbreaks of Yeast Infections
Number
Organism Reference Number of isolates/number of subjects Methods types
C. albicans 27 112 isolates/112 subjects REA 71

RAPD 58
PFGE 18
47 66 patients PFGE 34
52 32 isolates/32 patients RAPD 22
136 36 isolates/9 patients PCR 7/36, 8/8
8 isolates/8 reference cultures 27A probe 6/36, 8/8
137 14 isolates/14 patients PFGE 8
PFGE-REA 8
C. glabrata 138 23 isolates/20 patients PFGE 17
RAPD 8
mtDNA-REA 3
RAPD 6 or 9
C. krusei 56 7 isolates/7 patients REA 2
3 isolates/3 reference cultures 3
139 131 isolates/95 subjects PCR 95
C. parapsilosis 140 60 isolates/54 subjects PFGE-REA 8
PFGE 26
137 15 isolate/15 patients PFGE 10
PFGE-REA 5 or 8
C. tropicalis 48 8 isolates/6 patients ⫹ 1 nurse REA 1
9 isolates/9 control subjects 9
C. lusitaniae 142 47 isolates/33 patients PFGE-REA 25
PFGE 28
55 29 isolates/7 patients ⫹ 5 controls REA 8
C. inconspicua 143 5 isolates/3 patients ⫹ 1 reference REA 2
RAPD 2
S. cerevisiae 144 60 isolates/49 clinical ⫹ 11 other REA 41
145 15 isolates RAPD 6
PCR-REA 4
whereas 48 types were detected among 111 isolates of C. albicans. When REA
with one restriction enzyme fails to give satisfactory discrimination of isolates,
using a second enzyme can improve results. PFGE-REA with BssHII demon-
strated isolates of C. albicans to be identical, but they could be distinguished by
digestion with SfiI (50).
The ideal typing technique would be both fast and sensitive; however, REA
requires at least 2 days and PFGE can take at least 4 days if time for extraction
and electrophoresis is included. RAPD is a rapid and sensitive method, but suc-
Yeasts 513
cess depends heavily on the choice of primer and the organism being tested.
Greater discrimination was achieved for C. glabrata by PFGE than by RAPD,
but the reverse appeared to apply to studies of C. albicans (Table 3). Twenty-
two isolates of C. glabrata were divided into either six or nine types, depending
on the primer used (51). More usually several primers are screened for their
discriminatory ability, and the primer yielding the largest number of types is
selected for use. Indeed, Robert et al. (52) screened 12 primers before choosing
one to compare 32 isolates of C. albicans.
Once an appropriate technique has been selected and the conditions opti-
mized many different areas of microbial ecology and disease can be examined.
The possibility of a patient being infected at different sites with different strains
of the same yeast species was demonstrated in an AIDS patient with meningitis
and oral candidiasis caused by distinct strains of fluconazole-resistant C. albicans
(53). The dynamics of antifungal resistance within populations of C. albicans
causing recurrent oropharyngeal candidiasis has been examined and the develop-
ment of several different mechanisms of resistance identified (54). Simultaneous
oral carriage in HIV-positive and -negative patients of more than one type of
C. albicans and with more than one species has been shown (18). Nosocomial
transmission of yeasts such as C. lusitaniae (55) and C. krusei (56) within inten-
sive care settings has been demonstrated. Typing of Cryptococcus neoformans
revealed geographical groupings and also that patients acquired their infecting
strain from different locations and that the infection had remained dormant for
many months or years before diagnosis (57).
D. Probes that Demonstrate Microevolution

One of the first species-specific DNA probes used for epidemiological investiga-
tions was the C. albicans-specific probe 27A (58). This probe distinguished be-
tween isolates from different patients and between isolates from different anatom-
ical locations of individual patients. Minor differences, however, in band pattern
were observed in a proportion of colonies from four strains following unselected
laboratory passage on laboratory media and in the spontaneously laboratory-pro-
duced 5-fluorocytosine mutants of one strain. These results demonstrated that
genetic reorganization occurred and was detectable. Another C. albicans-specific
probe, Ca3, also used to assess strain relatedness, was found to provide stable
patterns for three strains tested through 400 generations (59). Lockhart et al. (60),
however, used the Ca3 probe to compare multiple isolates from individual pa-
tients and found that the patterns within each patient population could vary by
up to three bands. In addition, another probe derived from Ca3, the C1 fragment,
demonstrated single band differences in populations shown to be identical with
Ca3. Such results suggest the process of microevolution within the ecology of
an individual patient’s flora. Recently, similar studies have been conducted on
other Candida species. Two probes, Cg6 and Cg12, have been used to demon-
strate minor genetic changes in isolates of C. glabrata recovered from four vagi-
nitis patients over a 50-month period (61). Similarly, isolates of C. tropicalis
were compared by means of the Ct3 probe (62). This probe produced stable
patterns for three strains over 600 generations, but could distinguish minor differ-
ences in the hybridization patterns of sequential isolates of some patients and
not others. Neither of these studies involved multiple isolates recovered at each
sampling time to reveal the ecology at the site of isolation or the proportion
of genotypes present. Sequential minor changes in highly related hybridization
patterns, however, were demonstrated for isolates recovered from some patients
but not all, indicating that microevolution also occurs with C. glabrata and C.
tropicalis.
E. Molecular Methods in the Clinical Laboratory

Routine phenotypic and physiological identification of yeasts works well for the
majority of clinical specimens. As the numbers of immune-compromised and
immune-suppressed patients and the use of antifungals increase, however, so does
the array of yeast species with which patients can become colonized or infected.
Commercial yeast identification kits are commonplace in clinical laboratories,
and the efficiency of these systems is periodically evaluated. In 1994 the Vitek
Yeast Biochemical Card was reported to have failed to identify 42% (10/24) of
C. krusei isolates, four of five C. lambica isolates, and seven of eight Tri-
chosporon beigelii isolates (63). A new API Candida system was evaluated in
1996 and failed to identify 23% (4/17) C. famata, or any isolates of C. sphaerica,
C. sake, C. rugosa, C. pelliculosa, C. lipolytica, and C. intermedia that were not
included in the database (64). More recently the identification of 19 species of
yeasts by seven commercial systems varied from 59.6–80.8%, and all failed to
identify C. norvegensis, C. catenula, C. haemulonii, and C. dubliniensis (65).
Candida sake was a common misidentification by the ID 32C system of the re-
cently described species C. dubliniensis (20); however the accuracy of identifica-
tion of this species varies between commercial systems (66). Other species that
may be misidentified with biochemical methods are C. inconspicua, C. krusei,
and C. norvegensis. Molecular methods have been developed to aid in the identi-
fication of some of these species. Karyotyping by PFGE (67) and RAPD (4)
differentiated isolates of C. krusei and C. inconspicua, and PCR-REA differenti-
ated C. krusei, C. inconspicua, and C. norvegensis (34).
There is a variety of molecular techniques that can be used in the clinical
laboratory to aid in species identification. PCR-REA (68, 69) and RAPD (42,
70) provide characteristic fingerprints of organisms, while PCR reactions with
fungus-specific primers have been used for yeast identification (71) and to detect
yeasts in clinical fluids (72). Requiring more time and dedicated staff is the use
Yeasts 515
of species-specific probes for identification (73), although PCR combined with

an enzyme immunoassay using species-specific probes can reduce the overall
identification time (74). The application of probes to clinical specimens could
decrease the identification time of yeast species isolated from candidemias (75)
and has potential for use with in situ hybridization to histology sections (76).
The development of automated sequencing and array-based hybridization
schemes could lead to rapid identification of many yeast species as sequence
databases are constructed and made available (77).
VI. OCCURRENCE OF THE SPECIES AS PATHOGENS
The endomycetous and blastomycetous yeasts represent a diverse collection of

organisms that cause an equally diverse range of disease manifestations. The
most prominent genus is Candida, with at least 19 species capable of causing
infection in humans. As described in earlier sections of this chapter, however,
some of these species have teleomorphs that are in the endomycetous yeasts. In
this section, the teleomorphs of the holomorph will be described individually,
but the anamorphs, depending on the genus, will be considered as a group.
A. Arxiozyma
Arxiozyma telluris (anamorph, Candida pintolopesii, previously called Torulopsis
pintolopesii) is a rare agent of fungal infection. It has been isolated from the
pleura and lungs of a single patient and has been associated with oral leukoplakia
from one individual (78, 79).
B. Blastoschizomyces
B. capitatus (formerly Trichosporon capitatum) has been reported to cause sys-
temic infection, including endocarditis (80), onychomycosis (81), osteomyelitis
and discitis (82–84), urinary tract infections (85), possibly hepatitis (86), and
septicemia (86). The organism, which is widely distributed in nature, may be
considered an emerging cause of invasive disease in leukemia patients (87). It
has also been implicated in a fatal case of fungemia following infusion of organ-
ism-laden fluids (88).
C. Candida
Candida species infect a wide range of tissues and are, with the possible exception
of dermatophytes, the most common cause of fungal infections in humans. The
predominant diseases include vaginitis, oral thrush, and fungemia. Onycho-

mycosis caused by Candida species has been reported, but definitive evidence
that the species are the primary agents of the disease rather than secondary agents
or colonizers following dermatophytosis is lacking. Candida species are a com-
mon cause of infection in immunocompromised individuals, including patients
with hematogenous disorders and transplant recipients. Some species (e.g., C.
albicans, C. tropicalis, C. glabrata) are members of the normal microbiota of the
oral cavity, gastrointestinal tract, and vagina. Other species are normally found in
the environment.
Candida species represent the fourth leading cause of nosocomial blood-
stream infection (89, 90). Recent reports indicate that long-term azole therapy
(primarily fluconazole) may lead to selection in patients of fluconazole-resistant
pathogenic yeasts, such as C. glabrata, C. krusei, C. inconspicua, and C. dubli-
niensis (91, 92).
D. Clavispora
The two medically important species are Cl. capitatus and Cl. lusitaniae. The
former is the teleomorph of Blastoschizomyces capitatus and is discussed in Sec.
VI.B. Cl. lusitaniae, the teleomorph of Candida lusitaniae, is commonly identi-
fied as the anamorph in the clinical laboratory. Candida lusitaniae has been iso-
lated from cases of fungemia, meningitis, systemic disease, and urinary tract in-
fections (55, 93–95). A distinctive feature of the organism is its resistance to the
polyene amphotericin B. (For example, see Ref. 94.) Such resistance may not be
evident on initial testing in vitro, but may develop when the isolate is exposed
to the drug for extended periods.
E. Citeromyces
Citeromyces matritensis (anamorph, Candida globosa) has not been shown to
cause human disease, but it may be occasionally isolated from clinical specimens.
F. Cryptococcus
Combined with Candida species, Cryptococcus species represent ⬎90% of all
yeast infections in humans. The primary disease caused by Cr. neoformans is
meningitis, which was a relatively infrequent disease prior to the AIDS epidemic.
Cryptococcal meningitis occurs in individuals who are immunocompromised.
Four serotypes, A, B, C, and D, have been reported as pathogens, although addi-
tional serotypes may be involved (combinations of the serotypes). Serotype A
(Cr. neoformans var. grubii) and D (Cr. neoformans var. neoformans) are the
Yeasts 517
most common serotypes recovered from patients. Along with meningitis, Cr. neo-
formans can cause a variety of disease entities, most notably pulmonary and
cutaneous infections (96).
G. Debaryomyces
This genus is represented by D. hansenii, the teleomorph of Candida famata
(previously known as Torulopsis candida). D. hansenii has been isolated from
fresh droppings and cloacal samples of feral pigeons (97). In the clinical labora-
tory, the organism is typically reported as C. famata. This species has been associ-
ated with fungemia and endophthalmitis (98, 99). It is an infrequent cause of
both diseases. A recent study has revealed that several strains of Torulaspora
delbrueckii, a species with industrial applications, are actually Debaryomyces
spp., including one which may be D. hansenii (100).
H. Galactomyces
Galactomyces geotrichum is the teleomorph of Geotrichum candidum, which be-
longs to the order moniliales in the hyphomycetes. The anamorph does not pro-
duce blastoconidia and therefore does not belong in the blastomycetes. Geotri-
chum candidum has been reported to cause oral disease (101) and disseminated
disease (102). It is possible that true disease caused by this organism is rare; it
may have been reported as geotrichosis caused by Blastoschizomyces capitatus,
the recently revised genus-species of Geotrichum capitatum.
I. Hansenula
Hansenula anomala (anamorph Candida pelliculosa) is an emerging pathogen.
It has been associated with cases of fungemia, urinary tract infection, and endo-
carditis (103–106). Invasive disease caused by this organism other than those
already listed has also been suggested (107).
J. Issatchenkia
The medically important species I. orientalis (anamorph Candida krusei) has
been associated with cases of fungemia (108) and possibly sinusitis and pneumo-
nia (109). This organism is typically obtained from patients who are undergoing
treatment with fluconazole and from whom a fluconazole-resistant yeast is iso-
lated.
K. Kluyveromyces
Kluyveromyces marxianus (anamorph Candida kefyr; formerly Candida pseudo-
tropicalis) is a rare agent of fungemia and invasive disease in immunocompro-
mised patients (110).
L. Malassezia
The most common clinically important species in this genus are M. furfur, M.
pachydermatis, and M. sympodialis. Another four species have been isolated from
human specimens (32), however. Some of the species were previously considered
members of the genus Pityrosporum. The disease pityriasis versicolor is a com-
mon manifestation of superficial disease caused by Malassezia species. In addi-
tion, the species have been reported to cause folliculitis, fungemia (associated
with hyperalimentation fluids), dermatitis, and lung invasion (111–113).
M. Metschnikowia
Metschnikowia pulcherrima (anamorph, Candida pulcherrima) has been impli-
cated in onychomycosis and has been recovered in other clinical specimens, such
as sputum and cutaneous tissues (114). The overall significance of this organism
in disease is uncertain.
N. Pichia
This genus contains several medically important yeasts with anamorphs in the
genus Candida. Diseases caused by these species are manifold. The most frequent
species in clinical material are P. guilliermondii (anamorph, Candida guillier-
mondii) and P. norvegensis (anamorph, Candida norvegensis). Both species have
been reported to cause fungemia and invasive disease (115, 116).
O. Rhodotorula
The red yeasts Rhodotorula have basidiomycetous affinities. These organisms
have been reported as agents of fungemia, primarily associated with central ve-
nous catheters (117, 118). They have also been implicated in at least one case
each of peritonitis, meningitis, and extrinsic allergic alveolitis (95, 119, 120).
These organisms are found in the environment, particularly in water sources. The
primary species involved in human disease are R. rubra (synonymous with R.
mucilaginosa) and R. glutinis.
Yeasts 519
P. Saccharomyces
Saccharomyces cerevisiae is the primary medically important agent in this genus.
This organism has been associated with cases of vaginitis, oral disease, fungemia,
and empyema (121–123). The organism’s habitat is the environment, but it is
found in a number of basic foods (bread). When ingested, it can transiently colo-
nize the gastrointestinal tract, hence its isolation from GI-related sites may not
imply clinical significance.
Q. Sporobolomyces
This form genus, which has a teleomorph in the basidiomycetes, is an uncommon
agent of human disease. Several infectious manifestations have been reported,
including lymphadenitis, nasal polyps, fungemia, and dermatitis (124–126).
Sporobolomycosis is an opportunistic disease of immunocompromised individu-
als. At least three species have been isolated from clinical materials and include
S. salmonicolor, S. holsaticus, and S. roseus.
R. Stephanoascus
The anamorph of S. ciferrii, Candida ciferrii, has been reported as a possible
agent of onychomycosis in elderly patients (127, 128) and possibly otomycosis
(128). The clinical significance of the organism is doubtful (128), however.
S. Trichosporon
Trichosporon beigelii was once considered the only pathogenic species in this
genus, but recent revisions to the genus suggest that six species may be pathogens.
The epithet T. beigelii is considered by some authorities as invalid, as this species
actually contained multiple variants that have been reassigned to new species
(129, 130). The five species have been implicated as agents of superficial disease
(e.g., white piedra) and two species, T. mucoides and T. asahii, have been sug-
gested to cause disseminated infections in leukemia patients. The organisms are
resistant to amphotericin B, making therapeutic management difficult (131, 132).
‘‘T. beigelii’’ has been isolated from cases of fungemia (131–133). Trichosporon
cutaneum has been shown to colonize and subsequently cause fungemia in neo-
nates (134).
T. Yarrowia
Yarrowia lipolytica (anamorph, Candida lipolytica) has been isolated from a case
of fungemia and a case of sinusitis (135). Due to the difficulty of identifying this
organism using standard clinical tests, misidentifications may have occurred and
this organism may be more common than suspected.
APPENDIX: DIFFERENTIAL CHARACTERISTICS

OF THE GENERA
Arxiozyma
Sexual spores and morphologic features: Asci with 1–2, spheroidal to ellip-
soidal, verrucose to tuberculate, ascospores. Yeasts diploid, globose to
ellipsoidal.
Biochemical characteristics: Urease ⫺, CHX S, nitrate ⫺, inositol assim.
⫺, DBB ⫺, fermentation ⫹.
Medically significant species: A. telluris.
Blastoschizomyces
Sexual spores and morphologic features: Asexual genus. Produces arthro-
conidia and annelloconidia on tip of percurrently proliferating conidio-
genous cell; blastoconidia present in young cultures.
Biochemical characteristics: Urease ⫺, CHX R, nitrate ⫺, inositol assim.
⫺, DBB ⫺, fermentation ⫺.
Medically significant species: B. capitatus.
Candida
Sexual spores and morphologic features: Asexual genus. Budding cells,
pseudo- and true-septate mycelium possible.
Biochemical characteristics: Urease ⫺, CHX R/S, nitrate ⫾, inositol as-
sim. ⫺, DBB ⫺, fermentation ⫹.
Predominant medically significant species: C. albicans (including C. dubli-
niensis), C. ciferrii, C. famata, C. guilliermondii, C. glabrata, C. haemu-
lonii, C. kefyr, C. krusei, C. lipolytica, C. lusitaniae, C. norvegensis, C.
parapsilosis, C. tropicalis, C. utilis, C. viswanathii, C. zeylanoides.
Clavispora
Sexual spores and morphologic features: Asci with 1–4 conical or clavate
ascospores; budding cells, pseudomycelium possible.
Source: Refs. 1, 3, 129, 146–148.

Yeasts 521
Biochemical characteristics: Urease ⫺, CHX ⫾, nitrate ⫺, inositol assim.

Medically significant species: C. lusitaniae, C. capitatus.
Citeromyces
Sexual spores and morphologic features: Asci with one (rarely two) round,
warty ascospore; spheroidal to ellipsoidal budding cells, no hyphae.
Biochemical characteristics: Urease ⫺, CHX?, nitrate ⫹, inositol assim.
Medically significant species (isolated as contaminant, not pathogen): C.
matritensis.
Cryptococcus
Sexual spores and morphologic features: Asexual genus. Budding cells.
Encapsulated. Occasional pseudo- and true hyphae.
Biochemical characteristics: Urease ⫹, CHX S (C. laurentii occasionally
R), nitrate ⫾, inositol ⫹ (occasionally⫺), DBB ⫹, fermentation ⫺.
Medically significant species: C. neoformans.
Debaryomyces
Sexual spores and morphologic features: Asci with 1–4 round or oval,
warty ascospores. Conjugation occurs between cell and bud.
Biochemical characteristics: Urease ⫺, CHX S/R, nitrate ⫺, inositol ⫺,
DBB ⫺, fermentation ⫾.
Medically significant species: D. hansenii.
Galactomyces
Sexual spores and morphologic features: Spherical asci with 1 to 2 (rare)
ellipsoidal, echinate ascospores; ascospores may contain equatorial fur-
row. Produces arthoconidia.
Biochemical characteristics: Urease ⫺, CHX?, nitrate ⫺, inositol ⫺, DBB
⫺, fermentation ⫾.
Medically significant species: G. geotrichum.
Hansenula
Sexual spores and morphologic features: Asci with 1 to 4 hat-shaped asco-
spores; budding cells, pseudo- or true hyphae possible.
Biochemical characteristics: Urease ⫺, CHX S/R, nitrate ⫹, inositol ⫺,

Medically significant species: H. anomala.
Issatchenkia
Sexual spores and morphologic features: Asci with 1 to 4 roughened, round
ascospores; budding cells, pseudohyphae possible.
DBB ⫺, fermentation ⫹.
Medically significant species: I. orientalis.
Kluyveromyces
Sexual spores and morphologic features: Evanescent asci with 1 to typi-
cally 4, 8, or 16 ascospores (one species produces up to 60 ascospores/
ascus); ascospores are oval, crescent shaped, or reniform; budding cells
spheroidal to elongate.
Medically significant species: K. marxianus.
Malassezia
Sexual spores and morphologic features: Asexual genus; monopolar bud-
ding on a broad base (percurrent); hyphae may be produced.
Biochemical characteristics: Urease ⫹, CHX R, nitrate ⫹?, inositol ?, DBB
⫹, fermentation ⫺.
Medically significant species: M. furfur, M. globosa, M. obtusa, M. pachy-
dermatis, M. restricta, M. slooffiae, M. sympodialis.
Metschnikowia
Sexual spores and morphologic features: Club-shaped asci with one or two
needle-shaped ascospores/spheroidal to ellipsoidal budding cells; rudi-
mentary pseudomycelium usually present.
Biochemical characteristics: Urease ⫺, CHX ?, nitrate ⫺, inositol ⫺, DBB
⫺, fermentation ⫹.
Medically significant species: M. pulcherrima.
Yeasts 523
Pichia
Sexual spores and morphologic features: Generally dehiscent asci with 1
to 4, smooth, hat-shaped, hemispherical, saturnine, or spheroidal asco-
spores.
Medically significant species: P. guilliermondii, P. fermentans, P. jadinii,
P. membranaefaciens, P. norvegensis.
Rhodotorula
Sexual spores and morphologic features: Asexual genus; budding cells, red
or yellow carotenoids produced.
Biochemical characteristics: Urease ⫹, CHX S/R, nitrate ⫾, inositol ⫺,
DBB ⫹, fermentation ⫺.
Medically significant species: R. rubra, R. glutinis.
Saccharomyces
Sexual spores and morphologic features: Persistent asci with 1 to 4,
smooth-walled, globose to short ellipsoidal ascospores; budding cells,
globose to ellipsoidal; pseudohyphae may be formed.
Medically significant species: S. cerevisiae, S. exiguus.
Sporobolomyces
Sexual spores and morphologic features: Asexual genus. Budding cells
generally ellipsoidal but variable, percurrent budding. Pseudohyphae
present. Ballistoconidia on sterigmata. Produces salmon-pink carot-
enoids.
Biochemical characteristics: Urease ⫹, nitrate ⫾, inositol ⫾, DBB ⫹, fer-
mentation ⫺.
Medically significant species: S. salmonicolor, S. holsaticus, and S. roseu.
Stephanoascus
Sexual spores and morphologic features: Asci formed after hyphal conjuga-
tion, contain 1 to 4 hat-shaped ascospores when immature, hemispherical
when mature.
Biochemical characteristics: Urease ⫺, nitrate ⫺, inositol ⫹, DBB ⫺, fer-

mentation ⫺.
Medically significant species: Stephanoascus ciferrii.
Trichosporon
Sexual spores and morphologic features: Asexual genus. Budding may be
absent or present. Arthroconidia produced.
Biochemical characteristics: Urease ⫹, CHX S/R, nitrate ⫺, inositol ⫾,
DBB ⫹, fermentation ⫺.
Medically significant species: T. asahii, T. asteroides, T. cutaneum, T. inkin,
T. mucoides, T. ovoides.
Yarrowia
Sexual spores and morphologic features: Asci with 1 to 4 round, oval,
walnut-shaped, hat-shaped, or saturnoid ascospores; budding cells;
pseudohyphae and hyphae usually present.
Biochemical characteristics: Urease ⫹, nitrate ⫺, inositol ⫺, DBB ⫺, fer-
mentation ⫺.
Medically significant species: Y. lipolytica.
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9
Basidiomycetous Yeasts
Teun Boekhout
Centraalbureau voor Schimmelcultures, Institute of the Royal
Netherlands Academy of Arts and Sciences, Utrecht, The Netherlands
Eveline Guého
Mauves sur Huisne, France
I. INTRODUCTION
Yeasts are generally defined as unicellar fungi. Numerous yeasts are able to form
hyphae and/or pseudohyphae, however. Yeasts are polyphyletic in origin and
belong to the Ascomycetes and the Basidiomycetes (1). Within the Basidiomy-
cetes, they occur in all three main phylogenetic lines, namely the Hymenomycetes
[Cystofilobasidiales, Trichosporonales, Tremellales (jelly fungi), and Filobasidi-
ales], the Urediniomycetes (Sporidiales, and including the obligate plant parasitic
rust fungi), and the Ustilaginomycetes (Malasseziales, and including the plant
parasitic smut fungi; Fig. 1) (2). Medically important basidiomycetous yeasts
belong to the genera Cryptococcus Vuillemin, Trichosporon Behrend (both Hy-
menomycetes), and Malassezia Baillon (Ustilaginomycetes). Other basidiomyce-
tous yeasts that have been reported from patients occur in the genera Rhodotorula
FC Harrison and Sporobolomyces Kluyver & van Niel (both Urediniomycetes).
Yeasts, including the basidiomycetous ones, are usually identified by using medi-
cal physiological characteristics. Summarized physiological data of the most im-
portant basidiomycetous yeasts are presented in Tables 1 and 2.
II. CRYPTOCOCCUS VUILLEMIN
Cryptococcus is an anamorphic basidiomycetous yeast belonging to the Hymeno-

mycetes (2), comprising in its current circumscription 34 species. The genus con-
535
536 Boekhout and Guého
Figure 1 Phylogenetic tree of selected basidiomycetous yeasts based on the D1/D2

domain of the large subunit ribosomal DNA. Cystofilobasidiales are used as an outgroup.
Asterisks indicate bootstrap values of 70% and higher.
Basidiomycetous Yeasts 537
Table 1 Specific Characters of C. albidus, C. curvatus, C. laurentii,

and C. neoformans
C. albidus C. curvatus a C. laurentii C. neoformans

Tests CBS 142 CBS 570 CBS 139 NIH 271
ID 32C pattern b
Galactose ⫺ ⫹ ⫹ ⫹
0.01% cycloheximide ⫺ D ⫺ ⫺
DL-lactate ⫺ ⫹ ⫺ ⫺
N acetyl-glucosamine ⫺ ⫹ ⫹ ⫹
L-arabinose ⫹ ⫺ ⫹ ⫹
Cellobiose ⫹ ⫹ ⫹ ⫺
Raffinose ⫹ ⫹ ⫹ ⫺
Ribose ⫺ ⫹ ⫺ ⫹
Glycerol ⫺ ⫹ ⫺ ⫺
Erythritol ⫺ ⫺ ⫹ ⫹
Melibiose ⫺ ⫺ ⫹ ⫺
Gluconate ⫺ ⫹ ⫹ ⫹
Lactose ⫺ ⫹ ⫹ ⫺
Sorbose ⫺ ⫺ ⫹ ⫹
Urea hydrolosis c ⫹ ⫹ D ⫹
Phenoloxydase ⫺ ⫺ ⫺ ⫹
Growth at 37°C ⫹ D D ⫹/D
a
Characteristics were the same for five clinical isolates of C. curvatus, except that inositol was nega-
tive for the type culture CBS 570.
b
For the four species, growth was negative (⫺) on levulinate and positive (⫹) on the other substrates
of the ID 32C strips.
c
D ⫽ delayed.
tains C. neoformans, the most important basidiomycetous yeast from a clinical

perspective (3), as well as a number of other species encountered in the medical
literature. The name of the genus was conserved as Cryptococcus Vuillemin to
improve nomenclatural stability (4).
A. Cryptococcus neoformans
Cryptococcus neoformans is known in both its asexual (anamorph) and sexual
(teleomorph) states, for which the respective names Cryptococcus neoformans
and Filobasidiella neoformans Kwon-Chung are used. Estimates on the incidence
rate in AIDS patients range from 5–30%, with the highest numbers occurring in
sub-Saharan Africa (5). The fungus can cause serious infections, especially in
immunocompromised patients. The main sites of infection are the lungs and the
538
Table 2 Physiological Patterns of Clinical Basidiomycetous Yeasts Analyzed with ID 32C (bioMérieux, Marcy l’Étoile, France)
T. T. T. T. T. T. T. C. C. C. C. C. Filobasiditum R. R. Pseudozayma
asahii asteroides ovoides inkin cutaneum montevideense mucoides neoformans albidus curvatus humicola laurentii uniguttulatum glutinis mucilaginosa species
Esculine V ⫹ ⫺ ⫺ ⫹ ⫹ ⫹ V ⫹ ⫹ ⫹ ⫹ ⫺ ⫺ ⫺ ⫹
Glucosamine ⫹ v ⫹ v V ⫹ ⫹ ⫹ ⫺ ⫹ ⫹ ⫹ ⫹ ⫺ ⫺ ⫹
Sorbose ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ V V ⫺ ⫺ ⫹ ⫺ ⫺ ⫺ ⫹ ⫹
Glucose ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹
Inositol ⫺ ⫺ ⫺ ⫹ ⫹ ⫹ ⫹ ⫹ V ⫹ ⫹ ⫹ ⫹ ⫺ ⫺ ⫹
Lactose ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫺ ⫺ ⫹ ⫹ ⫹ ⫺ ⫺ ⫺ ⫺
Mannitol ⫺ ⫺ V ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ V ⫹ ⫹
Laevulinate ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫹ ⫺ ⫺ ⫺ ⫺ ⫺
Gluconate ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫺ ⫹ ⫹ ⫹ ⫺ ⫺ ⫺ ⫹
Melezitose V V ⫹ ⫹ V ⫹ V ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹
Glucuronate ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫺ ⫺ ⫹
Melibiose ⫺ ⫺ ⫺ ⫺ ⫹ ⫺ ⫹ ⫺ ⫺ ⫺ ⫹ ⫹ ⫺ ⫺ ⫺ ⫺
Erythritol ⫹ ⫹ ⫺ ⫹ ⫹ ⫺ ⫹ V ⫺ ⫺ ⫹ ⫹ ⫺ ⫺ ⫺ ⫹
Palatinose ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹
Rhamnose V ⫺ V ⫺ V ⫺ V ⫹ ⫹ ⫹ ⫹ ⫹ V ⫺ ⫺ ⫹
Glycerol V V V ⫺ ⫹ ⫹ ⫹ ⫺ ⫺ ⫹ ⫹ ⫺ ⫺ ⫹ ⫹ ⫹
Ribose ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫺ ⫹ ⫹ ⫹ ⫺ ⫹ ⫺ ⫹
D-Xylose ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹
Sorbitol ⫺ ⫺ ⫺ ⫺ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ V ⫹ ⫹
α-methyl-D-glucoside ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ V ⫹ ⫹ ⫹ ⫹ ⫺ ⫺ ⫹
2-Keto-D-gluconate ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫺ ⫺ ⫹
Trehalose V ⫹ ⫹ ⫹ V ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹
Maltose ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹
Raffinose ⫺ ⫺ ⫺ ⫺ V ⫺ ⫹ V ⫹ ⫹ ⫺ ⫹ ⫹ ⫹ ⫹ ⫹
Cellobiose ⫹ ⫹ ⫹ ⫹ V ⫹ ⫹ V ⫹ ⫹ ⫹ ⫹ ⫺ V ⫺ ⫹
L-Arabinose ⫹ ⫹ V ⫺ ⫹ ⫹ ⫹ ⫹ ⫹ ⫺ ⫹ ⫹ ⫹ ⫹ ⫺ ⫹
DL-Lactate ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫺ ⫺ ⫹ ⫹ ⫺ ⫺ ⫺ ⫺ ⫹
N-Acetyl-glucosamine ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫺ ⫹ ⫹ ⫹ ⫹ ⫺ ⫺ ⫹
Sucrose ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹
0.01% Cycloheximide ⫹ V ⫹ V ⫺ ⫹ ⫹ ⫺ ⫺ ⫹ ⫹ ⫺ ⫺ ⫺ ⫹ ⫺
Galactose ⫹ V ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫺ ⫹ ⫹ ⫹ ⫺ ⫹ ⫹ ⫺
T. ⫽ Trichosporon; C. ⫽ Cryptococcus; R. ⫽ Rhodotorula.

Boekhout and Guého
central nervous system, including cerebrospinal fluid (CSF ). In the brain it causes
meningitis, meningoencephalitis, and granuloma formation. Primary cutaneous
infections are rare, and most skin infections are due to disseminated systemic
infections (6, 7). Cryptococcus neoformans var. neoformans is encountered in
nearly all of the AIDS-related infections (7, 8).
According to the current classification, the species consists of three varie-
ties: C. neoformans var. neoformans (serotype D), C. neoformans var. grubii
(serotype A), and C. neoformans var. gattii (serotypes B and C). In contrast,
the teleomorph Filobasidiella neoformans var. neoformans corresponds to both
serotypes A and D, and F. neoformans var. bacillispora to serotypes B and C
(9–18).
The occurrence of recombinants between strains of C. neoformans var. neo-
formans and C. neoformans var. gattii, and the presence of genetic recombination
in the first filial generation (F1) suggested their varietal status (15, 19), but no
genetic analysis of the second generation (F2) has been made. In contrast, rather
low DNA-DNA reassociation values ranging between 55–63% (20) were ob-
served between isolates of the two varieties, suggesting a considerable genetic
divergence between the taxa. Both varieties differ in their karyotypes (21, 22),
DNA fingerprints (23, 24), and a number of physiological characteristics; for
example, assimilation of D-proline, D-tryptophane, and L-malic acid, regulation
of creatinine deaminase by ammonia production (25–28), and susceptibility to
killer toxins of C. laurentii CBS 139 (29). The varieties also differ in geographic
distribution and habitat. C. neoformans var. grubii and C. neoformans var. neo-
formans seem to occur worldwide in clinical and veterinary sources, bird drop-
pings, and occasionally in such substrates as fermenting fruit juice, drinking wa-
ter, wood, and air (5, 7, 30–31). Both varieties occur in AIDS patients, and
serotype D isolates seem to be relatively common in Europe (32). Recently, sero-
type A isolates were also isolated from trees in Brazil (33). C. neoformans var.
gattii seems to occur mainly in the tropics, the southern hemisphere, southern
Europe, and the southern United States. Clinical isolates are mainly known from
non-AIDS patients (e.g., CSF, skin, tumor) and animals (e.g., cats and goat, as
well as cows with mastitis). Saprobic isolates are usually associated with Euca-
lyptus species (3, 16, 34–36), but have been isolated from bat guano (33), and
more recently from almond trees (37–38) as well.
Selective isolation and presumptive identification of the species is usually
based on its ability to form melanin on plates containing niger seeds (Guizotia
abyssinica) (39) or dopamines such as L-DOPA or norepiniphrine (40), and the
presence of an extracellular capsule visible in india ink preparations. Other identi-
fication tests are based on its ability to hydrolyze urea (41, 42), but this is a
characteristic for all basidiomycetous yeasts, and urease negative strains of the
species have rarely been reported (43). Distinction between the varieties is usually
performed on 1-canavanine-glycine-bromothymol blue medium (CGB-medium;
14, 44), or by testing D-proline or D-tryptophan assimilation (26, 27). The four
serotypes can be identified by agglutination with polyclonal antibodies (45) or
by immunofluorescence with monoclonal antibodies (46). The four serotypes
have the same physiological growth characteristics as are usually evaluated in
clinical laboratories (4) with a commercial testing system (Table 1).
The observed genetic, biochemical, ecological, pathological, and geograph-
ical differences raise strong questions about the conspecific status of the varieties.
If they interbreed, it is hard to understand how all the observed differences are
being maintained, as one would expect homogenization to occur within the popu-
lation. Recent observations using amplified fragment length polymorphism
(AFLP) (47) and nucleotide sequences of the intergenic spacer (IGS) of the ribo-
somal DNA (48) seem to support the existence of two species, therefore the
presence of two separate teleomorphic species is proposed: F. neoformans Kwon-
Chung and F. bacillispora Kwon-Chung with their respective anamorphs C. neo-
formans (Sanfelice) Vuillemin and C. bacillisporus Kwon-Chung & Bennett (⫽
C. neoformans var. gattii Vanbreuseghem & Takashio).
Using molecular data, Franzot et al. (49) recently described a new variety
for serotype A isolates of C. neoformans as C. neoformans var. grubii. Our AFLP
analysis and IGS sequence data largely support the existence of this variety, but
also showed that the resulting genotypic clusters do not entirely correspond with
the serotype boundaries (47). The same can be concluded from the URA5 se-
quence and CNRE-1 results presented by Franzot et al. (50), as a serotype D
isolate was incidentally found to cluster among the serotype A isolates they stud-
ied. These observations suggest that serotyping alone cannot be used to distin-
guish C. neoformans var. neoformans from C. neoformans var. grubii. We ex-
plain these nonconcordant results by proposing the presence of sexual or
parasexual hybridization between serotype A and D isolates of C. neoformans
and between serotype B and C isolates in C. bacillisporus. So far we have only
evidence for the occurrence of hybridization within—and not between—the spe-
cies. Presently we favor the scenario in which the fungus uses both sexual and
asexual reproduction mechanisms. The role of the sexual Filobasidiella states in
the hybridization events is not yet clear. After recombination, strains with the
hybrid genotypes may disseminate clonally. Hybrid strains may have a selective
(dis)advantage toward changes that occur in the environment. Such environmen-
tal changes may have happened several times in the history of the species (51).
The hybrids may also differ in virulence or resistance to antibiotics. Our hypothe-
sis about the reproductive biology of the species differs from the previous pro-
posed strictly clonal reproduction of C. neoformans, as was deduced from linkage
disequilibrium studies in which multilocus electrophoretic enzyme patterns were
used (52, 53). Additional support for the previous proposal derived from the
concordance between different molecular parameters in geographically separated
populations, as well as the dominance of a few genotypes in the population (54).
We think, however, that the molecular markers used in these studies probably
yield lower levels of resolution than the AFLP markers we employed.
B. Other Cryptococcus Species

Several cryptococci other than C. neoformans have been reported from clinical
sources. In a critical discussion of the literature, Krajden et al. (55) noticed the
problems related with a correct interpretation of earlier clinical reports on crypto-
coccal non-C. neoformans infections. This uncertainty is due to the possible con-
tamination with saprobically growing yeasts, unreliable identifications, and the
fact that most isolates were not deposited in public culture collections. Summa-
rized physiological data of some of the clinically reported Cryptococcus species
are given in Tables 1 and 2.
Cryptococcus luteolus was reported from pulmonary cryptococcosis (56).
The limited physiological data given, however, suggested a proper identification
of this isolate as C. neoformans, thus supporting the suggestion of Krajden et al.
(55, 57).
Clinical isolates of C. albidus have mainly been reported to occur in immu-
nocompromised patients. Isolates have been reported from AIDS patients and
leukemic patients with pemphigus foliaceus, pulmonary cryptococcosis, and
meningitis, as well as a nosocomial infection (58–67). Wieser (68) reported the
presence of C. albidus in cerebrospinal fluid of five patients with meningitis. The
identity of an isolate described by Krumholz (59) was strongly questioned by
Gordon (69). In most of these cases the identity of the isolates should be inter-
preted with care. The limited data presented in the literature usually hamper a
reliable reidentification, and in most cases no isolates have been preserved in a
public culture collection. To complicate the situation, recent molecular taxonomic
investigations of C. albidus suggest that many species occur in this complex (70).
Cryptococcus curvatus was reported to occur in the cerebrospinal fluid
(CSF ) of a 30-year-old male AIDS patient (71). Clinical isolates of this species
present in the CBS yeast collection (72) were obtained from sputum, urine, feces,
and the uterus of a cow.
Another cryptococcal species, C. laurentii, has been reported from pulmo-
nary lesions, peritoneal fluid, fungemia occurring in a bone marrow transplant
patient, a cutaneous infection and granulomas, endophalmitis, CSF of an AIDS
patient, and from a catheter-associated fungaemia (67, 73–80). The isolates from
two cases of pulmonary lesions showed a cross-reaction with fluorescent antibod-
ies against C. neoformans, and maintained their fluorescence after absorption
with a C. laurentii antigen. It was therefore concluded that the disease very likely
was not caused by C. laurentii but by C. neoformans (55).
Cryptococcus ater has been isolated from an ulcer of a leg of an 18-year-
old man in Portugal. No proof of its pathogenicity was presented (81), however.
Cryptococcus humicola has been isolated from skin and bronchial excretion, as
well as soil and mushrooms. Isolates of Filobasidium uniguttulatum, the perfect
state of Cryptococcus uniguttulatus (82), have been obtained from an infected
fingernail, bronchus, sputum, the sole of a foot, and white patches in the mouth
(72). No proof for pathogenicity exists for the latter two species, however. Filo-
basidium uniguttulatum does not grow at human body temperature in vitro.
III. TRICHOSPORON BEHREND
The genus Trichosporon consists of anamorphs for which no sexual state (teleo-
morph) has been described. In 1992, the genus was limited to yeastlike fungi
combining a layered cell wall ultrastructure, the presence of a dolipore, positive
urease and Diazonium Blue B (DBB) reactions, and the ability to both produce
true filaments (hyphae) and multiply asexually by arthroconidia and under certain
conditions blastoconidia (83). Behrend had created the genus in 1890 with the
species T. ovoides to accommodate a fungus isolated from mustache white piedra
nodules (84). T. ovoides was reintroduced as the type species of the genus to
replace the name T. beigelii (83). The epithet beigelii first appeared in 1867 when
Küchenmeister and Rabenhorst (85) observed round cells composing white piedra
nodules on ‘‘postiche chignon’’ hair. They named the micro-organism Pleurococ-
cus beigelii, because they thought that these nodules were composed of algae.
They did not get a culture or provide a description, however. In contrast, the
description given by Behrend (84) for T. ovoides was macroscopically and micro-
scopically well detailed. Unfortunately, his original culture was not maintained,
but a neotype culture of the species with the same characteristics as the original
material was selected and deposited in culture collections (86, 87). In 1992 all
isolates preserved in culture collections and differing from this type of culture
were shown to represent other known species or undescribed species [e.g., see
T. mucoides described in 1992 (83)]. The genus was enlarged to 19 species.
Trichosporon pullulans, which resembles the other species only morphologically,
needs to be recombined in another genus, as it belongs to a distinct phylogenetic
lineage. This species belongs to the order Cystofilobasidiales, which contains
psychrophilic genera such as Mrakia and Cystofilobasidium. The genus Tri-
chosporon sensu stricto is phylogenetically related to the Tremellales and the
Filobasidiales, and has recently been classified in the Trichosporonales (2, 88–
90). The species of the genus are serologically similar to Cryptococcus neo-
formans (91). The psychrophily of T. pullulans (no growth above 20°C) precludes
any pathogenicity of this species in humans or other warm-blooded animals. The
rare papers describing T. pullulans as a pathogen may be based on misidentified
isolates (92). Contrary to the other Trichosporon species, T. pullulans is able to
grow with nitrates as a nitrogen source.
The results of the revision of 1992 were confirmed by subsequent authors

(93), which transferred three taxa recognized as species to the varietal level. T.
faecale and T. coremiiforme were made varieties of T. asahii, and T. laibachii
a variety of T. loubieri (93). Recently, several species, either from humans or
the environment, have been described (94–97). Consequently, the genus pres-
ently again includes 19 species, and more species are expected to be discovered
in the near future. The genus is not as large as Candida or Cryptococcus, but
the lack of well-defined key characteristics makes recognition of its species diffi-
cult. Any identification of a medical Trichosporon isolate should involve the
following characteristics: macromorphology (colony texture and rate of growth)
and micromorphology with specific features, such as appressoria, fusoid or meri-
stematic cells, and globose or ovoid blastoconidia, obtained after growth of the
organism in slide cultures on 2% malt extract agar or cornmeal agar; growth at
37°C or higher temperatures; and cycloheximide sensitivity at 0.01 and 0.1%.
The assimilation tests must include a sufficient number of substrates (Table 2).
Also, the origin of isolates is important since each species occupies its preferential
ecological niche (83, 98, 99).
The second most important source of Trichosporon yeast isolates has gener-
ally been the human skin. Since the description of T. beigelii, and its synonym
T. cutaneum (85), the two names were used for most Trichosporon isolates irre-
spective their origin (83, 86). In 1970, Watson and Kallichurum (100) published
a case of deep-seated infection (brain abscess) caused by a species of Tri-
chosporon (in this case named T. cutaneum). Unfortunately, no isolate was main-
tained in a culture collection. During the following three decades many cases of
trichosporonosis were published, most of them listing T. beigelii as the causative
agent (101, 102). In the 1992 taxonomic revision six species adapted to humans
were included (83, 98, 102). Among these species, only three were recognized
as occurring in deep-seated trichosporonosis, and five were found in nodules of
white piedra (103, 104). In order of decreasing frequency and virulence, the six
species infecting humans are T. asahii, T inkin, T. mucoides, T. ovoides, T. aster-
oides, and T. cutaneum. It is recommended that clinical laboratories should iden-
tify at least the three agents of deep-seated trichosporonosis, T. asahii, T. inkin,
and T. mucoides. Because it is difficult to distinguish the cutaneous species, a
PCR detection assay for T. asahii was developed by using sequences of the inter-
nal transcribed spacer (ITS) regions of the ribosomal DNA (105). Later, the same
methodology was extended to the five other clinically relevant species, and a
database was constructed (106, 107). According to recent findings T. montevi-
deense, isolated from nails of a patient with AIDS (E. Guého, unpublished data),
and the new species T. domesticum (94) should be considered of clinical impor-
tance as well. All these species show irregular hyphae and conidia which appear
as pseudohyphae and blastoconidia in primary cultures, and all are able to hy-
drolyze urea in liquid urea-indol medium at 37°C if heavily inoculated. None of
them uses nitrates as a nitrogen source, but all are able to use most of the carbon
substrates commonly used in yeast identification (83).
A. Trichosporon asahii
Although originally isolated as a contaminant from psoriatic nails, this species
is becoming recognized as the major causative agent of deep-seated trichospor-
onosis (102, 108), in particular in immunocompromised patients. It is also iso-
lated from cutaneous lesions, nails, and white piedra nodules, although not from
capital white piedra (103, 104). It is able to grow at 37°C, but the typical colonies,
measuring 16 to 24 mm in diameter in 10 days and with a wrinkled white farinose
surface, are better developed at 25–28°C on Sabouraud glucose agar (SGA) with-
out antibiotics. Hyphae disarticulate into regular arthroconidia (Fig. 2a), which
become barrel-shaped with age on slide cultures. Appressoria and blastoconidia
are missing (83). Its physiological pattern is characterized by lack of assimilation
of raffinose and sorbitol, which are substrates present in most commercial kits
for yeast identification (98, 104). It should be noted, however, that since the
physiological data presented in the fourth edition of The Yeasts: A Taxonomic
Study (99) are based on relatively long incubation periods, the results may differ
from those obtained with commercial kits. This discrepancy in assimilation pat-
terns may cause confusion to medical microbiologists. Consequently, only a few
clinicians such as Mahal and coworkers (109) have risked publishing any of the
correct, newer names instead of the obsolete T. beigelii. T. asahii was proven to
correspond to the serotype II standard culture, one of the causative agents of
summer-type hypersensitivity pneumonitis (110). The species as currently de-
fined includes previously recognized variants, such as T. infestans (83, 99), now
Figure 2 Microscopic morphology of Trichosporon mucoides and T asahii. (a) Arthro-

conidia of T. asahii; (b) Hyphae, arthroconidia, and lateral refringent blastoconidia of T.
mucoides.
considered a synonym, and retains the former T. faecale and T. coremiiforme as

varieties (93).
B. Trichosporon inkin
The fungus was first isolated from scrotal skin lesions. Most isolates from the
genital area isolates, including those from white piedra on pubic hairs, were
shown to belong to the same genetic entity. The fungus may be particularly well
adapted to the genital microniche (83, 87, 103, 104). The species is also found
in urine, however, and occasionally causes deep-seated trichosporonosis (102).
Reports with the right identification of T. inkin (111) are rare, and therefore its
clinical importance is not yet known.
Because of the presence of sarcinae, round multiseptate cells that occur in
the cutaneous tissues of scrotum, T. inkin has been classified as Sarcinomyces
inkin or Sarcinosporon inkin. These monotypic genera are presently considered
to be synonyms of T. inkin (E. Guého, unpublished data), however. Consequently,
the report of a Sarcinosporon inkin invasive infection (112) must be attributed
to T. inkin. The species was also thought to be related to algae of the genus
Prototheca, because the sarcinae are reminiscent of the morphology of this genus,
and to Fissuricella filamenta multiplying by meristematic cleavage. (See T.
asteriodes.) Sarcinae are restricted to the parasitic phase of T. inkin, however,
and are formed in vitro on culture media with high sugar contents (Fig. 3a).
Figure 3 Micromorphology of Trichosporon inkin: (a) sarcina-like cells formed on high

sugar containing media; (b) arthroconidia and appressoria formed on solid substrates (e.g.,
glass slides); (c) primary culture from urine with uninformative arthroconidia and blasto-
conidia-like cells, which can be observed in other species as well.
Hyphae and arthroconidia are similar to those of other Trichosporon species, and
appressoria similar to those of T. ovoides (98, 113) (Figs 3b, c). Colonies on
SGA measure only 9 to 12 mm in diameter. They are dry and finely cerebriform
with a white farinose covering, do not have a marginal zone, and often crack the
agar medium. Appressoria, mostly borne laterally on long arthroconidia are pres-
ent in slide cultures (Fig. 3b). The species grows at 37°C, and L-arabinose and
raffinose are not assimilated (83, 98, 104).
C. Trichosporon mucoides
Several strains were found to be identical with the agent of a cerebral trichospor-
onosis (114), but did not agree with any known species. Therefore a new species,
T. mucoides, was described from the characters of a strain isolated from a brain
as its type culture (83). It is unknown if the first reported agent of a deep-seated
trichosporonosis from a brain abscess and a case of chronic meningitis (83, 100,
114) corresponded to this species. Presently it is clear that T. mucoides is medi-
cally very important (102), and it seems to be the only Trichosporon species able
to cross the cerebral barrier. Besides, it has been isolated from lesions of skin
(115), nails, and from pubic white piedra (103, 104). In Japan, it has been found
to occur in the environment of a patient with summer-type hypersensitivity pneu-
monitis and was identified as serotype I standard culture (110). The species is
able to grow at 37°C. Colonies at 25–28°C reach 13 to 17 mm in diameter, and
are mucoid, cerebriform at the center, and have deep furrows toward the margin.
Morphologically, T. mucoides is characterized by hyphae that can disarticulate
into arthroconidia, but the species also produces lateral blastoconidia that at the
mature state are thick-walled and refringent (Fig. 2b). The species utilizes most
carbon substrates, including L-arabinose and raffinose (98). Its physiological pro-
file is similar to that of Cryptococcus humicola. This latter species, however, was
never found to be implicated in any pathology, nor does it produces arthroconidia.
Moreover, in contrast with T. mucoides it assimilates levulinate present in the
ID 32C identification strips (bioMérieux, Marcy l’Etoile, France; Table 2).
D. Trichosporon ovoides
White piedra on the head and beard hair shaft is nowadays rare, and so is its
causative agent, T. ovoides (83, 104). Only a few isolates were included in the
taxonomic revision (83), but Sugita and co-workers recently isolated T. ovoides
from the urine of a patient with trichosporonosis (108). The species grows very
slowly at 37°C. Colonies at 25–28°C resemble those of T. asahii, while arthro-
conidia and appressoria are similar to those of T. inkin. Physiologically, T.
ovoides is also intermediate between these two species. Like T. asahii, it does
not grow with raffinose, and unlike T. inkin it grows weakly with L-arabinose.
Phylogenetically, T. ovoides clusters together with T. inkin and the new species
T. japonicum (95) at a short distance from T. asahii.
E. Trichosporon asteroides
This species is mainly known from skin, but incidentally was also isolated from
blood (83). The distinction from the phylogenetically closely related T. asahii is
difficult. Growth at 37°C is weak, but colonies at 25–28°C are very similar to
those of T. asahii. Appressoria are not present, and arthroconidia are often thick-
walled and septate. Assimilation reactions, although much slower, are the same
as in T. asahii. Fissuricella filamenta, which morphologically differs from T.
asahii, was found to be conspecific because of high DNA/DNA reassociation
values and rRNA sequence similarity (83). Colonies of F. filamenta are dry, finely
cerebriform, and brownish, and measure only 5 to 6 mm in diameter after 10
days. Growth of the filamentous phase is very brief and typical meristematic
packs of cells quickly formed (98).
F. Trichosporon cutaneum
This species, long synonymized with T. beigelii (83), occurs on skin and hairs
(98, 104, 108). It does not grow at 37°C or in the presence of cycloheximide.
Its colonies are similar to those of T. mucoides, and produce hyphae, arthroconi-
dia, and lateral blastoconidia. The primary cultures may be strictly yeastlike,
however, and may be confused with C. neoformans. Like the latter species, T.
cutaneum is also urease-positive, but it lacks a visible capsule when stained with
india ink.
G. Trichosporon montevideense and T. domesticum

Trichosporon montevideense has recently been isolated from human nails (E.
Guého, unpublished data), and T. domesticum from skin (94). They are close
relatives (94, 107), but T. domesticum grows at 37°C and T. montevideense does
not. Both species produce only hyphae and arthroconidia. Trichosporon montevi-
deense was represented by only two strains in the taxonomic revision (83), but
it does not seem to be a rare species since it also corresponds to the standard
culture of serotype III, another causative agent of the summer-type hypersensitiv-
ity pneumonitis (110).
IV. MALASSEZIA BAILLON
The genus Malassezia belongs to the basidiomycetous yeasts because of its multi-
layered cell wall, enteroblastic budding, urease activity, and a positive staining
reaction with DBB. Phylogenetically it belongs to the Ustilaginomycetes, where
it forms a well-defined cluster, classified as the Malasseziales R.T. Moore emend.

(Fig. 1), which is closely related to the Exobasidiales and Ustilaginales (2, 116).
The recognition of species in Malassezia has caused considerable confusion
(117). The genus was created in 1889 by Baillon (118) with the species M. furfur
to accommodate the filamentous fungus observed in scales of the human skin
disease pityriasis versicolor (PV). In fact, filaments and round, yeastlike cells
had been observed to occur in PV scales much before, and earlier names were
proposed for the yeastlike cells (117). Pityrosporum (119) was proposed as an
alternative generic name, but unfortunately no living culture was preserved. Be-
cause the name Malassezia was published before Pityrosporum, the former has
nomenclatural priority. For a long time the genus remained limited to M. furfur
and M. pachydermatis. The first species concept included Malassezia isolates
that were lipophilic and lipid-dependent and that occur only in humans. M. pachy-
dermatis, still regarded as a valid single species, is lipophilic but not lipid-
dependent, and usually occurs on animals (120). In keeping with this taxonomy,
for many years all pathologies caused by M. furfur sensu lato, particularly disor-
ders of the skin (PV, dandruff, seborrhoeic dermatitis, and folliculitis), were as-
cribed to a single species (121). Only the recent recognition of new clinically
important species changed this approach (122).
Ribosomal RNA sequences published in 1995 revealed seven genetic enti-
ties (123), which later were described as species with distinct morphological and
physiological key characteristics (117). These characteristics were established in
liquid medium (117), but a practical identification scheme based on tests perform-
able on agar media was established for the laboratory routine (124). The recent
addition of esculin and cremophor EL (castor oil) tests (125) further improved
this identification scheme. Primary tests for the identification of Malassezia spe-
cies are catalase and β-glucosidase activities (esculin tubes), followed by evalua-
tion of growth with Tweens 20, 40, 60, 80, and cremophor EL by using the
diffusion method on Sabouraud glucose agar. In this method each compound is
deposited in a well cut into an agar plate or on a paper disk placed on the plate
(Figs. 4a, b). Because three species have their maximum temperatures for growth
at 37°C, all tests are performed at 32–34°C. The ability to grow at 37°C is evalu-
ated as a useful but not indispensable characteristic for identification (126). Fortu-
nately, easy-to-apply molecular techniques, such as PCR-restriction fragment
length polymorphism (PCR-RFLP) (126a,b) and amplified fragment length poly-
morphism (AFLP) (126c) analysis, have shortened and facilitated considerably
the identification of Malassezia yeasts, in particular that of the strongly lipid-
dependent species.
A. Malassezia furfur
This species is represented by two neotype cultures, namely CBS 1878 from a
scalp and CBS 7019 from lesions of PV. The two cultures proved to be conspe-
Figure 4 Plates showing growth of M. furfur (a), and Malassezia globosa (b) on media
with Tween 20, Tween 40, Tween 60, and Tween 80 (start at black bar and clockwise)
and cremophor EL (castor oil) (center). M. furfur shows distinct growth with all substrates,
whereas M. globosa shows weak or no growth with these substrates. (Notice the white
precipitate around Tween 40 and 60.)
cific, and M. furfur is maintained as the generic type species (117). Several ques-
tions remain about this species, however. All strains retained as M. furfur showed
high percentages of DNA/DNA reassociation and high ribosomal RNA similarity
(123), but two karyotypes were observed (127). Moreover, the species is morpho-
logically heterogeneous with globose, oval, or cylindrical yeast cells, even though
it is physiologically homogeneous. Malassezia furfur can be routinely identified
by the combined characteristics of its ability to grow at 37°C, a strong catalase
reaction, absence or a very weak β-glucosidase activity, and equal growth in the
presence of Tweens 20, 40, 60, 80 and cremophor EL as sole lipid sources (126)
(Fig. 4a). Some strains are able to produce filaments, either spontaneously or
under particular culture conditions (128) (Fig. 5a). These filaments should be
interpreted as pseudohyphae, not as true hyphae, because they lack septa with a
central pore. Strains of the species originate from various hosts, sites, and dis-
eases. M. furfur was not observed in recent epidemiological surveys of healthy
persons and patients with PV or seborrhoeic dermatitis (SD) (129) and healthy
volunteers (130). This absence may perhaps be caused by the isolation protocols
used, or may arise from competition between different skin-inhabiting species
of Malassezia (131). When the same protocol is used, however, the species has
been isolated from systemic and mucosal sites, such as urine, vagina, and blood
(127), or exposed sites such as nails (E. Guého, unpublished data). As a prelimi-
nary conclusion, we can state that the species is a pathogen, but its role in disease
remains to be elucidated. Because M. furfur is mildly lipid-dependent, it sur-
vives—particularly in collections—better than the other species. It has also been
isolated from animals (132–134).
Malassezia slooffiae, one of the four new species, may be misidentified as
Figure 5 Micromorphology of M. furfur (a), and Malassezia globosa (b): (a) gram-
stained oval yeast cells and filaments of M. furfur CBS 7019 originally isolated from
pityriasis versicolor; (b) scales of pityriasis versicolor with parker ink-stained globose
yeast cells and short filaments of M. globosa.
M. furfur. It can be differentiated from the latter species, however, by its unique
absence of growth with cremophor EL (126). The species is regularly isolated
from human skin and is mostly found in association with M. sympodialis or M.
globosa. Its role as human pathogen is likely very weak, but it may be better
adapted to animals, especially pigs (132, 133).
Malassezia obtusa, another new species, resembles M. furfur morphologi-
cally but differs physiologically. It does not grow at 37°C, nor with any of the
five lipids used in the tests. It darkens esculin medium (126), however. It is a
very rare species that so far is known to occur only in the healthy skin of humans.
B. Malassezia pachydermatis
Malassezia pachydermatis is the only lipophilic Malassezia species able to grow
without supplementation of long-chain fatty acids or their esters. So far, the non-
lipid-dependent Malassezia yeasts are considered to represent a single species,
for which the epithet pachydermatis has priority (135). The species may be in
the process of speciation (123), however, likely in relation to host specificity
(136). In contrast to M. furfur, it has a constant karyotype (127), and minor pheno-
typic differences seen among isolates correlate with differences observed in
rDNA genotypes. All isolates grow well at 37°C, and some primary cultures
show a certain lipid dependence (137, 138), therefore epidemiological surveys
of Malassezia yeasts from any animal or human source should utilize lipid-
supplemented media. The weakly lipid-dependent isolates have smaller colonies
than those that lack a response to lipid supplements. Differences in catalase and
β-glucosidase expression, in Tweens 20, 40, 60, 80, and cremophor EL reactivity
occur in all rDNA genotypes (126). These different compounds, particularly
Tween 20 and cremophor EL, may be more or less inhibitory, as growth may
occur only at some distance from the compounds (139). Even if these phenotypic
differences are found to be reproducible, however, they may not be sufficient to
discriminate consistent separate species among the non-lipid-dependent isolates.
M. pachydermatis is rare in humans, although it has been found to cause septic
epidemics, usually in infants as a complication of prematurity (140, 141). In one
case, the contamination was linked to a nurse’s dog (142). M. pachydermatis is
well known as a normal cutaneous inhabitant of numerous warm-blooded ani-
mals. Seborrhoeic dermatitis and otitis associated with this lipophilic yeast are
now commonly recognized, especially in dogs (143). So far, however, it has not
been possible to relate differences in pathogenicity to phenotypic or genotypic
traits (136, 144).
C. Malassezia sympodialis
This lipophilic and lipid-dependent Malassezia yeast was described in 1990
(145), but only the comparison of a large number of strains resulted in a more
accurate circumscription of the species (117). It correlates to the former M. furfur
serovar A (146) and can be characterized in routine identifications by a strong
β-glucosidase activity, which deeply darkens the esculin medium in 24 hr. The
species grows at 37°C. Cremophor EL as a unique lipid supplement does not
allow good growth (as also seen in M. slooffiae, but this species does not split
esculin). Primary isolates may develop a ring of tiny colonies at some distance
from the cremophor EL source. Morphologically, the yeast cells are small and
ovoid, and they are not able to form filaments in culture. The species is commonly
isolated from healthy as well as diseased skin (129). Its role as a pathogen is not
yet elucidated. Indeed, in skin lesions M. sympodialis is often present, but usually
associated with the more abundantly occurring M. globosa (130). The species
has also been isolated from healthy feline skin (147).
D. Malassezia globosa
This species has a stable micromorphology in that its yeast cells remain spherical
even after several transfers (117). Buds are also spherical and emerge from the
mother yeast through a narrow site, contrary to the patterns seen in other Malas-
sezia species. The species corresponds to the original description of Pityrosporum
orbiculare obtained from a PV case (148). It seems likely, however, that many
data published in the past as P. orbiculare correspond in large part to other spe-
cies. Like M. furfur, M. globosa is able to produce filaments. Particularly in
primary cultures, a certain number of yeasts produce germination tubes of various
lengths (Fig. 5b), but this ability disappears after transfer. M. globosa correlates
to former M. furfur serovar B (146). The yeast does not grow at 37°C or does
so poorly, does not grow on the five lipid substrates (Fig. 4b), and does not split
esculin. Malassezia globosa is the most important species in PV (148a), either
alone or associated with other species, particularly M. sympodialis (131). The

species is present in other types of cutaneous lesions as well, but more studies
are necessary to examine its role in different pathologies, alone or in synergy
with other species. Malassezia globosa has also been isolated from a cat (147),
but it mainly occurs on humans (122, 131, 148a).
E. Malassezia restricta
This is the only lipid-dependent species lacking catalase activity (117). Like M.
globosa, M. restricta lacks β-glucosidase activity, does not grow at 37°C, and
is strongly lipid-dependent. Growth of the colonies is very restricted. The species
is isolated almost exclusively from the head, including scalp, neck, and face
(130), and it corresponds to serovar C (146). Because of its localization on the
human head and its small oval to round yeast cells, M. restricta resembles the
former concept of Pityrosporum ovale (149). Unfortunately, no culture has been
preserved to represent the latter name. Malassezia restricta does not produce any
filaments. Although M. restricta is very fastidious, more studies are needed to
understand its implication in Malassezia-associated diseases, in particular dan-
druff and seborrhoeic dermatitis. This species is not known to occur in animals
(132, 133).
V. RHODOTORULA HC HARRISON
The genus Rhodotorula is an anamorphic genus with 34 species of mainly red-

pigmented basidiomycetous yeasts, belonging to the Urediniomycetes (2, 150).
All species are considered to be nonpathogenic or of low pathogenicity, but may
cause severe infections after gaining access to normally sterile regions of the
human body, mainly through indwelling catheters (151–155). During a 29-month
survey Rhodotorula yeasts comprised 0.71% of the total of 8062 fungal speci-
mens cultured at a clinical center (156). In one year, sepsis due to Rhodotorula
species occurred in 7 out of 47 patients at another clinical center after implanta-
tion of central venous catheters. From 1985 onwards an increase of the incidence
of catheter-related infections with Rhodotorula yeasts was observed (157). In 5
years 36 patients were found to be infected, and all had underlying diseases such
as tumors, leukemia, and AIDS, and almost all had catheters. Of the 23 clinically
relevant cases 22 were caused by Rhodotorula mucilaginosa (⫽ Rh. rubra), and
one was caused by Rh. minuta (155).
Rhodotorula mucilaginosa (⫽ Rh. rubra) is the only species considered to
be clinically relevant (151). Isolates of this species present in the CBS yeast
collection have been obtained from a wide variety of sources, including lung,
nail, lymph nodes, skin, and feces (72), although by no means were all of these
isolations etiologically significant. The species is mainly reported from patients
receiving total parenteral nutrition, but also from patients with AIDS, carcinoma,
endocarditis, meningitis, sepsis, and keratitis (158–164).
Another species, Rhodotorula glutinis var. glutinis, has been reported from
patients receiving nutrition or antibiotics through indwelling catheters, and also
from keratitis (165–167). Rhodotorula minuta has been reported from an AIDS
patient (168). Unidentified Rhodotorula yeasts were isolated from patients suffer-
ing from endocarditis, carcinoma, leukemia, corneal infection, or diabetes, or
receiving neurosurgery (151–154, 169–171).
VI. SPOROBOLOMYCES KLUYVER & CB NIEL
The genus Sporobolomyces is an anamorphic yeast genus containing 21 species

and belonging to the Urediniomycetes (2, 172). All species are considered to
be nonpathogenic, but may be involved in AIDS-related infections (173, 174).
Sporobolomyces holsaticus was reported to cause dermatitis, and Sporobolo-
myces roseus was isolated from a case of madura foot and from mycotic lesions
of mucous membranes (72, 175, 176). The ballistoconidia of Sporobolomyces
species may cause respiratory allergy (177). Isolates of S. salmonicolor (including
S. johnsonii and S. holsaticus) are known from infected skin, but also from cere-
brospinal fluid, and S. roseus has been isolated from a mycotic lesion on skin
and a case of madura foot (72). As these yeasts widely occur in nature, however,
and produce air-borne conidia, these isolates may represent contaminants.
VII. TILLETIOPSIS DERX EX DERX AND PSEUDOZYMA

BANDONI EMEND. BOEKHOUT
These yeastlike fungi represent anamorphs of the plant pathogenic smut fungi
(Ustilaginomycetes) (2, 116, 178, 179). Tilletiopsis species from ballistoconidia,
whereas acropetally branched chains of fusiform to cylindrical blastoconidia oc-
cur on sterigmalike outgrowths in Pseudozyma (180, 181). Tilletiopsis albescens
is known from a lesion of colostomy, and T. minor has been isolated from the
human cervix and urethra and also from a puncture from an eye of a 70-year-
old human (178). Pseudozyma strains have been isolated from diverse substrates,
including skin and blood (182). Pathogenicity has not been proven in any of these
cases, however, and the isolates may represent contaminants.
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162. H Papadogeorgakis, E Frangoulis, C Papaefstathiou, A Katsambas. Rhodotorula
rubra fungaemia in an immunosuppressed patient. J Eur Acad Derm Venereol 12:
169–170, 1999.
163. AY Lui, GS Turett, DL Karter, PC Bellman, JW Kislak. Amphotericin B lipid
complex therapy in an AIDS patient with Rhodotorula rubra fungemia. Clin Infec
Dis 27:892–893, 1998.
164. OH Gyaurgieva, TS Bogomolova, GI Gorshkova. Menigitis caused by Rhodotorula
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166. G Bertoli, F Rivasi, U Fabio. Rhodotorula glutinis keratitis. Int Ophthal 16:187–
190, 1992.
167. C Casolari, A Nanetti, CM Cavallini, F Rivasi, U Fabio, A Mazoni. Keratomycosis
with an unusual etiology (Rhodotorula glutinis): A case report. Microbiologica 15:
83–88, 1992.
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169. PF Shelburne, RJ Carey. Rhodotorula fungemia complicating staphylococcal endo-
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10
Dematiaceous Hyphomycetes
Wiley A. Schell
Duke University Medical Center, Durham, North Carolina, U.S.A.
I. DESCRIPTION AND NATURAL HABITATS
Dematiaceous fungi comprise a heterogeneous group that is characterized by the

presence of a melanin compound within the cell walls of the hyphae or spores
(or both). Cell coloration varies from hyaline (colorless) to pale or mid-brown,
depending on the concentration of melanin within individual cells. This causes
colony coloration to range from gray, olive, light to dark brown, to black. The
presence of melanin traditionally has been used to group these fungi as a practical
basis for their identification. Dematiaceous fungi are represented in the Hypho-
mycetes, Ascomycetes, Coelomycetes, and Zygomycetes. Most are pathogens of
plants or saprobes of senescent and decaying matter, and can be found in soil.
They are widely disseminated in the environment, primarily via dispersal of their
spores, and most can be encountered in the laboratory setting as insignificant
isolates from nonsterile clinical specimens or as outright laboratory contaminants.
In addition, they can cause opportunistic infections in humans and animals
(1–5).
II. CLINICAL OVERVIEW
Dematiaceous fungi can cause chromoblastomycosis, mycetoma, phaeohypho-

mycosis, and sporotrichosis in normal and compromised hosts. In most cases,
infection occurs following implantation of the fungus during abrasion or penetrat-
ing injury to the host. In contrast, cases of fungal sinusitis and at least some cases
of pulmonary or disseminated infection are presumed to begin after conidia are
565
566 Schell
inhaled. Chromoblastomycosis is a chronic, localized infection of skin and subcu-

taneous tissue that is caused mainly by three species of molds. When in host
tissue, these molds undergo a morphologic conversion into subglobose, often
multicellular (muriform) bodies. These bodies propagate by splitting along their
septa. The finding of muriform bodies in cutaneous or subcutaneous tissue is
pathognomonic of chromoblastomycosis, although very rarely they can be seen
in other mycotic infections as well (1). In some cases, dematiaceous hyphae are
present. Because all agents of chromoblastomycosis have the same morphology
in host tissue, the identity of the fungus in a particular case can be determined
only by culture.
Phaeohyphomycosis similarly begins with traumatic implantation or inhala-
tion of cells, but the fungus appears in tissue as dematiaceous yeastlike cells,
pseudohyphae, hyphae, or any combination of these forms. Most agents of phaeo-
hyphomycosis form at least some dark cells in infected tissue, but some, such
as species of Alternaria, Bipolaris, and Curvularia, often are hyaline in tissue
because of scant melanin production. Still, these species are considered agents
of phaeohyphomycoses because they clearly are dematiaceous when grown in
culture. More than 100 fungi are documented causes of phaeohyphomycoses (2),
and because the morphology of these agents is similar in infected tissue, they
must be recovered in culture media before identification is possible. Historical
usage and distinctions for the terms phaeohyphomycosis, chromoblastomycosis,
and chromomycosis have been reviewed (1).
Sporotrichosis is caused only by Sporothrix schenckii. In host tissue, this
mold converts to budding yeast cells. Most cases begin with implantation of the
fungus, resulting in chronic lesions of skin and subcutaneous tissue that tend to
spread via the lymphatic system. Musculoskeletal involvement and disseminated
infection sometimes occur, and nasal and pulmonary infections following inhala-
tion of conidia also have been documented (6–9). Sporotrichosis can be a zoo-
notic disease as well, as shown by numerous infections acquired from cats or
other animals (1,10,11).
Mycetoma is a chronic, localized granulomatous infection of subcutaneous
and cutaneous tissue that can spread to and destroy adjacent bone tissue. Infection
follows traumatic implantation of the fungus. At least 26 species of fungi cause
mycetoma, and several of them are dematiaceous molds (12,13).
III. IDENTIFICATION
Taxonomy, nomenclature, and identification of some dematiaceous fungi are

problematic because of their phenotypic plasticity, and because of complexities
and uncertainties in their life cycles. Application of molecular sequencing tech-
niques coupled with powerful analytic algorithms has done much to establish
Dematiaceous Hyphomycetes 567
apparent phylogenetic relationships among described taxa, as well as determining

the homogeneity of isolates within various taxa. Still, in most settings, especially
clinical laboratories, the traditional morphocentric approach remains the primary
if not sole means by which dematiaceous fungi are identified.
Some species of dematiaceous fungi readily form a teleomorph during cul-
ture, and the identification accordingly is based on the teleomorph whether or
not an anamorph is present (14). Other species are able to form only an anamorph,
and the identification necessarily is based on this asexual morph. Still other spe-
cies lack a teleomorph but are able to form multiple asexual forms, termed syna-
namorphs. Each of these synanamorphs may be referred to by a separate name
as needed. One approach to identifying these synanamorphic fungi bases the
name on the anamorph that is judged to be the predominant or most distinctive.
An accompanying synanamorph then can be referred to, if need be, by using a
genus-level name. For example, in the case of the Fonsecaea pedrosoi, the name
is based on the synanamorph that forms a compactly sympodial, branched conid-
iophore. A Rhinocladiella synanamorph and a Phialophora synanamorph also
may be seen, but these are not essential to the identification of an isolate as F.
pedrosoi (15).
IV. DESCRIPTIONS
A. Acrophialophora Edward 1959
Acrophialophora fusispora (Saksena) M.B. Ellis 1971, has been identified as
causing keratitis (16). Colonies are dense, matted, and feltlike at the base, with
floccose aerial hyphae, initially dull white, becoming grayish-brown. Colony re-
verse is black. Conidiophores are erect, septate, smooth or roughened, thick-
walled and brown at base, becoming pale toward apex (Fig. 1). Phialides are
flask-shaped and swollen near base, with tapered neck, borne singly, in pairs or
verticils on conidiophores or sometimes on vegetative hyphae. Conidia are one-
celled, colorless to pale brown, broadly ellipsoidal to limoniform, with fine echi-
nulations formed in spiral bands. Conidia are borne in long basipetal chains.
Compare with Paecilomyces (17, 18).
B. Alternaria Nees 1816

Alternaria species have been reported in infections of bone, cutaneous tissue,
ears, eyes, paranasal sinuses, and the urinary tract (19,20). The great majority of
infections are caused by A. alternata (synonym A. tenuis) (21). A. chlamydospora
has infected hyponychium (22) and skin (23), both A. infectoria and A. longipes
have caused cutaneous phaeohyphomycosis (24,25), and A. chartarum has in-
fected the nasal septum (26). Cutaneous infections have been attributed to A.
568 Schell
Figure 1 Acrophialophora fusispora. Source: Ref. 26a.
tenuissima (27–29). Colonies are rapidly growing, floccose, white to gray, be-
coming brown, reverse brown to black. Conidiophores are erect, dark, septate,
simple, or branched. Conidia are muriform, obclavate, with beak (tapering apex),
darkly pigmented, smooth or rough, in simple or branched acropetal chains. Com-
pare with Ulocladium, Stemphilium, Embeliasia (4,5,17,30).
1. Alternaria alternata (Fr.) Keissler 1912

A teleomorph is not known. Condidiophores are pale yellow-brown to mid-
brown, usually unbranched, mostly 30 to 50 µm long and one-to-three septate,
bearing a single apical scar, sometimes with one to two subterminal scars (Fig. 2).
Figure 2 Alternaria alternata.
Conidia are smooth to minutely roughened, obpyriform or obclavate, sometimes

ovoid, 20–63 (37) µm long, 9–18 (13) µm wide, three to eight transverse septa,
one or more longitudinal septa, apical beak when present not exceeding one-
fourth to one-third total length of conidium (Fig. 2). Conidia sometimes are soli-
tary, but usually form in acropetal chains that sometimes branch (17).
2. Alternaria chlamydospora Mouchacca 1973

A teleomorph is not known. Conidia mostly are 20–50 µm ⫻ 7–20 µm, obclavate
to obpyriform, and lack a defined beak (Fig. 3). Cells of most conidia enlarge
and bulge in a manner reminiscent of chlamydospore development, resulting in
distorted conidia. Conidia sometimes arise directly from hyphae (31).
570 Schell
Figure 3 Alternaria chlamydospora. Source: Ref. 31.
3. Alternaria longipes (Ellis & Everhart) Mason 1928

This species is one of the small-spored species, and can be confused with A.
alternata. The most prominent difference is the tendency of the conidia to form
a beak that immediately continues its development into a conidiophore (Fig. 4).
This transitional region of the conidium often is marked by a slight constriction
(31). A teleomorph is not known.
4. Alternaria tenuissima (Kunze ex Persoon) Wiltshire 1933

This species is characterized by conidia that are straight or curved, obclavate or
tapering to a beak, and solitary or borne in short chains (Fig. 5). The beak usually
Figure 4 Alternaria longipes. Source: Ref. 31.
is swollen at the tip, but can be pointed. The beak can be as long as one-half the
length of the conidium (17,31).
C. Aureobasidium Viala & Boyer 1891

Aerobasidium pullulans reportedly has caused infections of the peritoneum, nail,
skin, and subcutaneous and deeper tissues (32–39). Hormonema species often are
mistaken for Aureobasidium species, in part because they have been illustrated in
the past under the name Aureobasidium. It is likely that some infections attributed
to A. pullulans instead were caused by misidentified isolates of Hormonema spe-
cies. A. pullulans differs from Hormonema spp. by having conidia that arise syn-
chronously, and differs from Phaeococcomyces spp. by the lack of dematiaceous
yeast cells. Compare with Hormonema, Scytalidium (5,17,30,40).
1. Aureobasidium pullulans (de Bary) Arnaud 1910

A. pullulans is the most common of the several described species. Colonies are
smooth and moist, off-white, cream, light pink, or light brown, finally becoming
dark brown from formation of dematiaceous arthroconidia. Conidiogenous cells
are undifferentiated from hyphae; intercalary or terminal (Fig. 6). Conidia are
572 Schell
Figure 5 Alternaria tenuissima, left center; A. longipes, top; A. alternata, bottom.

Source: Ref. 31.
hyaline, smooth, straight, ellipsoidal, one-celled, ranging in size from (7.5–) 9–

11 (⫺16) ⫻ (3.5–) 4–5.5 (⫺7) µm (40). Secondary blastoconidia often are pro-
duced. Large, dark one-to-two-celled, thick-walled arthroconidia (i.e., a Scytali-
dium synanamorph) usually are present. In addition, conidia sometimes can be
formed endogenously in clusters within hyphal cells. The teleomorph of A. pullu-
lans might be Discosphaerina fulvida (41).
D. Bipolaris Shoemaker 1959

Several species, including B. australiensis, B. hawaiiensis, and B. spicifera, have
caused meningitis; paranasal sinusitis; and subcutaneous, eye, pulmonary, and
Figure 6 Aureobasidium pullulans.
disseminated infections (42–46). Species of Drechslera, Exserohilum, and Hel-

minthosporium were confused with Bipolaris species in the past, but subsequent
taxonomic studies established useful criteria for separating species of the genera.
Exserohilium is distinguished by a protuberant hilum on each conidium. Drech-
slera differs from Bipolaris by its ability to germinate from any cell of its conidia
rather than just the two end cells, and further by its conidial germ tube arising
at a strong angle to the main axis of the conidium (43, 47). Colonies are rap-
idly growing, woolly, gray to black. Conidiophores are dark, erect, simple or
branched, septate, and geniculate. Conidia are multidistoseptate, cylindrical to
oblong, dark, with hila protruding only slightly. Conidia germinate only from end
cells (with little exception), and the germination hyphae initially grow parallel to
the long axis of the conidium. Compare with Curvularia, Drechslera, Exsero-
hilum, Helminthosporium, Nakateae (4,5,43,47,48)
1. Bipolaris australiensis (Ellis) Tsuda & Ueyama 1981

The teleomorph of this species is Cochliobolus australiensis and is heterothallic.
Conidia are similar to B. spicifera except they (rarely) can have as many as four
to five distosepta, are mostly 10 or fewer µm wide (most range from 18–33 µm
⫻ 8–10 µm), and do not exhibit a pale region just above the hila (Fig. 7).
2. Bipolaris hawaiiensis (Ellis) Uchida & Aragaki 1979

The teleomorph is Cochliobolus hawaiiensis and is heterothallic. Conidia mostly
have five distosepta but can have as few as two and as many as seven distosepta
(Fig. 8). Conidial size usually ranges from 12–37 µm ⫻ 5–11 µm.
574 Schell
Figure 7 Bipolaris australiensis. Source: Ref. 17.
3. Bipolaris spicifera (Banier) Subramanian 1971

The teleomorph of this species is Cochliobolus spicifera and is heterothallic.
Conidia consistently have three (rarely four) distosepta (Fig. 9). Distosepta are
evenly colored, none being darker than others. Most conidia are less than 40 µm
long and more than 10 µm wide (typical size range is 30–36 µ ⫻ 11–13 µm).
Mature conidia are straight and pale brown in color except for a small region
just above the hila.
Figure 8 Bipolaris hawaiiensis.

Figure 9 Bipolaris spicifera.
E. Botryomyces de Hoog & Rubio 1982

1. Botryomyces caespitosus de Hoog & Rubio 1982
The teleomorph is unknown. Skin infections have been reported, and its tissue
forms are consistent both with phaeohyphomycosis and chromoblastomycosis
(49,50). Colonies are black, restricted in growth, raised, and dry in texture. Hy-
phae are absent. Cells are unicellular to muriform, subglobose, hyaline when
young, brown in age, 7.5 to 13 µm. Budding is absent, and cells instead adhere
in aggregations and become septate, with portions of the aggregates eventually
separating (Fig. 10). Compare with Sarcinomyces.
576 Schell
Figure 10 Botryomyces caespitosus.
F. Cladophialophora Borelli 1980

The genus Cladophialophora Borelli (1980) now accommodates certain species
previously classified in other genera, including Cladosporium and Xylohypha
(51). More notable species in Cladophialophora are Cladophialophora carrionii
(formerly Cladosporium carrionii) and Cladophialophora bantiana (synonyms
Cladosporium bantianum, Xylohypha bantiana, Cladosporium trichoides). This
taxonomic revision reflects phylogeny inferred from molecular sequencing data.
(52,53). Xylohypha emmonsii in 1995 was regarded as conspecific with Clado-
phialophora bantiana based on nuclear DNA homology analysis (52). New data,
however, have led to a reversal of this disposition, and the binomial Xylohypha
emmonsii has been reintroduced (54). Cladophialophora bantiana has caused
dozens of cerebral infections (53) and also has been involved occasionally in
cutaneous and subcutaneous infections (55–57). Because most cases are cerebral
infection with no evidence of cutaneous lesions, it is assumed that the organism
can gain entrance via the lungs. For this reason it has been recommended that
isolates be manipulated only within a biological safety cabinet (58). Recently an
isolate from a cerebral infection was reidentified and described as a new species,
Cladophialophora modesta (59). Cladophialophora carrionii is a leading agent
of chromoblastomycosis in Africa, Australia, and Madagascar (1,60). Cladophia-

lophora emmonsii is known from cutaneous and subcutaneous tissue and from
spleen, but not from brain (54,56). Compare with Cladosporium, Taeniolella.
1. Cladophialophora bantiana (Saccardo) de Hoog et al.

1995
C. bantiana conidiophores are hyphalike, poorly differentiated, pale brown. Co-
nidia occur in very long sparsely branched chains that in most isolates are poorly
differentiated from conidiophores and vegetative hyphae (Fig. 11). Conidia of
Figure 11 Cladophialophora bantiana.

578 Schell
C. bantiana measure 2–2.5 ⫻ 4–7 µm (sometimes larger). All isolates tested

thus far possess a 558 base pair intron at position 1768 of the small subunit rDNA
gene (54). Isolates of C. emmonsii tested thus far lack this distinctive intron.
Morphologically, C. emmonsii differs by having conidia that are asymmetric to
sigmoid, sometimes two-celled, occurring in shorter chains, and failing to grow
at 42–43°C. Compare with Cladosporium (4, 56, 61).
2. Cladophialophora carrionii (Trejos) de Hoog et al. 1995

Colonies are velvety, olivaceous gray to black, slow to moderate in growth rate.
Gelatin hydrolysis is negative (rarely weak), there is growth at 37°C, and isolates
are not susceptible to cycloheximide. Conidiophores are short, lateral or terminal,
and bear long, infrequently branched chains of conidia that tend not to disarticu-
late completely (Figs. 12, 13). Conidia are one-celled, approximately 2.2–2.6 ⫻
4.5–6.0 µm, elliptical, bilaterally symmetrical, mainly uniform in size and shape.
A Phialophora anamorph can be seen when grown on nutritionally poor media
(Fig. 13) (4).
3. Cladophialophora devriesii (Padhye & Ajello) de Hoog et al.

1995
This species was described as a new species in 1984 from a case of subcutaneous
phaeohyphomycosis (62). Diagnosed in 1981, the infection was refractory to ther-
apy and subsequently disseminated to the liver, killing the patient in 1988 (63).
Morphology of the species is similar to that of Cladosporium cladosporioides
Figure 12 Cladophialophora carrionii.

Figure 13 Phialophora synanamorph (left) of Cladophialophora carrionii.
and C. sphaerospermum, but the conidia taper more sharply toward both ends
in such a way that the spores are lemon-shaped to fusiform and their connectives
often are narrow and elongated (Fig. 14). Spore walls remain smooth. Further in
contrast to C. cladosporioides and C. sphaerospermum, colonies will grow at
37°C. There is no growth at or above 40°C. Gelatin hydrolysis is negative and
urease is formed. Only one isolate has been described. Subsequently described
was a species, Cladophialophora arxii (64), that seems to have no significant
morphologic difference from C. devriesii, but that grows at 40°C and exhibited
differences in partial 26S rRNA sequence. Recently an isolate similar in morphol-
ogy to C. arxii and C. devriesii, but differing in molecular makeup was described
(65).
4. Cladophialophora emmonsii (Padhye et al.) de Hoog &

Padhye 1999
This species closely resembles C. bantiana. It differs by conidia that are asym-
metric to sigmoid, one- (rarely two-) celled, occurring in shorter chains than seen
with C. bantiana, and failing to grow at 42–43°C (Fig. 15). Also, molecular
analysis shows that it lacks a distinctive intron found in C. bantiana (54).
G. Cladosporium Link 1816

Cladosporium species are widespread in the environment, and there is little evi-
dence that they can cause infection in humans. Reported cases consist of cutane-
580 Schell
Figure 14 Cladophialophora devriesii. Source: Ref. 62.
ous, subcutaneous, and eye infections (5,66–71), and colonization of a pre-

existing lung cavity (72). Species of Cladosporium, especially C. cladosporioides
and C. sphaerospermum, are among the most common dematiaceous mold con-
taminants recovered by clinical laboratories. Colonies are rapidly growing, vel-
vety or cottony, olive-gray to olive-brown or black. Conidiophores are dark, erect,
long, often septate and branching. Conidia are one-celled (several-celled in some
species), smooth or rough, with dark prominent hila, occurring in long, fragile,
profusely branched acropetal chains. Ramoconidia are one-to-three-celled, usu-
Figure 15 Cladophialophora emmonsii.
ally shield-shaped. Usually no growth at 37°C; gelatin hydrolysis usually posi-

tive. Compare with Fonsecaea, Cladophialophora (4,5,17,30,62,73).
1. Cladosporium cladosporioides (Fresen.) de Vries 1952

Conidia are smooth in most strains, ellipsoidal to lemon-shaped, one- (rarely
two-) celled, mostly 3-7 ⫻ 2-4 µm with one or more hila (Fig. 16). No teleo-
morph is known.
2. Cladosporium herbarum (Pers.) Link ex S.F. Gray 1821

Conidia are pale brown to brown, more or less verrucose, mostly one-to-two-
celled with some up to four-celled. One-celled conidia mostly are 4–5 ⫻ 4.5–
582 Schell
Figure 16 Cladosporium cladosporioides.
11 µm. Conidiophores are up to 225 µm long, often nodose, and somewhat genic-
ulate (Fig. 17). No teleomorph is known.
3. Cladosporium sphaerospermum Penz. 1882

Conidia rough-walled, mostly globose to subglobose and one-celled, 3–4.5 µm
in diameter. Ramoconidia smooth or rough-walled, one-to-three-celled, with one
or more hila (Fig. 18). No teleomorph is known.
H. Curvularia Boedijn 1933

Collectively, Curvularia species are leading agents of fungal sinusitis and kerati-
tis and may cause endocarditis, mycetoma, pulmonary infection, cerebral in-
fection, and subcutaneous phaeohyphomycosis as well (42,74–78). Curvularia
Figure 17 Cladosporium herbarum. Source: Ref. 17.
Figure 18 Cladosporium sphaerospermum.

584 Schell
brachyspora, C. clavata, C. geniculata, C. lunata, C. pallescens, C. senegalensis,

and C. verruculosa specifically have been identified from infections, with C. lu-
nata being most common by far. Descriptions by Ellis (17,30,79) should be con-
sulted if a Curvularia isolate must be identified to species. Colonies are rapidly
growing, woolly, gray to grayish-black or brown. Conidiophores are dark, erect,
and geniculate due to sympodial development. Conidia are multiseptate, usually
curved, with central cell larger and darker than end cells, thickness of septa and
outer cell wall approximately the same; hilum dark. Some species will form large,
erect, cylindrical, sometimes branched, black stromata when grown on rice grains
and sometimes on potato dextrose agar (PDA). Compare with Alternaria, Bipo-
laris, Drechslera, Exserohilum, Nakataea.
1. Curvularia brachyspora Boedijn 1933

Conidiophores often are nodose (Fig. 19). Conidia are three-septate; middle sep-
tum is median, occurs at widest part of conidium, and is the thickest and darkest
septum. Conidia mostly are somewhat curved, broadly fusiform, end cells are
subhyaline to very pale brown, and intermediate cells are brown, smooth-walled,
19–26 µm ⫻ 10–14 µm. Conidia are borne in apical clusters and in verticils at
the conidiophore nodes. Stromata are formed in rice grains, sometimes on PDA.
2. Curvularia geniculata Nelson 1964

The teleomorph is Cochliobolus geniculatus and is heterothallic. Conidia usually
are curved, rather fusiform in shape, 18–37 ⫻ 8–14 µm, uniformly five-celled,
Figure 19 Curvularia brachyspora. Source: Ref. 79.

Figure 20 Curvularia geniculata.
end cells subhyaline or only pale brown, remaining cells plainly brown (Fig. 20).
Stromata are not formed on rice or PDA.
3. Curvularia lunata (Wakker) Boedijn 1933

The teleomorph is Cochliobolus lunatus Nelson & Haasis 1964, and is heterothal-
lic. Stromata can be present on rice or PDA and are large, black, and sometimes
branched. Conidiophores often are geniculate (Fig. 21). Conidia are straight to
curved, smooth, 18–30 ⫻ 8–15 µm, mostly four-celled; some septa may be thick
and dark, and the third cell from the base often is darker and larger. End cells
of the conidia usually are paler; hila not protuberant.
4. Curvularia pallescens Boedijn 1933

The teleomorph is Cochliobolus pallescens and is heterothallic. Conidia are
straight to slightly curved, 17–32 ⫻ 7–12 µm, smooth, all cells pale, end cells
sometimes more so, four-celled, with third cell from bottom often being the wid-
est; hila not protuberant (Fig. 22). Stromata are absent.
5. Curvularia senegalensis (Spegazzini) C.V. Subramanian

1956
The teleomorph is not known. Conidia mostly are curved, 19–30 ⫻ 10–14 µm,
mostly five-celled, smooth, end cells colorless to pale brown, other cells plainly
brown, hila not protuberant (Fig. 23). Stromata are not formed.
6. Curvularia verruculosa Tandon & Bilgrami 1962

Conidia are straight or curved, ellipsoidal to clavate, 20–35 ⫻ 12–17 µm, four-
celled, basal cell or both end cells smooth and colorless to pale brown; central
586 Schell
Figure 21 Curvularia lunata.
cells are brown and verrucose (Fig. 24). Stromata are formed on rice and some-
times PDA. No teleomorph is known.
I. Dactylaria Saccardo 1880

Dactylaria is a genus noted in medical mycology because of the binomial D.
gallopava, which was proposed for a species described originally as Diplorhino-
trichum gallopavum (80,81). In addition to causing epizootic encephalitis in poul-
try flocks, this species is a well-documented cause of disseminated mycoses in
immunocompromised humans (82–84). Two varieties of this binomial were pro-
posed (85): D. constricta var. gallopava and D. constricta var. constricta. Be-
cause the genus Dactylaria is characterized in part by conidia that are released
cleanly from their conidiophore through an enzymatic (rhexolytic) process, how-
ever, and because the conidia of gallopava separate from their conidiophores by
cell wall breakage (schizolytic), it was suggested that Dactylaria cannot satisfac-
torily accommodate this species (Fig. 59). As a result, the new combination Och-
roconis gallopava was proposed (86). The distinction between the genera Ochro-
conis and Scolecobasidium is not clear, however, and so an alternative binomial,
Figure 22 Curvularia pallescens. Source: Ref. 79.
Figure 23 Curvularia senegalensis. Source: Ref. 79.

588 Schell
Figure 24 Curvularia verruculosa. Source: Ref. 79.
Scolecobasidium gallopavum (87), probably would be more satisfactory. Recent

data from molecular sequencing also would support this disposition (88). The
matter remains unsettled and multiple names continue to be used. See Sec. IV.
A′ for further discussion.
J. Dissitimurus Simmons, McGinnis & Rinaldi 1987

A teleomorph is not known in this monotypic genus. A single isolate named
D. exedrus was cultured from nasopharynx and turbinate lesions in a human (89).
The colony is blackish-brown, and growth rate at 20–25°C is 1 cm per week.
Growth is subsurface except for long conidiophores that develop slowly over 2
to 4 weeks. Conidiophores are sympodial to geniculate, with conidia borne singly
or in chains (Fig. 25). The genus is quite similar to dysgonic isolates of Alternaria
except for a distinctive disjunctor that occurs between conidia borne in chains
(Fig. 26). Compare with Alternaria.
K. Drechslera Ito 1930

Only one species of Drechslera is known to have caused infection in a human
host (90). Several Bipolaris species that can cause infection in humans were
Figure 25 Dissitimurus exedrus. Source: Ref. 89.
accommodated briefly in the genus Drechslera before being returned to Bipolaris.

Compare with Bipolaris, Curvularia, Exserohilum, Helminthosporium, Nakateae.
1. Drechslera biseptata (Saccardo & Roumeguère)

Richardson & Fraser 1968
Colonies are rapidly growing, velvety to lanose, gray becoming brown to black-
ish. Conidia are straight, usually obovoid to slightly clavate, without a protuberant
hilum, mostly less than 40 µm long and more than 10 µm wide, with two to
three distosepta, pale to mid-brown (Fig. 27). Conidia can germinate from middle
as well as end cells. Germ tubes arise at a large angle to the main axis of the
conidium.
590 Schell
Figure 26 Dissitimurus exedrus. Source: Ref. 89.
L. Exophiala Carmichael 1966

Exophiala jeanselmei is a leading etiologic agent of the subcutaneous phaeohy-
phomycoses and has caused mycetoma and peritonitis (91–93). Exophiala moni-
liae, E. pisciphila, and E. spinifera also have been reported as agents of phaeohy-
phomycoses (94–96). Exophiala werneckii, the cause of ‘‘black palm,’’ has been
renamed Phaeoannellomyces werneckii (pro parte Hortae werneckii). The species
known most recently as Wangiella heteromorpha and as E. jeanselmei var. heter-
omorpha is reported to have been the cause of human infections, but at least
some of these isolates were reidentified as Wangiella dermatitidis (97). This fun-
gus appears by molecular analysis to be different enough to warrant species sta-
tus, but opinion is divided as to whether the appropriate genus is Wangiella or
Figure 27 Drechslera biseptata. Source: Ref. 17.
Exophiala (98,99). Reidentification of isolates to which pathogenicity has been

attributed is warranted.
The colony morphology of Exophiala species is varied. Most isolates ini-
tially grow in the form of a brown yeast (Phaeoannellomyces synanamorph) that
is succeeded by the hyphal Exophiala synanamorph. As a result, colonies are
moist and yeastlike at first, becoming velvety to woolly with age, and pale brown
to black in color. Some isolates that consist predominantly of the Phaeoannello-
myces synanamorph of E. jeanselmei may remain yeastlike in colony texture.
Conidiogenous cells are annellides. Conidia are one-celled (one-to-three-celled
in E. salmonis), hyaline to pale brown, accummulating in balls at apices of the
annellides. For all species described below, the teleomorph is not known, a
592 Schell
Figure 28 Exophiala castellanii.
Phaeoannellomyces synanamorph is present, potassium nitrate is assimilated, and

conidia are one-celled (with the exception in E. pisciphila).
Phaeoannellomyces werneckii (syn. Exophiala werneckii) (100) is dis-
cussed under the genus Phaeoannellomyces. Compare with Phaeoannellomyces,
Phaeococcomyces, Rhinocladiella, Wangiella (4,40,101–107)
1. Exophiala castellanii Iwatsu, Nishimura & Miyaji 1984

Exophiala castellanii is characterized by poorly differentiated conidiogenous
cells having inconspicuous annellations (Fig. 28). There is no growth at 40°C.
It has been suggested that E. mansonii is conspecific with E. castellanii (108).
2. Exophiala jeanselmei (Langeron) McGinnis & Padhye 1977

E. jeanselmei is the most common species in the genus. Two varieties are recog-
nized. Exophiala jeanselmei var. lecanii-corni bears annellations arising directly
from micronematous annellides (Fig. 29). Exophiala jeanselmei var. jeanselmei
bears well-developed, erect, lageniform to cylindrical annellides (Fig. 30), and
in addition may exhibit some micronematous annellides. There is no growth at
Figure 29 Exophiala jeanselmei var. lecanii-corni.
Figure 30 Exophiala jeanselmei var. jeanselmei.

594 Schell
Figure 31 Exophiala moniliae.
40°C. It has been suggested that these two organisms might be regarded as sepa-
rate species (109).
3. Exophiala moniliae de Hoog 1977

The species is morphologically distinguished from E. jeanselmei by annellides
that are swollen or bulging in shape, and that exhibit a prominently elongated
annellated apical zone (Fig. 31). Conidia are 2.5–4 ⫻ 1.5–2.5 µm, broadly ellip-
soidal, sometimes curved cylindrical. Growth at 40°C is variable. Analysis of
mitochondrial DNA suggests that E. moniliae may be conspecific with E. jeansel-
mei (110).
4. Exophiala pisciphila McGinnis & Ajello 1974

This species is very similar to E. jeanselmei, differing by its inability to grow at
37°C and by its larger conidia 3–8 µm ⫻ 2–4 µm, which rarely may be one-
septate (Fig. 32). It was noted without further details that the single isolate from
man grew at 35°C but not at 41°C.
5. Exophiala spinifera (Nelson & Conant) McGinnis 1977

The teleomorph is unknown. Conidiophores are distinctly spinelike, multicellular,
often are darker than vegetative hyphae, and terminate in a prominent, annellated,
sporogenous tip (Fig. 33). Annelloconidia are ellipsoidal, 2–4 ⫻ 3–4 µm. A
Phialophora synanamorph bearing small (1.5 µm) globose conidia may be
Figure 32 Exophiala pisciphila.
Figure 33 Exophiala spinifera.

596 Schell
Figure 34 Phialophora synanamorph of Exophiala spinifera.
present (Fig. 34). A Phaeoannellomyces synanamorph always is present (111).

There is growth at 37°C and a few isolates have shown weak growth at 40°C
(112).
M. Exserohilum Leonard & Suggs 1974

Phaeohyphomycosis of skin, subcutaneous tissue, cornea, and nasal sinuses has
been documented (42,43,113–116). Exserohilum contains three recognized op-
portunistic pathogens: E. longirostratum, E. mcginnisii, and E. rostratum. Mem-
bers of the genus previously had been confused with species of Bipolaris, Drech-
slera, and Helminthosporium, but ensuing taxonomic studies have clarified the
distinctions (43,47). Colonies are rapidly growing, woolly, gray to black. Conid-
iophores are dark, erect, geniculate due to sympodial development. Conidia are
multiseptate, cylindrical to oblong, dark, each with a strongly protruding hilum.
Compare with Bipolaris, Drechslera, Helminthosporium, Nakateae.
1. Exserohilum longirostratum (Subramanian) Sivanesan 1987

This species is characterized by a dichotomy in the size of the conidia. The
smaller conidia are very much like those of E. rostratum. The larger conidia
range from 100 to 400 µm in length, exhibit as many as 21 distosepta, and are
rostrate (beaked) in shape (Fig. 35). Because of morphologic overlap between
E. longirostratum and E. rostratum it has been suggested that the two might be
conspecific (5,43,48).
Figure 35 Exserohilum longirostratum.
2. Exserohilum mcginnisii Padhye & Ajello 1986

This species is similar to E. rostratum but differs by the lack of thick-walled
distosepta and by the presence of unevenly roughened cell walls in age (Fig. 36)
(117).
3. Exserohilum rostratum (Drechslera) Leonard & Suggs 1974

The teleomorph of this fungus is Setosphaeria rostrata Leonard 1976 and is heter-
othallic. Conidia are rostrate, straight or slightly curved, cylindrical or sometimes
ellipsoidal, usually having 6–8 (⫺16) distosepta (Fig. 37). The distoseptum clos-
est to each end of the conidium is dark and thick-walled, and each end cell is
pale compared to the other cells. Conidia commonly measure 60–90 ⫻ 11–20
µm and are smooth-walled.
N. Fonsecaea Negroni 1936

Fonsecaea pedrosoi (Brumpt) Negroni 1936 and F. compacta Carrión 1940 are
the only species in the genus, and both can cause chromoblastomycosis. Fonse-
caea pedrosoi is the leading agent worldwide, while F. compacta is extremely
rare (1,118,119). Fonsecaea pedrosoi also has caused phaeohyphomycosis in a
few cases (120–123). Isolates are pleomorphic and no teleomorph is known. They
are characterized by the formation of one-celled conidia on erect, dark, compactly
sympodial conidiophores. These conidia directly give rise to a second level of
598 Schell
Figure 36 Exserohilum mcginnisii.
conidia, and usually a third level is formed similarly. This limitation to two or
three levels of conidia contrasts with Cladosporium spp. and constitutes the dis-
tinctive morphology for the genus. In addition, Rhinocladiella and Phialophora
synanamorphs can be present. Colonies of both species are slow-growing, vel-
vety, olivaceous black. Conidiophores are pale to mid-brown, usually erect, with
slight apical swelling of main axis. Conidia are one-celled, pale to mid-brown.
Fonsecaea compacta (Fig. 38) differs from F. pedrosoi (Fig. 39) by conidia that
are subglobose, broadly attached, and formed in compact conidial heads. Both
species grow at 37°C. Molecular analysis has shown substantial ITS sequence
diversity among isolates in this genus (99, 124). Compare with Cladosporium,
Rhinocladiella (4,15)
O. Hormonema Lagerberg & Mellin 1927

Hormonema species often have been identified incorrectly as Aureobasidium spe-
cies even though the mechanism of sporogenesis differs between the two genera.
In Hormonema species, conidia arise by percurrent succession from micronema-
tous loci on the surface of hyaline to dematiaceous hyphae (Fig. 40). In contrast,
Aureobasidium species produce conidia synchronously. Brown arthroconidia de-
velop in most isolates of both Hormonema dematioides and Aureobasidium pullu-
lans. Cutaneous phaeohyphomycosis and peritonitis have been reported from
Hormonema dematioides (125,126). Colonies are yeastlike, white or slightly
pinkish in color, becoming dark brown centrally, at the margin, in sectors, or
Figure 37 Exserohilum rostratum.
entirely. True hyphae are present with age, and are most easily found submerged
within the agar. Hyphal formation and observation of sporulation can be facili-
tated by inoculating an agar plate and then placing a cover glass over the inocu-
lum. The teleomorph of Hormonema dematioides is Sydowia polyspora. Compare
with Aureobasidium, Scytalidium (40).
P. Madurella Brumpt 1905

Madurella mycetomatis is the world’s leading cause of mycotic mycetoma with
more than 1,000 cases having been reported, while Madurella grisea has been
600 Schell
Figure 38 Fonsecaea compacta.
reported fewer than 100 times (127). Madurella grisea colonies are slow-grow-
ing, velvety, folded and heaped, dark brown with grayish tints, reverse dark
brown without diffusing pigment. Isolates of M. grisea are sterile, though some
reportedly have formed pycnidia. Conidia are not present. Growth is better at
30°C than at 37°C. Sucrose is assimilated; lactose assimilation is variable. Colo-
nies of M. mycetomatis are slow-growing, cream-colored, glabrous, folded,
tough; with age becoming velvety, dark brown, staining the agar with a diffusing
brown pigment. Growth is enhanced at 37°C; lactose is assimilated but sucrose
Figure 39 Fonsecaea pedrosoi.
is not. Colonies are sterile on routine media; sporulation is present in about 50%
of M. mycetomatis isolates grown on nutrient-poor media. Conidia are subglobose
to pyriform, 3 to 5 µm, with truncate basal scar, occurring in balls, rarely in
fragile chains. Phialides are variable, usually 9 to 11 µm long, tapering, often
with collarette; but range from long (15 µm) and tubular to short (3 µm) and
integrated. Occasionally two or three phialides may be borne upon a single
Figure 40 Hormonema dematioides.

602 Schell
branch. Large vesicles, terminal and intercalary, often are present. Sclerotia may
be present. No teleomorph is known for either species (12).
Q. Mycocentrospora acerina (Hartig) Deighton 1972

Colonies initially are hyaline, becoming green or grayish, finally olivaceous
black. Hyphae in culture mostly are colorless except for dark brown swollen
cells. Conidiophores are 25 to 50 µm long, sympodial and geniculate (Fig. 41).
Conidia have four to 24 (mostly 8–11) septa, 60-250-150 µm long, 5 to 7 µm
wide. Middle cells of conidia often are dark, swollen, thick-walled. Apical cells
of conidia taper to form a long, filiform appendage. Basal cell of some conidia
forms a filiform appendage up to 150 µm in length. One case of disseminated,
fatal phaeohyphomycosis has been reported (128). The isolate from this case
Figure 41 Mycocentrospora acerina. Source: Ref. 17.

grew better at 30°C than at 25°C, but did not grow at 37°C. No teleomorph is
known. Compare with Cercospora, Vermispora (17).
R. Mycoleptodiscus Ostazeski 1968

1. Mycoleptodiscus indicus (Sahni) Sutton 1973
Colonies initially are spreading with long, thin aerial hyphae, yellow-brown to
tan in color, becoming grayish-black. Conidial formation is delayed. Conidia are
approximately 15 ⫻ 7 µm, hyaline, one-celled, reniform, and sharply tapered to
both ends, with a hairlike appendage at either end (Fig. 42). Conidia arise from
brown ampulliform phialides with collarettes. Phialides may or may not be orga-
nized as part of sporodochia. Dark brown ellipsoidal appressoria may be present.
A teleomorph (Omnidemptus) has been reported in the genus. At least two cases
Figure 42 Mycoleptodiscus indicus.

604 Schell
of phaeohyphomycosis are known, one of which is published (129). Compare

with Ciliphora (30,130).
S. Phaeoacremonium W. Gams, Crous & M.J. Wingf. 1996

The genus Phaeoacremonium is morphologically intermediate between Acremo-
nium and Phialophora. Its morphology is quite similar to that of Acremonium
and differs by hyphae that become dematiaceous in part. Phialides range from
micronematous to macronematous and their morphology is quite variable, but
the most distinctive shapes are elongate and taper steadily from base to tip in an
awllike manner. The collarette usually is fairly well developed but is thin-walled
and somewhat inconspicuous; collarette walls are parallel to slightly divergent.
Teleomorphs are not known. Six species have been proposed for the genus, three
of which are known to cause opportunistic infections in humans. Prior to the
1996 revision all three were considered to be isolates of Phialophora parasitica.
Phaeoacremonium parasiticum is a well-documented agent of phaeohypho-
mycosis (131–133). The identification of some of these reported isolates might
be changed under current taxonomy. Phaeoacremonium inflatipes and P. rubri-
genum recently have been reported as agents of phaeohyphomycosis (134,135).
Compare with Acremonium, Lecythophora, Phialemonium, Phialophora.
1. Phaeoacremonium parasiticum (Ajello et al.) W. Gams et al.

1996
Colonies are buff-colored, almost glabrous when young, becoming velvety during
maturity. Colonies become brown either in sectors or entirely except for the mar-
gin. Phialides are variable in length, some isolates forming extremely long phi-
alides swollen near their bases, with prominent wartlike cell wall encrustations
on the lower section of the conidiophore and the adjoinining hyphal sections (Fig.
43). Conidia are elliptical to cylindrical, often curved, range in size from
2–6 ⫻ 1–2 µm, and accumulate in a mass at the phialide tip (136). An inconspicu-
ous yeastlike synanamorph arises from conidia that do not immediately germinate
but instead enlarge and begin to sporulate in a phialidic manner. These yeastlike
forms are one-to-two-celled and bear a collarette at the sporogenous orifice.
2. Phaeoacremonium inflatipes W. Gams, Crous & M.J. Wingf.

1996
This species is quite similar to P. parasiticum. It differs by phialides that have
a more pronounced bulge at their base, and larger conidia that are 3.0–5.0(⫺7.0)
⫻ (1.0) 1.5–2.0(⫺2.5) µm (136).
Figure 43 Phaeoacremonium parasiticum.
3. Phaeoacremonium rubrigenum W. Gams et al. 1996

This species is highly similar to P. inflatipes but differs by its vinaceous red
reverse on malt extract agar. Conidia are the same size range as with P. inflatipes,
although rarely may be very slightly larger (136).
T. Phaeoannellomyces McGinnis & Schell 1985

The genus Phaeoannellomyces was established for dematiaceous yeast morphs
that are characterized by annellidic sporulation (100). A teleomorph is not known.
The two clinically important species are P. werneckii and P. elegans. Both are
agents of phaeohyphomycosis (121,122,137,138). The otherwise identical genus
Phaeococcomyces differs by forming conidia in a holoblastic manner. Compare
with Phaeococcomyces, Exophiala, Wangiella.
1. Phaeoannellomyces elegans McGinnis & Schell 1985

This species usually is found as a yeast synanamorph accompanying isolates of
various species of Exophiala. In such circumstances, colonies at first are mucoid,
slow-growing, smooth, yeastlike, pale brown to black. As the mold synanamorph
606 Schell
Figure 44 Phaeoannellomyces elegans.
develops and becomes predominant, the colonies become filamentous. Some iso-
lates of P. elegans, however, have little or no development of a hyphal synana-
morph (139). Phaeoannellomyces elegans exhibits one-celled annellated yeast
cells that are subhyaline to pale brown (Fig. 44). Pseudohyphae may be formed.
Compare with Exophiala, Phaeococcomyces, Wangiella (100,106).
2. Phaeoannellomyces werneckii (Horta) McGinnis & Schell

1985 (syn. Cladosporium werneckii, Exophiala werneckii,
Hortaea werneckii pro parte)
This species is the cause of a superficial phaeohyphomycosis known as black
palm (6). The species exhibits a distinctive one-to-two-celled, very broadly annel-
lated yeast morph that is the predominant and most stable synanamorph seen in
this pleomorphic species and that provides the basis of the name (Fig. 45). The
binomial Hortaea werneckii has been proposed for the mold synanamorph that
sometimes is present (Fig. 46). This hyphal synanamorph, however, often is
poorly developed and thus is not of primary importance to a morphologically
based identification of isolates.
U. Phaeotrichonis Subramanian 1956

1. Phaeotrichonis crotalariae (Salam & Rao) Subramanian 1956
This species was reported from a case of keratitis (140, 141). Colonies are lanose,
dark gray to grayish-brown, sometimes with sclerotia forming. Conidiophores
Figure 45 Phaeoannellomyces werneckii.
are macronematous, multicellular, thick-walled, brown, sympodial, at times ge-

niculate (Fig. 47). Conidia are borne singly, three to eight (mostly 5–6) septate,
obclavate, with prominent filiform beak 30 µm or more in length (17). A teleom-
orph is not known. Compare with Bipolaris, Drechslera, Exserohilum.
V. Phialophora Medlar 1915

Phialophora verrucosa long has been known as a leading cause of chromoblasto-
mycosis, and has caused a few cases of keratitis and phaeohyphomycosis (1,
142,143). Phialophora americana has been regarded by many mycologists as a
synonym of P. verrucosa, but now molecular sequence data seem to correlate
well with the proposed morphologic distinction (144). Phialophora americana
has caused chromoblastomycosis. Phialophora bubakii, P. repens, and P. rich-
ardsiae are agents of phaeohyphomycosis (5,145–147). Other reported infections
include endocarditis, keratitis, osteomyelitis, atypical eumycetoma, and opportu-
608 Schell
Figure 46 Phaeoannellomyces werneckii, showing the filamentous synanamorph (ge-

nus Hortaea).
nistic infections in AIDS patients (148–151). Two additional agents of phaeohy-

phomycosis previously classified in Phialophora have been transferred to the
genus Lecythophora as L. hoffmannii and L. mutabilis (152). (See Chap. 11.)
Phialophora parasitica has been reclassified as Phaeoacremonium parasiticum
(136). Colonies are moderate in growth rate, cottony to velvety, olive-gray to
black. Conidiophores (if present) usually are short and pale brown. Conidiogen-
ous cells are phialides with distinct collarettes. Conidia are one-celled, hyaline
to pale brown, accumulating in balls at the phialide apices. Capronia semiim-
mersa has been shown as the teleomorph of Phialophora americana (144); other-
wise, teleomorphs are not proved. Compare Phialophora with Acremonium, Le-
cythophora, Phaeoacremonium, Phialemonium (4,5,30,73,131,136,145,152).
1. Phialophora americana (Nannfeldt) S. Hughes 1958

This species produces prominent flask-shaped phialides with dark collarettes that
are deeper than wide, resulting in a vaselike shape (Fig. 48). Conidia are elliptical.
Phialophora americana is extremely similar in morphology to P. verrucosa, dif-
fering only in the shape of the collarette. Colonies are olivaceous black.
2. Phialophora repens (Davidson) Conant 1937

This species shows a wide variation in the size and shape of its phialides, similar
to that seen with Phaeoacremonium parasiticum. Phialides can be adelophialides,
or can be up to 20 µm long and subtended by a supporting cell. Phialides mostly
are cylindrical to slightly lageniform with a delicate collarette (Fig. 49). Very
Figure 47 Phaeotrichonis crotalariae. Source: Ref. 26a.
rarely they can occur in branched clusters (Fig. 50). Conidia are cylindrical and
often curved.
3. Phialophora richardsiae (Nannfeldt) Conant 1937

This species produces phialides of variable size and shape. Some phialides are
long with flattened, saucer-shaped collarettes, and these give rise to conidia that
are almost spherical and 2.5 to 3.5 µm in diameter (Fig. 51). The remaining
610 Schell
Figure 48 Phialophora americana.
Figure 49 Phialophora repens.

Figure 50 Phialophora repens clustered phialides on branched conidiophores.
Figure 51 Phialophora richardsiae.

612 Schell
Figure 52 Phialophora verrucosa.
phialides are adelophialides or lageniform phialides of short length (12 µm or

less), bearing an inconspicuous collarette, that form ellipsoidal to cylindrical,
often curved conidia. Melanin concentration in those phialides having prominent
collarettes, the hyphae associated with those phialides, and the globose conidia
is high, giving these cells a mid- to dark reddish-brown color as compared with
the remaining cells.
4. Phialophora verrucosa Medlar 1915

This species produces prominent flask-shaped phialides with dark collarettes that
are not deeper than wide, resulting in a cuplike or funnellike shape (Fig. 52).
Conidia are elliptical. Phialophora americana is extremely similar in morphology
to P. verrucosa, differing only in the shape of the collarette, which is deeper than
wide, resulting in a vaselike shape.
W. Pseudomicrodochium B. Sutton 1975

1. Pseudomicrodochium suttonii Ajello et al. 1980
This species has caused phaeohyphomycosis in a dog (153), and was isolated
from subcutaneous lesions in a human (154). A teleomorph is not known. Colo-
Figure 53 Pseudomicrodochium suttonii.
nies are velvety, olivaceous black with olivaceous gray aerial hyphae. Conidio-
genous cells are monophialidic, typically micronematous and integrated, often
with collarettes, not separated from the hyphae by a septum (Fig. 53). Erect lateral
or terminal phialides are rarely present. Conidia are holoblastic, schizolytic, sub-
hyaline to pale brown, zero to three septate, acerose to falcate when mature, 14–
30 ⫻ 1–1.8 µm. Anastomosis between adjacent conidia occurs readily, and co-
nidia sometimes function as phialides by directly producing phialoconidia from
a lateral focus.
X. Ramichloridium Staehl ex de Hoog 1977

A teleomorph is not known. Colonies are velvety, pale brown, brown, or oliva-
ceous brown, sometimes with a yellow to orange hue on the reverse. Ramichlori-
dium has no distinct morphologic difference compared with Rhinocladiella. If
these genera were to be regarded as congeneric, Rhinocladiella would retain
priority. The most medically significant species, Ramichloridium obovoideum,
was described originally as a species of Rhinocladiella (i.e., Rhinocladiella obo-
voidea Matsushima 1975). Compare with Exophiala, Fonsecaea, Rhinocladiella,
Veronae (15,17,30,155).
614 Schell
Figure 54 Ramichloridium obovoideum. Source: Ref. 156.
1. Ramichloridium obovoideum (Matsushima) de Hoog 1977

Several brain infections have been caused by R. obovoideum (156). Most of these
were attributed to a newly described species, R. mackenziei. It is not clear, how-
ever, that this species is different from the previously described Ramichloridium
obovoideum. Further study is needed, and for the purposes here the two will be
treated as synonyms. Conidiogenous cells are macronematous, sympodial, almost
geniculate at times, and somewhat thick-walled in the lower portion (Fig. 54).
Conidia are borne on prominent denticles, are obovoid in shape, 3–5 ⫻ 5–12
µm, pale brown, with basal scar sometimes protuberant. Growth is very slow at
25°C and much faster at 30–35°C (156,157).
Y. Rhinocladiella Nannfeldt 1934

Colonies are rapidly growing, velvety, blackish-brown to olive-black on obverse
and reverse. Conidia from sympodial cells are one-celled, ellipsoidal to obovoid
to fusiform. A similar genus, Veroneae, differs by conidia that often are two-
celled, and one report of skin infection has been noted for V. botryosa (5). Com-
pare with Exophiala, Fonsecaea, Rhamichloridium, Veronae (15,17,30).
1. Rhinocladiella aquaspersa (Borelli) Schell et al. 1983

The teleomorph is not known. Rhinocladiella aquaspersa has been reported only
rarely as causing chromoblastomycosis, these cases being from Brazil, Mexico,
Colombia (158), and Venezuela (155,159,160). Colonies are velvety, elevated,
olivaceous black, hyphae are mid- to pale brown. Conidiophores usually are
darker than vegetative hyphae, well developed, 21–82 ⫻ 1.3–3.5 µm, zero to
four septate, macronematous, erect, unbranched, often tapering slightly (Fig. 55).
Sporulation is sympodial; fertile region is long and scarred with partially to prom-
Figure 55 Rhinocladiella aquaspersa.

616 Schell
inently raised denticles. Conidia are fusiform, ellipsoidal, or obovate, one-celled,

smooth, mid-brown, 4–9 ⫻ 1.3–2.6 µm. An Exophiala synanamorph and a
Wangiella synanamorph can be present.
2. Rhinocladiella atrovirens Nannfeldt 1934

Rhinocladiella atrovirens was suspected of causing cerebral infection in an AIDS
patient (161) and mycetoma of the foot (162). Isolates sometimes are confused
with Fonsecaea pedrosoi. There often is an annellated yeast (Phaeoannellomyces
elegans) synanamorph inconspicuously present, however, and this is not the case
with F. pedrosoi. Also, R. atrovirens differs by its sympodial conidiophore that
tends to resume growing after sporulating and then sporulates again at the new
apex (Fig. 56). A Phialophora synanamorph can occur rarely (Fig. 57) and an
annellidic anamorph (as seen in Exophiala spp.) may be present. Sympodial co-
nidiophores are pale to mid-brown, erect, and usually bear distinct crowded scars.
Conidia are one-celled, ellipsoidal to obovate to fusiform, pale brown, with a
flat basal scar. Conidia occur along the elongating conidiophore. Occasionally a
conidium will give rise to a second conidium, allowing potential confusion with
Fonsecaea pedrosoi. A teleomorph is not known.
Z. Sarcinomyces Lindner 1898

1. Sarcinomyces phaeomuriformis Matsumoto et al. 1986
A teleomorph is unknown. Colonies are black, restricted, raised, dry, friable,
mulberrylike in texture. Hyphae are absent. Cells are unicellular to muriform,
Figure 56 Rhinocladiella atrovirens.

Figure 57 Phialophora synanamorph of Rhinocladiella atrovirens, as seen in paratypus

specimen No. 59.36 (160:7).
subglobose, hyaline when young, brown with age, forming multilateral holo-
blastic buds, often adhering in large masses (Fig. 58). Skin infections have
been documented (163,164). Compare with Botryomyces, Phaeococcomyces,
Phaeoannellomyces, Wangiella.
A′. Scolecobasidium Abbot 1927

A teleomorph is not known. Distinction between Scolecobasidium and Ochro-
conis de Hoog & von Arx 1973 has been uncertain, and the two genera will be
considered here as synonyms (87,165). This disposition also is supported by re-
cent molecular sequencing data (88). The species gallopava can cause dissemi-
nated (often cerebral) phaeohyphomycosis in immunocompromised persons (53,
82–84), and localized infection has been reported (166). Colonies are flat, oliva-
ceous-gray to brown, usually with brown to reddish-brown diffusing pigment
evident. Conidiophores are hyaline, erect, sympodial, occasionally geniculate
(Fig. 59). Conidia are two-celled, dark, cylindrical to oblong, with basal frill
resulting from rhexolytic dehiscence (breakage of the supporting cell wall). One-
celled, globose phialoconidia may be present in young colonies. The species
grows at 45°C, but is inhibited on media containing 5% NaCl when incubated
at 30°C (88). Scolecobasidium constrictum does not grow at or above 37°C, but
does grow on media containing 5% NaCl when incubated at 30°C. No naturally
occurring infections are known. A teleomorph is not known. Compare with Dac-
tylaria (4,85,167).
618 Schell
Figure 58 Sarcinomyces phaeomuriformis. Source: Ref. 163.
B′. Sporothrix Hektoen & Perkins 1900

Colony color varies from white to shades of brown, and texture varies from gla-
brous to granular or floccose. Conidiogenous cells are sympodial, and the fertile
zone in some of the species is restricted to the apex of the cell. Conidia are one-
celled, hyaline, elliptical, ovoid, clavate, or curved cylindrical. No teleomorph
has been proved.
Figure 59 Species gallopava.

Sporothrix has accommodated a heterogeneous group of morphologically

similar species. One of these, known in the medical literature as S. cyanescens,
was removed from the genus because its septal ultrastructure suggests affinity to
basidiomycetes (168). This species, distinctive for its whitish colony and a pur-
ple-blue diffusing pigment, has been recovered in culture of clinical specimens,
but virulence studies in animals have shown no pathogenicity (169–171). More
than one dozen species of Sporothrix are described, but only S. schenckii (in
two varieties, S. schenckii var. schenckii and S. schenckii var. luriei) is a known
pathogen. Subcutaneous–cutaneous infection is most common, but any part of
the body (including lung) can be infected (172–174).
1. Sporothrix schenckii Hektoen & Perkins 1900

Colonies of most isolates initially are cream-colored, smooth, moist, yeastlike;
gradually turning brown in irregular patches; becoming velvety to lanose as aerial
hyphae develop. Conidiophores are elongate, compactly sympodial, with swollen
apex; bearing one-celled, hyaline, elliptical to obovate conidia in a radial ‘‘ro-
sette’’ appearance (Fig. 60). Conidia of a second kind arise directly from the
vegetative hyphae, are one-celled, dematiaceous, thick-walled, and oval (rarely
triangular) (Fig. 61). These second conidia are chiefly responsible for the dark
color of the colony. Sporothrix schenckii can be converted to a budding yeast
form at 35–37°C by using enriched media such as brain–heart infusion agar,
chocolate agar, or brain–heart infusion both containing 0.1% agar (Fig. 62). Mor-
phologic identification of an isolate as S. schenckii requires this demonstration
of dimorphism. Some isolates are difficult to convert and might require multiple
subcultures and extended incubation time. Conversion of all cells is not neces-
sary, and a mixture of hyphal and budding cells is commonly observed. Several
Sporothrix sp. isolated from environmental samples during investigation of a spo-
Figure 60 Sporothrix schenckii sympodial conidiophores.

620 Schell
Figure 61 Sporothrix schenckii thick-walled dematiaceous conidia attached directly to

vegetative hyphae.
Figure 62 Sporothrix schenckii transformed to budding yeast.

rotrichosis outbreak appeared to have the morphology of Sporothrix schenckii,

except for their lack of thick-walled, dematiaceous conidia borne on vegetative
hyphae. These isolates did convert to the yeast form at 35–37°C, but were aviru-
lent in mice. It was thus suggested that the presence of these conidia might consti-
tute a significant identification criterion for S. schenckii (175).
2. Sporothrix schenckii var. luriei

Three isolates of Sporothrix schenckii var. luriei have been reported (176–178).
Its appearance differs from S. schenckii var. schenckii only by its morphology
in host tissue. In culture, whether as mold or yeast, the two varieties are indistin-
guishable. Assessment of mitochondrial DNA seems to confirm that S. schenckii
var. luriei is markedly different from S. schenckii (179). The fungus exhibits
large (10–30 µm), subglobose to elongated, thick-walled (up to 2 µm) cells. Each
of these forms a single septum that precedes its separation into two cells. During
separation, a fragment of the cell wall previously shared by the two cells briefly
remains attached in a bridgelike manner, giving a distinctive appearance of eye-
glasses. Sometimes a second septum forms, giving the fungus a muriform appear-
ance. It is not known whether there might be serologic, therapeutic, or prognostic
differences associated with this variety. Sporothrix schenckii and P. parasiticum
can be confused because of highly similar colony coloration and the presence of
a (phialidic) yeast synanamorph in P. parasiticum. Compare with Rhinocladiella
and Phaeoacremonium parasiticum.
C′. Taeniolella Hughes 1958

Both T. stilbaspora and T. exilis reportedly have caused cutaneous and subcutane-
ous lesions (5). A teleomorph is not known. Colonies are filamentous, immersed
or superficial, brown to olivaceous black. Conidiophores are little differentiated
from hyphae (Fig. 63). Conidia sometimes are solitary, but more often are borne
in acropetal moniliform, unbranched, or sparsely branched chains. Conidia are
brown, thick-walled, cylindrical, truncate, not readily released from chains (17).
Taeniolella stilbaspora Hughes 1958 is characterizd by conidia that are 7 to 11
µm wide and have 3 to 24 septa. Taeniolella exilis Hughes 1958 is distinguished
by conidia that are 12 to 15 µm thick and composed of two to four cells. Compare
with Cladophialophora, Xylohypha, Septonema.
D′. Tetraploa Berkeley & Broome 1850

1. Tetraploa aristata Berkeley & Broome 1850
The teleomorph is not known. Colonies are velvety, dark grayish-brown. Conid-
iophores are micronematous, inconspicuous with conidia borne singly (Fig. 64).
622 Schell
Figure 63 Taeniolella exilis.
Figure 64 Tetraploa aristata.

Figure 65 Thermomyces lanuginosus. Source: Ref. 26a.
Conidia usually are brown, roughened, six- to 16-celled, consisting of four bun-
dled rows of two or four cells per bundle, with a slight furrow between rows.
Conidia measure 14–29 ⫻ 25–39 µm and are further distinguished by a long
(12–80 µm), filiform, multicellular appendage at the apex of each bundle (17).
Keratitis and subcutaneous infection have been reported (180,181). Compare to
Hughesinia.
E′. Thermomyces Tsiklinsky 1899

1. Thermomyces lanuginosus Tsiklinsky 1899
A teleomorph not known. Colonies are velvety to lanose, grayish-white, becom-
ing greenish, then brown to black, often with a pink or vinaceous diffusing pig-
ment. Conidia are borne singly on short stalks and are one-celled, dark brown,
subglobose, with a roughened surface, 6–12 µm in diameter (17) (Fig. 65). Endo-
carditis has been reported (182). Compare to Harzia, Acremoniula.
F′. Ulocladium Preuss 1851

1. Ulocladium chartarum (Preuss) Simmons 1967
A teleomorph is not known. Colonies are velvety to floccose, rapidly growing,
olivaceous brown to black. Conidia are obovoid to short ellipsoidal, golden brown
to blackish-brown, roughened, with one to five oblique or longitudinal septa and
one to five lateral septa, borne singly or in short chains from sympodial conidio-
624 Schell
Figure 66 Ulocladium chartarum.
phores (21) (Fig. 66). Subcutaneous infection has been attributed (5). Compare
to Alternaria, Stemphilium, Embeliasia.
G′. Wangiella McGinnis 1977

1. Wangiella dermatitidis (Kano) McGinnis 1977
This species caused phaeohyphomycoses of cutaneous and subcutaneous tissue,
and infections of the eye, brain, joint, and catheter-related fungemia also have
been documented (97,164,183–185). Most isolates initially grow in a yeast form
(Phaeococcomyces synanamorph) with development of toruloid hyphae that is
succeeded by the hyphal W. dermatitidis synanamorph. Colonies are slow-grow-
ing, initially yeastlike, smooth, viscous, pale brown to black, becoming filamen-
tous to velvety. Conidia are one-celled, subglobose to elliptical or obovoid, sub-
hyaline to pale brown, not catenate. Growth occurs at 40°C; potassium nitrate is
not assimilated. A teleomorph has not been proved. Conidiogenous cells are phi-
alides that lack collarettes (Fig. 67) (97,186). Some conidiogenous cells exhibit
a group of slightly raised, truncate protrusions at their apices that occur as a
result of a polyphialidic development. In addition, annellides of the kind seen in
Exophiala may be formed rarely, and this has led some mycologists to propose
that the fungus be reclassified as Exophiala dermatitidis. Results from molecular
Figure 67 Wangiella dermatitidis.
sequencing analyses have not yet resolved this question (52,144). A Phialophora
synanamorph may be present (Fig. 68). Another fungus known lately as Exophi-
ala jeanselmei var. heteromorpha has been reevaluated by morphologic criteria,
and a new combination for it, W. heteromorpha, has been proposed (98). (See
Exophiala.) Compare with Phaeoannellomyces, Phaeococcomyces, Phialophora
(3,104,187).
Figure 68 Phialophora synanamorph (upper right) of Wangiella dermatitidis.

626 Schell
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11
Miscellaneous Opportunistic Fungi
Microascaceae and Other Ascomycetes,
Hyphomycetes, Coelomycetes, and
Basidiomycetes
Lynne Sigler
University of Alberta, Edmonton, Alberta, Canada
I. INTRODUCTION
The fungi treated in this chapter have different phylogenetic affinities. Pathogenic
members of two ascomycete families—Microascaceae and Chaetomiaceae—are
treated first. The family Microascaceae includes Scedosporium anamorphs and
teleomorphs in Pseudallescheria and Petriella in addition to Scopulariopsis ana-
morphs and teleomorphs in Microascus. The remaining fungi represent diverse
ascomycetes and basidiomycetes that are recognized in culture usually by their
conspicuous conidial stages or by vegetative mycelial features. The conidia are
produced from conidiogenous cells borne diffusely on the mycelium, on mono-
nematous or synnematous conidiophores, or in sporodochial or pycnidial conidi-
omata. These species traditionally have been classified in the Hyphomycetes and
the Coelomycetes. Although connections to teleomorphs have been established
for many of the species, they are described here under their better-known anamor-
phic names. The hyphomycetous fungi included in this chapter are predominantly
moniliaceous; that is, they produce lightly colored conidia. Fungi producing
darkly pigmented conidia are treated in Chap. 10.
Isolates that are initially sterile in culture may be ones that form fruiting
bodies representative of the ascomycetes, the basidiomycetes, or the coelomy-
637
638 Sigler
cetes. Sexually producing fungi may be heterothallic, and the teleomorph will not
be obtained unless compatible mating strains are crossed. Isolates of homothallic
species will form fruiting bodies (ascomata or basidiomata) when grown on
a suitable medium or under appropriate conditions of light or temperature. Me-
dia that stimulate sporulation include oatmeal-salts agar, cornmeal agar, and
Takashio agar or low-glucose media amended with natural substrates (e.g., carna-
tion leaves, wood shavings, hair, or other keratins) (1). It is often necessary to
try more than one medium and to hold cultures for extended time periods (4–8
weeks or longer) to obtain mature structures. Inspection of plates with a dissecting
microscope (with illumination from above and below) is of great value in locating
pycnidia or ascomata that may be embedded in the agar or hidden under mats
of mycelium and/or exudate droplets, and for observing the presence of ostioles,
appendages, or setae and the manner of spore release (e.g., ascospores in cirrhi,
conidia in slime).
II. FAMILY MICROASCACEAE (ORDER MICROASCALES)

A. Taxonomic Concepts
The Microascaceae were formerly classified with the Ophiostomataceae in the
Microascales, but recent molecular phylogenetic analyses have placed these fami-
lies in separate orders (reviewed in Ref. 2). These are the Microascales and Ophi-
ostomatales (3–6). This separation is supported by physiological characters, as
the Ophiostomataceae demonstrate a tolerance to cycloheximide (7), while the
Microascaceae are tolerant of benomyl (8, 9). Members of the Microascaceae
produce ostiolate or nonostiolate membranaceous ascocarps (perithecia and cleis-
tothecia) that are small (usually less than 300 µm in diameter), brown to black,
smooth or hairy, and contain asci irregularly arranged within the centrum. Asci
are eight-spored, ovoid, and prototunicate; that is, they have thin walls that lyse,
allowing for passive discharge of the ascospores. Ascospores are one-celled,
smooth, yellowish to reddish-brown (straw to copper-colored), dextrinoid when
immature, and commonly have one or two germ pores. Teleomorphic genera
included in the Microascaceae are Microascus, Pseudallescheria, Petriella, Pi-
thoascus, Kernia, and Lophotrichus (5). Anamorphs are present in many species
and are placed in the genera Scopulariopsis, Scedosporium, Graphium (in part),
Cephalotrichum (⫽Doratomyces), Trichurus, Wardomyces, Wardomycopsis, and
Echinobotryum. Conidia are produced from annellidic conidiogenous cells borne
on mononematous or synnematous conidiophores or are solitary. The main patho-
genic genera are Microascus and Pseudallescheria, and these are distinguished
by ascomatal type and ascospore and conidial features. Species of Microascus
produce perithecia, yellowish to reddish-brown asymmetrical, reniform, heart-
shaped or triangular ascospores bearing a single pore (10–12). As perithecia age,
Miscellaneous Opportunistic Fungi 639
ascospores are commonly extruded in a long column of spores (a cirrhus). Co-

nidia are produced in dry chains and the anamorphs are placed in the genus
Scopulariopsis. Members of the genus Pseudallescheria produce cleistothecia,
yellowish-brown ellipsoid ascospores having two germ pores (12, 13). Conidia
are slimy and anamorphs are placed in Scedosporium and Graphium.
B. Species of Medical Relevance

1. Pseudallescheria boydii (Shear) McGinnis, Padhye & Ajello,
Mycotaxon 14:97, 1982 (Figs. 1a–c, 2, 3)
Synonyms: Petriellidium boydii (Shear) Malloch, Mycologia 62:739, 1970;
Allescheria boydii Shear, Mycologia 14:242, 1922
Synanamorphs: Scedosporium apiospermum (Saccardo) Castellani & Chal-
mers, Manual of Tropical Medicine Ed. 3, p 1122, 1919; Graphium eu-
morphum Saccardo, Michelia 2:560, 1881
Occurrence as a Pathogen. Clinical problems mimic those presented by
Aspergillus fumigatus (14–18). Patients have similar risk factors and symptomol-
ogy. It can be difficult to distinguish between colonization and invasive infection,
and infections are often refractory to antifungal therapy. As well, hyphae in tissue
resemble those of Aspergillus. Disease occurs in the healthy or immunocompro-
mised host, and the spectrum of clinical syndromes includes otomycosis, sapro-
phytic pulmonary colonization, allergic sinusitis and bronchopulmonary mycosis,
fungus ball, keratitis, endocarditis, osteomyelitis, keratitis, endophthalmitis, inva-
sive sinusitis, and pulmonary or disseminated disease. Pseudallescheria boydii
may affect the brain, causing cerebral abscesses or meningitis. It also causes
white grain mycetoma. Persistent respiratory tract colonization is a problem for
patients with cystic fibrosis and chronic suppurative lung disease who may be
excluded for consideration of a lung transplant due to therapeutic difficulties (19).
Description. Colonies grow moderately rapidly, and are pale to dark
brownish-gray (‘‘mouse-gray’’), with light to olivaceous brown reverse. Yellow
to brown diffusible pigment is often produced. Isolates grow well at 37°C, and
many isolates grow at 40°C. Growth is equivalent or somewhat inhibited on me-
dium containing cycloheximide. In culture, this homothallic ascomycete may pro-
duce three states. The diffuse Scedosporium apiospermum anamorph is produced
by all isolates, but the Graphium synanamorph and the Pseudallescheria teleom-
orph are produced only by some. Ascomata are nonostiolate (cleistothecia), dis-
crete, solitary, globose, brownish-black, wall of textura epidermoidea, 55 to 180
µm in diameter, and often immersed in the agar. Asci are globose to ovoid, eva-
nescent, and eight-spored. Ascospores are ellipsoidal, symmetrical, yellowish-
brown, smooth with two germ pores, and measure 6 to 8.5 µm long by 3.5 to
640 Sigler
5.5 µm wide. Ascospores characteristically have a de Bary bubble. Conidia of

Scedosporium apiospermum are oval or ellipsoidal, 5 to 11 µm long by 3 to 6.5
µm wide, dilute yellow-brown, and are solitary on the side of the hypha or formed
in slimy masses from short, usually unswollen conidiogenous cells (annellides).
Graphium conidia are cylindrical or clavate, 3 to 11.5 (22.5) µm long by 1.5 to
3.5 (6.5) µm wide, dilute yellow-brown, and formed in slimy heads from annel-
lides borne at the tip of a synnema.
Comments. This ubiquitous, soil-borne thermotolerant fungus and its rela-
tive, Scedosporium prolificans, are the most important pathogenic species of the
Microascaceae. Scedosporium prolificans is distinguished by annellidic conidio-
genous cells that are basally swollen, and by the absence of a teleomorph and a
Graphium stage. Development of ascomata and synnemata in P. boydii isolates
is enhanced on oatmeal-salts agar or other medium. Examination with a dissecting
microscope and basal illumination may reveal ascomata that are often immersed
in the agar.
Comparison of ribosomal internal transcribed spacer (ITS) region se-
quences revealed considerable genetic variability within Pseudallescheria boydii
and confirmed its distinction from S. prolificans (20, 21). Pseudallescheria an-
gusta, P. ellipsoidea, and P. fusoidea, known thus far only from soil (12), are
considered synonyms based on their grouping within a clade of clinical and envi-
ronmental isolates of P. boydii (21), as had been suggested by morphological
similarity (13). Molecular typing (19, 21) has been found useful to characterize
individual strains but has not yet elucidated the source of infection and means of
transmission of this species (19). Patients with chronic suppurative lung disease
demonstrated persistent carriage of the same molecular type over long periods
(19), but isolates of different types could be obtained from individual patients
(19) and from identical environmental sites (21).
2. Scedosporium prolificans (Hennebert & Desai) Guého &

de Hoog, J Mycol Med 118:9, 1991 (Fig. 1a)
Synonyms: Lomentospora prolificans Hennebert & Desai, Mycotaxon 1:
47, 1974; Scedosporium inflatum Malloch & Salkin, Mycotaxon 21:249,
1984
Occurrence as a Pathogen. Although the spectrum of disease is similar
to that of S. apiospermum, S. inflatum is more commonly associated with osteo-
myelitis and arthritis in humans and animals (16, 22, 23). Idigoras et al. (24)
reviewed 18 Spanish cases that occurred over a 10-year period and grouped clini-
cal conditions into three types: (1) pulmonary colonization, particularly in pa-
tients with cystic fibrosis, AIDS, or transplant recipients; (2) localized infection
in immunocompetent and immunocompromised patients involving the bone, skin,
Figure 1 Pseudallescheria boydii and Scedosporium prolificans. (A) Scedosporium

apiospermum conidiogenous cells (annellides). (B) Scedosporium apiospermum annel-
lides. (C) Graphium synnema. (D) Scedosporium prolificans annellides. Note: A, C—bar
⫽ 20 µm. B, D—bar ⫽ 5 µm.
eye, lung, heart valve, and peritoneum; and (3) disseminated infection in immuno-
compromised patients. They reported the frequent isolation from blood cultures.
The species is notorious for its antifungal drug resistance in vitro, and this is
correlated with clinical therapeutic failure, especially if there is no reversal of
the underlying deficiency.
Description. Colonies grow slightly slower than S. apiospermum and are

denser and darker gray-brown to dark brown. Isolates grow well at 37°C. Growth
is inhibited on medium containing cycloheximide. Conidiogenous cells (annel-
lides) are borne singly on the hypha or in a cluster of two to six at the apex of
642 Sigler
Figure 2 Pseudallescheria boydii. (A) Ascomata (cleistothecia). (B) Immature asci. (C)
Wall of ascoma. (D) Ascospores. Source: Reprinted from Ref. 119, p. 96, by courtesy of
Marcel Dekker, Inc.
a short conidiophore, and are distinctly basally swollen and tapered at the tip.
The tip elongates as successive conidia are formed, and conidia often remain
attached laterally. Conidia are formed in slimy masses and are subhyaline to light
brown, ovoid, and 5.5 to 8 µm long by 3.5 to 5.5 µm wide.
Comments. S. prolificans is distinguished from S. apiospermum by annel-
lides with inflated bases commonly borne in a cluster of two to four at the ends
of short conidiophores, an absence of synnematal and sexual stages, and inhibi-
tion by cycloheximide. Although there was initial disagreement, it is now ac-
cepted that Lomentospora prolificans is an earlier name for Scedosporium infla-
tum (25). No teleomorph has been found, but molecular data show a relationship
with species of Petriella (26), a genus traditionally separated from Pseudalles-
Figure 3 Pseudallescheria boydii ascospore. Note: bar ⫽ 2 µm.
cheria on the basis of ostiolate perithecia but sharing similar anamorphs. Isolates
of S. prolificans demonstrate less genetic variability than those of S. apiospermum
(20, 21). Clinical isolates have been typed using randomly amplified polymorphic
DNA analysis (27,28).
3. Scopulariopsis brevicaulis (Saccardo) Bainier, Bulletin

Societé Mycologique de France 23:99 (Fig. 4)
Synonym: Scopulariopsis koningii (Oudemans) Vuillemin, Bulletin Societé
Mycologique de France 27:143, 1911
Teleomorph: Microascus brevicaulis S.P. Abbott, Mycologia 90:298, 1998
Occurrence as a Pathogen. S. brevicaulis mainly causes onychomycosis,
usually of the toenail, sometimes in mixed infections with dermatophytes (29,
30), and exceptionally involves plantar skin (30, 31). Sporadic reports include
endocarditis (32, 33), keratitis (34), and deep tissue invasion in immunocompe-
tent and immunocompromised individuals (18, 35–39).
Description. Colonies are sandy-tan to avellaneous, rapid growing, and
coarsely powdery. Growth is lightly to moderately inhibited on medium with
cycloheximide and at 37°C. Conidiogenous cells are annellides borne on penicil-
lately branched conidiophores, and are cylindrical to slightly ampulliform, 10 to
25 µm long by 3 to 5 µm wide. Conidia are borne in dry chains and are globose
to subglobose or lemon-shaped with broadly truncate base, coarsely roughened,
occasionally smooth or finely roughened, and measure 6 to 9 µm long by 5.5 to
9 µm wide. Heterothallic with perithecia forming in mated strains and uncom-
644 Sigler
monly in wild type isolates having both mating types (40, 41). Perithecia are
subglobose to globose, with a papilla or short neck, have a wall of textura angu-
laris, and are black and not hairy. Asci are 8 to 10 µm in diameter, subglobose,
and evanescent. Ascospores are broadly reniform (plano-convex to concavo-con-
vex), 5 to 6 µm long by 3.5 to 4 µm wide in face view, and flattened 2.5 to 3
µm in end view. They are subhyaline to orange in mass and smooth. De Bary
bubbles, guttules, and germ pores are not evident (40, 41).
Comments. Morton and Smith (10) described six species within the
‘‘Scopulariopsis brevicaulis series’’ as having annellides of similar shape; that
is, cylindrical to slightly ampulliform and broad at the apex. These differed, how-
ever, in conidial ornamentation and colony color (white avellaneous, or fuscous).
Mating studies have confirmed that S. brevicaulis, S. candida, and S. asperula
are anamorphs of M. brevicaulis, M. manginii, and M. niger, respectively (41).
There are occasional reports of S. koningii in the medical literature, mainly con-
cerning onychomycosis. This species has been shown to be a synonym of S.
brevicaulis, however, and such reports should be considered as probably referring
to S. brevicaulis variants that have conidia that roughen tardily (10, 41).
4. Scopulariopsis brumptii Salvanet-Duval, Thèse Fac Pharm

Paris 23:58, 1935
Occurrence. Reports of onychomycosis by this species appear to be vali-
dated both by positive direct microscopy showing nondermatophytic filaments
and by repeat isolation (30, 42). Other reports are more difficult to evaluate. Few
details of the fungus are given in a report concerning brain abscess in a liver
transplant (43). Microascus species may be confused with S. brumptii if not
grown on a medium to induce the sexual stage.
Description. Colonies are slow-growing, reaching a diameter of 2 to 3
cm in 21 days, and are charcoal gray to grayish-black, usually with a narrow
white margin. Growth occurs at 37°C, but isolates vary in their ability to grow
on medium with cycloheximide (22). Annellides are borne in clusters on branched
conidiophores, are basally swollen, and are 5 to 11 µm long by 2 to 2.5 µm wide
at the base. Conidia are ovoid to subglobose, smooth but often surrounded by
mucilage or a thin membrane, and 3 to 5 µm long by 2 to 4 µm wide.
Comment. Colonies of S. chartarum are similar in color but faster-grow-
ing, and annellides are mostly solitary. See also comments under Microascus
cinereus.
5. Scopulariopsis candida Vuillemin, Bulletin Societé

Mycologique de France 27:143, 1911 (Figs. 5, 6)
Teleomorph: Microascus manginii (Loubière) Curzi, Bollettino Stazione
di Patologia Vegetale di Roma (NS) 11:60, 1931
Figure 4 Microascus brevicaulis. Anamorph Scopulariopsis brevicaulis. (A) Conidia

and ascospores (bar ⫽ 10 µm). (B) Ascoma (bar ⫽ 75 µm). (C) SEM of conidia and
ascospores (bar ⫽ 3 µm). (D) Ascoma (bar ⫽ 25 µm).
Occurrence as a Pathogen. Scopulanopsis candida is an uncommon

agent of onychomycosis (29, 30). It was reported to cause invasive sinusitis in
a girl with non-Hodgkin’s lymphoma (44).
Description. Colonies are rapid-growing, coarsely powdery, white to yel-
lowish-white, sometimes darkening centrally with the development of infertile
black perithecia (‘‘sclerotia’’). Growth is tolerant of cycloheximide and restricted
at 37°C. Conidiogenous cells are annellides that are solitary or borne on clusters
on short conidiophores. Annellides are cylindrical to slightly ampulliform, and
646 Sigler
Figure 5 Microascus manginii. Anamorph Scopulariopsis candida. (A) Conidia. (B)

Ascomata (perithecia). (C) Asci. (D) Ascospores. (E) Conidiogenous cells (annellides).
Source: Reprinted from Ref. 119, p. 97, by courtesy of Marcel Dekker, Inc.
10 to 25 µm long by 2.5 to 4 µm wide. Conidia are borne in chains and are

subglobose or broadly ovate, usually rounded, sometimes tapered at the apex,
broadly truncate at the base. They are smooth and measure 5 to 8 µm long by
4 to 7 µm wide. Scopulariopsis candida is heterothallic with perithecia forming
in mated strains and uncommonly in wild type isolates having both mating types;
infertile ascomata are sometimes formed (10, 41). Perithecia are black, globose,
and papillate, with a wall of textura angularis, and are not hairy. Asci are 8 to
Figure 6 Microascus manginii. (A) Ascospores. (B) Scopulariopsis candida conidia

(bar ⫽ 4 µm).
12 µm in diameter, subglobose to ovoid, and evanescent. Ascospores are pale

reddish-brown, smooth, broadly reniform to heart-shaped (markedly concavo-
convex), 5 to 6 µm long by 4 to 6 µm wide, and extruded in a cirrhus in age
(10, 11, 41).
Comments. S. candida is distinguished from S. brevicaulis by its smooth
conidia and white to yellowish-white colonies.
6. Microascus cinereus (Émile-Weil & Gaudin) Curzi, Bollettino

Stazione de Patologia Vegetale di Roma 11:60, 1931 (Figs.
7, 8)
Anamorph: Scopulariopsis
Occurrence as a Pathogen. Émile-Weil and Guidin (45) first described
this fungus as the cause of onychomycosis, and it is considered a confirmed but
rare agent of nail infection (29). Infections also include maxillary sinusitis in
which biopsy specimens demonstrated characteristic perithecia (46), endocarditis
in a prosthetic valve recipient (47), skin lesions in a patient with chronic granulo-
matous disease (48), and a brain abscess in a bone marrow transplant recipient
(49). Marques et al. (48) and Baddley et al. (49) reviewed other reports concern-
ing this species.
Description. Colonies are moderately fast-growing (approximately 2.5 to
3.5 cm in 14 days). They are velvety becoming granular, initially pale becoming
mid- to dark gray or brownish-black, sometimes appearing reddish-brown, espe-
cially with development of ascospores. Tested isolates are tolerant of cyclohexi-
648 Sigler
Figure 7 Microascus cinereus. Anamorph Scopulariopsis. (A) Conidia. (B) Ascospores.

Source: Reprinted from Ref. 119, p. 99, by courtesy of Marcel Dekker, Inc.
mide (9) and grow at 40°C (49). Conidiogenous cells are annellides borne solitary
on the hyphae or on short conidiophores in verticils or in a penicillately branched
arrangement. Annellides are slightly swollen near the middle and measure 4 to 9
µm long, narrowing to 1.5 to 2 µm wide at the tip. Conidia are formed in chains,
and are brown, smooth, oval, slightly pointed at the tip and truncate at the base,
and measure 3.5 to 4 µm long by 2.5 to 4 µm wide. Perithecia are smooth to finely
hairy, black, globose, and usually with a small nipplelike protuberance, sometimes
extending to form a neck up to 100 µm long. The ascospores are plano-convex
to slightly concavo-convex (almost flattened on one side and hemispherical on
Figure 8 Microascus cinereus. Ascospores (bar ⫽ 10 µm).

other side), with two germ pores. They are pale reddish-brown, measuring 5 to 7
µm long by 3 to 4 µm wide, usually extruded in a cirrhus in age (10–12, 29).
Comments. There are many Scopulariopsis species with darkly pig-
mented conidia, and it can be difficult to distinguish among them on the basis
of anamorph alone. Isolates of M. cinereus and M. cirrosus will fruit in culture
if grown on a suitable medium such as oatmeal-salts or other suitable agar (1).
This approach is also beneficial in uncovering isolates that form synnemata.
Darkly pigmented synnematous isolates are classified in the genus Cephalotri-
chum (synonym Doratomyces).
M. cirrosus differs from M. cinereus in having fruiting bodies with short
necks and ascospores that are broadly reniform to almost heart-shaped. Pseudal-
lescheria boydii forms cleisothecia (globose ascomata without an opening), and
conidia are produced in slimy masses.
7. Microascus cirrosus Curzi. 1930. Bollettino. Stazione de

Patologia Vegetale di Roma 10:308 (Figs. 9, 10)
Anamorph: Scopulariopsis
Occurrence as a Pathogen. Microascus cirrosus, originally reported un-
der the name M. desmosporus is a confirmed but rare agent of onychomycosis
(29). An invasive cutaneous infection in a bone marrow transplant recipient has
also been documented (50).
Description. Colonies and Scopulariopsis anamorph are as described for
M. cinereus. Perithecia are black, globose, with short, well-developed necks up
to 80 µm long. Ascospores are concavo-convex (curved on one side and hemi-
spherical on the other), irregular to almost heart-shaped, 4.5 to 6 µm long and
3 to 4.5 µm wide (10–12, 29).
Comments. Isolates described as M. desmosporus by Morton and Smith
(10) represent M. cirrosus. (See also discussion under M. cinereus.)
8. Additional Comments
The species listed above are reliably documented as agents of onychomycosis,
but reports concerning other species (51, 52) have not been validated by stringent
criteria, including observation of nondermatophytic filaments by direct micros-
copy and isolation of the same species from repeat specimens (29). Scopulari-
opsis chartarum was reported as a cause of disseminated disease involving the
brain in a dog (53), but the case isolate was reidentified as Acrophialophora
fusispora (54). (See Chap. 10 of this volume.) Scopulariopsis acremonium was
cited as the cause of invasive sinusitis in a patient with leukemia, but no details
of the fungus were given (55). Colonies of S. acremonium are white to cream,
650 Sigler
Figure 9 Microascus cirrosus. Anamorph Scopulariopsis. (A) Ascomata with asco-

spores in cirrhi viewed under dissecting microscope. (B) Ascoma (perithecium) with short
neck and ruptured at the base (bar ⫽ 20 µm). (C) Conidia (bar ⫽ 20 µm). (D) Ascospores
(bar ⫽ 10 µm).
like those of S. candida, which has also been reported from sinus (44), but conidia
of the former are elongate and tapered to pointed at the tip. Neglia et al. (56)
reported Scopulariopsis species as the cause of persistent ear infection and dis-
seminated disease involving the brain in two leukemic patients. One of the iso-
lates was later identified as S. candida, but without explanation (38). Pneumonia
in patients with leukemia (57), brain abscess following bone marrow transplanta-
tion (58), and nasal disease in a normal host (59) have been attributed to unidenti-
Figure 10 Microascus cirrosus. Ascospore (bar ⫽ 2 µm).
fied species. Guarro (60) has pointed out that a new genus Ascosubramania was
described for a misidentified Microascus isolate.
III. FAMILY CHAETOMIACEAE (ORDER SORDARIALES)

A. Taxonomic Concepts
Approximately 300 species of Chaetomium have been described, but only about
100 are currently recognized and species identification is often difficult (61). The
genus Chaetomium is recognized by the distinctive lateral and terminal setae or
hairs covering the perithecia and by the single-celled, brown, typically lemon-
shaped (lemoniform) ascospores with one (sometimes two) germ pores (61). The
shape and branching pattern of the setae and differences in ascospore size, shape,
and symmetry have been considered important characters in distinguishing spe-
cies (61). Perithecia are usually subglobose to ovoid and lack a neck. Similar
fungi lacking hairs on the perithecia have been assigned to the genus Achaetom-
ium, but the relationship between these genera and to other ascomycetes has not
been clear (2). Cannon (62) accepted Achaetomium, but transferred A. strumarium
to Chaetomium. Small subunit rDNA sequence analysis did not support this trans-
fer (63).
Colonies are fast-growing, commonly filling a petri dish within 10 days at
25°C, and are yellowish-green to yellowish-gray. Isolates may remain sterile on
rich media. Most species are homothallic and will usually fruit in culture if grown
on oatmeal salts, cornmeal agar, or other appropriate medium (1). These fungi
652 Sigler
are vigorous cellulose decomposers and may fruit on sterilized filter paper or
cellophane membrane. Phialidic or solitary conidia are produced in a few species.
About 20 cases of infection have been described, and these mainly concern three
species (17, 64, 65). It is significant that two species, C. atrobrunneum and A.
strumarium, which demonstrate a higher optimum temperature for growth, are
neurotropic (64).
B. Species of Medical Relevance

1. Chaetomium globosum Kunze ex Fries, Syst Mycol
3:226, 1829
Occurrence as a Pathogen. C. globosum is a rare cause of onychomycosis
(29, 64, 65). An isolate of C. globosum reported to cause brain abscess (66) was
reidentified as C. atrobrunneum (64). An isolate of C. perpulchrum from nail
(67) was determined to be C. globosum (64, 65). The pathogenic role of C. globo-
sum in a bone marrow transplant recipient with sepsis was unclear due to the
recovery of a single colony from sterile fluids and lack of evidence of tissue
invasion (68).
Description. Colonies on cornmeal agar are yellowish or grayish-green
and measure 4 to 5 cm in diameter after 5 days at 25°C. Growth is similar at 35°C,
and no growth occurs at 42°C (64). Perithecia are 175 to 280 µm in diameter and
have olivaceous green, unbranched, undulate or wavy, thick-walled hairs (setae
or appendages). Ascospores are lemon-shaped, bilaterally flattened, with a single
germ pore. They are brown and 8.5 to 11 µm long by 7 to 8.5 µm wide.
Comments. Although C. globosum is the more commonly reported spe-
cies in the medical literature, it is difficult to evaluate its role in deep infections
because some literature reports are based on misidentified isolates. (See the dis-
cussion above.) C. globosum is known to produce the mycotoxins chaetoglobos-
ins and chaetomin (Fig. 11).
2. Chaetomium atrobrunneum Ames, Mycologia 41:641, 1949

(Fig. 11A–B)
Occurrence as a Pathogen. Three cases of fatal cerebral infection have
been reported: a leukemic patient (69), a diabetic male who received a kidney
transplant (66), and a bone marrow recipient with multiple myeloma (70, 71).
The latter patient had concomitant pulmonary abscess.
Description. Colonies on cornmeal agar are dark gray to black and
slower-growing than C. globosum, measuring 1.5 to 2 cm in diameter at 25°C.
Growth is faster at 35°C and 42°C (colonies 3 to 3.5 cm and 4 to 4.5 cm diameter)
Figure 11 A–B: Chaetomium atrobrunneum. (A) Ascoma (perithecium) with straight

hairs. (B) Ascospores. C–D: Achaetomium strumarium. (C) Ascoma covered with fine
hairs. (D) Ascospores. Note: A, C—bar ⫽ 50 µm. B, D—bar ⫽ 10 µm.
(64). Perithecia are 70 to 150 µm in diameter and have few hairs, which are
dark brown, straight, and occasionally branched in age. Ascospores are brown,
narrowly fusoidal, with a single slightly subapical germ pore. They are 9 to 11
µm long by 4.5 to 6 µm wide.
Comments. Both C. atrobrunneum and A. strumarium cause brain abscess
with high mortality. These species differ from C. globosum in the smaller size
of the ascocarps, the nature of the ascomatal hairs, and in their ability to grow
at 42°C (64).
654 Sigler
3. Achaetomium strumarium Rai, Tewari & Mukerji, Can J Bot

42:693, 1964 (Fig. 11c–d)
Synonyms: Chaetomium strumarium (Rai et al.) Cannon, Trans Brit Mycol
Soc 87:65, 1986; Achaetomium cristalliferum Faurel & Locquin-Linard,
in Locquin-Linard, Cryptogamie Mycologie 1:235, 1980
Occurrence as a Pathogen. Three cases of fatal cerebral infection in
males with prior histories of intravenous drug use have been reported under the
name Chaetomium strumarium (64).
Description. Colonies on cornmeal agar are white with sparse aerial my-
celium, developing patches of pale yellowish-green to yellow hyphae in which
ascomata form, and typically developing an intense pink to pinkish-brown diffus-
ible pigment. The colony diameter is 4.5 to 5.5 cm at 25°C and ⬎9 cm at 35°C
after 5 days (64). Growth at 42°C is similar to that at 35°C, but ascomata are
produced more abundantly at 35°C. Perithecia are 100 to 250 µm in diameter,
subglobose, pale brown, and have few thin-walled straight or flexuous (curved),
finely roughened hyphalike setae (hairs). Ascospores are smooth, brown, fusoidal,
and rarely inequilateral, with single apical germ pore. They are 13 to 17.5 (21)
µm long by 8.5 to 11 µm wide. Crystals are produced on hyphae or associated
with ascomata. An anamorph is usually present on sporulation medium. Small
conidia (up to 6 µm long by 3 µm wide) are produced from short, solitary phi-
alides.
Comments. Cannon (62) retained the genus Achaetomium for the type
species but transferred A. strumarium and A. luteum to Chaetomium based on
similarities of asci and ascospores. Although these species share a common ances-
tor with Chaetomium species, A. strumarium and A. macrosporum grouped to-
gether suggest their separate placement was warranted (64). The rapid growth at
37° and 42°C, development of pink diffusing pigment on most media, and the
perithecia with less distinct hairs distinguish this neurotropic species from C.
atrobrunneum.
Yeghen et al. (72) identified C. globosum as the cause of fatal pulmonary mycosis
in a leukemic patient, but their illustration suggests a different species, possibly
C. atrobrunneum (18, 60). Schulze et al. (73) reported a similar case, except
that Aspergillus fumigatus was also isolated. Their isolate was identified as C.
homopilatum, but the length of the hairs and shape of the ascospores appear
dissimilar to published descriptions for this species (61, 62) and more similar to
C. atrobrunneum.
IV. MISCELLANEOUS ANAMORPHIC FUNGI

A. Moniliaceous Hyphomycetes
1. Arthrographis kalrae (Tewari & MacPherson) Sigler & JW
Carmich, Mycotaxon 4:360, 1976 (Figs. 12–13)
Teleomorph: Eremomyces langeronii (Arx) Malloch & Sigler, Can J Bot
66:1931, 1987
Synonyms: Pithoascus langeronii Arx, Persoonia 18:24, 1978; Pithoascina
langeronii (Arx) Valmaseda, Martinez, Barrasa, Can J Bot 65:102, 1987
Occurrence as a Pathogen. This thermotolerant fungus was first de-
scribed from chronic skin infection and has been implicated in pulmonary lesions
and keratitis (18, 74). Other reports include keratitis with severe photophobia in
a healthy woman (75) and sinusitis and meningitis in an AIDS patient (76).
Description. Colonies are slow-growing and variable in texture and color.
Initial growth is yellowish-white and pasty (yeastlike), but colonies become pow-
dery or fasciculate with development of aerial mycelium and are yellowish-white,
buff, or tan. Isolates are cycloheximide-tolerant and most will grow at 45°C.
Initial growth consists of budding cells, but these may not be formed by all iso-
lates and may be influenced by the medium. Small cylindrical arthroconidia are
formed by schizolytic dehiscence of fertile branches borne in a dendritic (treelike)
arrangement at the ends of short conidiophores. Vegetative hyphae also fragment
to form longer arthroconidia. Solitary conidia are usually produced in the sub-
merged mycelium and may be observed in slide culture preparations. The teleom-
orph is rarely produced. Cleistothecia are globose, brown, 75 to 160 µm in diame-
ter, and are usually submerged in the agar. Ascospores are phaseoliform (shaped
like orange sections), hyaline to yellowish-brown, smooth, lacking germ pores,
and 2.7 to 5 µm long by 1.8 to 2.6 µm wide.
Comments. Although the yeast phase is sometimes transitory and may
not be present in cultures that have been maintained for long periods, it is regu-
larly present in primary isolates from soil and human sources (Sigler, unpublished
data). Isolates may be mistaken for a yeast and subjected to methods normally
used for yeast identification (18, 76). Subculture onto cereal or oatmeal-salts (or
other) agar (1), however, yields the typical hyphal growth and arthroconidial
development. A. kalrae is a common fungus in north temperate soil, and soil
contamination of a contact lens was thought to contribute to infection in one
instance (75). The teleomorph is classified in the Eremomycetaceae which ap-
pears to belong in the Eurotiales. The connection between anamorph and teleo-
morph has been questioned, but sequence analysis is required to help resolve the
uncertainty (74, 77).
656 Sigler
Figure 12 Eremomyces langeronii. Anamorph Arthrographis kalrae. (A) Arthroco-

nidia. (B) Solitary conidia. (C) Wall of ascoma. (D) Asci and ascospores. Source: Re-
printed from Ref. 119, p. 101, by courtesy of Marcel Dekker, Inc.
Figure 13 Arthrographis kalrae arthroconidia (bar ⫽ 20 µm).
2. Beauveria bassiana (Bals.) Vuillemin, Bull Soc Bot Fr 59:

40, 1912
Occurrence. Mycotic keratitis in humans (78, 79) and pulmonary infec-
tion in alligator and tortoise (80, 81) have been reported, but human pulmonary
infection has not been substantiated (18).
Description. Colonies are moderately fast-growing, yellowish-white,
dense, and cottony to somewhat powdery. Conidiogenous cells are borne in dense
clusters (sporodochia) and are basally swollen, tapering at the apex and proliferat-
ing sympodially to form a zigzag (geniculate) rachis. Conidia are single-celled,
subglobose, and 2 to 3 µm long by 1.5 to 2.5 µm wide.
Comment. Beauveria bassiana is an insect pathogen with a wide distribu-
tion. Engyodontium album is closely related (17) and was formerly placed in the
genus Beauveria. (See comments under E. album.)
3. Engyodontium album (Limber) de Hoog 1978 Persoonia

10:53, 1978
Synonyms: Beauveria alba (Limber) Saccas, Revue Mycol 13:64, 1948;
Tritirachium album Limber, Mycologia 32:27, 1940
Occurrence. E. album is a rare agent of eye infection (82) and native
valve endocarditis (83).
658 Sigler
Description. Colonies are slow-growing, white, and velvety. Conidio-

genous cells are solitary or in whorls of one to three, and are cylindrical, tapering
at the apex and proliferating sympodially to form a narrow zigzag rachis from
7 to 15 µm long and 1 µm wide. Conidia are single-celled, oval with an apiculate
base, and 1.8 to 2 µm long by 1 to 1.5 µm wide.
Comment. E. album has had a complicated nomenclatural history, with
various authors disagreeing about its correct generic placement (83). Although
it was transferred from Beauveria to the new genus Engyodontium by de Hoog
in 1978, it was not selected as type species. On the basis of 18S rDNA sequences,
E. album has been shown to be a close relative of Beauveria and Tolypocladium
species, but its relationship with other members of the genus Engyodontium was
not elucidated (17).
4. Lecythophora Species
Lecythophora was reintroduced for some species, formerly placed in Phialo-
phora, that form slimy conidia from peglike or reduced phialides (adelophialides)
lacking a basal septum. Adelophialides are often conical in shape, and the collar-
ette is usually distinct (84). Phialides may also be longer, slender, and resemble
those of Acremonium. Teleomorphs are in the genus Coniochaeta (84).
a. Lecythophora hoffmannii (Beyma) Gams & McGinnis, Mycologia 75:
985, 1983
Synonym: Phialophora hoffmannii (Beyma) Schol-Schwarz, Persoonia
6:79, 1970
Occurrence. Reports concerning Lecythophora hoffmannii may refer to
Phaeoacremonium species. (See Chap. 10 of this volume.) Phaeoacremonium
was erected in 1996 for Phialophora parasitica, a more commonly reported
pathogen, and some other species occasionally involved in infection. Rinaldi et
al. (85) described a patient with subcutaneous abscess of the buttock, but the
features of the case isolate, including cinnamon-colored colonies on Sabouraud
dextrose agar, initial yeastlike growth, and phialides darkening to pale brown,
are suggestive of a Phaeoacremonium species. An isolate from the sinus of a
male patient with AIDS (86) was reidentified as P. parasiticum (18).
Description. Colonies are pale salmon to orange, sometimes creamy
white in degenerate strains, moist but becoming hairy with development of hy-
phae. Conidia are oval or ellipsoidal to slightly curved and measure 3.5 to 7 µm
long by 1 to 2.5 µm wide.
b. Lecythophora mutabilis Nannf. in Melin & Nannf. in Svenska
Skogsvardsforen Tidskr 32:432, 1934
Occurrence. Endocarditis, eye infections, and peritonitis in a patient on

chronic ambulatory peritoneal dialysis have been reported (16, 17, 87, 88).
Description. Colonies are initially white to yellowish-white, but darken
to gray with the development of brown chlamydospores. Conidia are oval to
ellipsoidal and 2.5 to 4.5 µm long by 1 to 1.5 µm wide. Chlamydospores are
brown, thick-walled, solitary, terminal or lateral, 0–1 septate, 7 to 9 µm long by
4 to 5 µm wide, or intercalary and in short chains.
c. Additional Comments. Ramani et al. (89) reported Tilletiopsis minor,
a member of the Ustilaginales, as the cause of subcutaneous cyst in an immuno-
compromised man, but the case isolate appears to be a Lecythophora species.
Colonies become dark and have yellowish-brown mycelium but lack the pig-
mented chlamydospores of L. mutabilis. Conidia are formed from phialides as
illustrated in the case report (89) and are not ballistic.
5. Metarhizium anisopliae (Metschnikov) Sorokin, Plant

Parasites of Man & Animals, p 267, 1883 (orthographic
variant ‘Metarrhizium’) (Fig. 14)
Occurrence. Cases include keratitis (90) and sinusitis (91) in immuno-
competent hosts and invasive infection manifesting with skin lesions in a boy
with leukemia (92). An otherwise healthy cat developed invasive rhinitis with
extension through the nasal bones (93).
Description. Colonies are moderately fast-growing, initially floccose and
white, turning olivaceous green or buff with the development of conidia, usually
sporulating heavily. The optimum temperature is 25–28°C, with a maximum near
37°C (92). Conidiogenous cells are cylindrical phialides borne on verticillately
or irregularly branched conidiophores formed in sporodochia. Conidia are formed
in adherent columns and are cylindrical, smooth, yellowish-green, and measure
6.5 to 8 µm long by 2 to 3 µm wide. Irregularly shaped appresoria are formed
and may be observed in slide culture preparations.
Comments. Known as the green muscardine fungus, M. anisopliae is an
important insect pathogen worldwide and has commercial application as a biolog-
ical control agent (92).
6. Myceliophthora thermophila (Apinis) Oorschot, Persoonia 9:

403, 1977 (Fig. 15)
Synonyms: Sporotrichum thermophile Apinis, Nova Hedwigia 5:74, 1962;
Chrysosporium thermophilum (Apinis) Klopotek, Arch Microbiol 98:
366, 1974
660 Sigler
Figure 14 Metarhizium anisopliae. (A) Conidiogenous cells (phialides) in sporodo-

chium. (B) Appressoria (bar ⫽ 20 µm).
Teleomorph: Corynascus heterothallicus (Klopotek) von Arx, Sydowia 34:

25, 1981
Occurrence. Aortic vasculitis with fatal outcome occurred in two pa-
tients, secondary to invasive pulmonary infection in a leukemic patient (94), and
to idiopathic cystic medial necrosis (95). Secondary infection of a bacterial cere-
bral abscess developed after trauma (96).
Description. M. thermophila is thermophilic. Colonies are fast-growing
at optimum temperature of 37–45°C, and are pink to buff, then cinnamon-brown.
Pink to cinnamon-brown diffusible pigment is usually present. One to three co-
nidia are formed on small denticles borne on the sides or the ends of short stalks,
Figure 15 Myceliophthora thermophila conidia (bar ⫽ 20 µm).
which are usually slightly swollen. Conidia are yellowish to reddish-brown oval,
and rough-walled. Ascomata (cleistothecia) are produced only in mated cultures.
Comments. Found in molding silage, pulp, and soil, this species was de-
scribed originally in Sporotrichum, now used for anamorphs of some basidiomy-
cetes. It was transferred to Chrysosporium because of its formation of solitary
aleurioconidia. (See Chap. 6 of this volume for a discussion on Chrysosporium.)
Placement in Myceliophthora is appropriate because this genus includes ana-
morphs of Corynascus (Sordariaceae) that are cellulolytic and form conidia on
swollen stalks and excludes keratinolytic dermatophytoids (97).
7. Phialemonium Species
Species intermediate between Acremonium and Phialophora are placed in Phia-
lemonium, but distinction from Lecythophora can be difficult. Slimy conidia are
formed from short cylindrical lateral phialides (adelophialides lacking a basal
septum) or from longer Acremonium-like phialides. Collarettes are lacking or
inconspicuous (84).
a. Phialemonium obovatum Gams & McGinnis, Mycologia 76:978, 1983
Occurrence. Disseminated infection in a burned child, peritonitis in a re-
nal transplant recipient, and mycetoma, subcutaneous, and disseminated infec-
tions in dogs have been reported (16, 17, 98).
662 Sigler
Description. Colonies are slow-growing, creamy white, and initially

moist, becoming grayish, velvety to hairy. Conidia are obovoid (egg-shaped),
hyaline, smooth, and 3.5 to 5 µm long by 1.2 to 1.7 µm wide.
b. Phialemonium curvatum Gams & McGinnis, Mycologia 76:980, 1983
Occurrence. Endocarditis, subcutaneous cyst in a renal transplant recipi-
ent, and fungemia have been reported (16, 17, 99).
Description. Colonies are white, sometimes developing light brown to
grayish patches, and glabrous (smooth) to hairy. Conidia are cylindrical to curved
(allantoid), hyaline, and 2.5 to 4.5 (10) µm long by 0.8 to 1.5 µm wide.
Comment. P. dimorphosporum is considered a synonym based on PCR-
RFLP banding patterns (99).
8. Scytalidium Species
Scytalidium dimidiatum is the synanamorph of Nattrassia mangiferae. S. hyali-
num is a white variant. (See description under Sec. B.3.)
B. Coelomycetes
Plant-associated anamorphic fungi forming conidia within a conidioma have tra-
ditionally been assigned to the Coelomycetes. Sutton (100) re-evaluated 370 gen-
era and recognized six suborders based on conidial ontogeny and structure of
conidiomata—that is, whether pycnothyrial, pycnidial, sporodochial, or acervular
conidiomata. Nag Raj (101) used a similar approach to redescribe 142 genera
forming conidia with appendages. Coelomycetes are recognized as occasional
agents of infection, but there are complexities in identifying clinical isolates since
conidiomatal structures produced in culture may be less elaborate than those
formed on the plant host, and descriptions of many species lack cultural details.
Also, important information concerning the plant host is unavailable. Synopses
of the identifying features of medially important species are found in several
sources (17, 102–104); therefore only some species are treated here. Several spe-
cies are reported from single cases of infection. In order to authenticate some of
these reports, it would be prudent to verify the identity of case isolates; however,
in many instances these have not been retained in culture collections.
1. Colletotrichum Species (Fig. 16)

Teleomorph: Glomerella
Occurrence. C. dematium, C. gloeosporioides, C. coccodes, and C. gram-
inicola have been implicated in eye infection (103), while C. gloeosporioides
and C. crassipes were the cause of subcutaneous infections (105, 106).
Figure 16 Colletotrichum gloeosporioides. (A) Sporodochium and setae. (B) Conidia

and appressoria (bar ⫽ 20 µm).
Description. Conidiogenous cells are phialides formed in sporodochial

conidiomata (acervular on plant host) or occasionally solitary. Thick-walled,
brown septate setae are formed in conidiomata by many species. Sclerotia are
sometimes present and may bear setae. Appressoria are usually present and are
brown, smooth, mostly solitary, and oval, clavate, or irregularly shaped and lobate
to crenate. Conidia are hyaline, straight, and cylindrical or fusiform to falcate
(curved and tapered at the ends).
Comments. Species are distinguished by the size and shape of conidia
(straight or falcate), shape and size of appressoria, and presence of setae (17,
100, 105). The most common species, C. gloeosporioides, has conidia that are
664 Sigler
straight with tapered ends, 9 to 24 µm long by 3 to 4.5 µm wide. Appressoria

are clavate to irregular and 6 to 20 µm long by 4 to 12 µm wide (100).
2. Lasiodiplodia theobromae (Pat.) Griffon & Maublanc, Bull

Trimest Soc Mycol Fr 25:57, 1909
Synonym: Botryodiplodia theobromae Pat., Bull Trimest Soc Mycol Fr
8:136, 1892
Teleomorph: Botryosphaeria rhodina (Berk. & Curt.) Arx, The Genera of
Fungi Sporulating in Pure Culture p 143, 1970
Occurrence. Mainly causing eye and nail infection, this fungus is rarely
seen in temperate areas in patients who have immigrated from tropical and sub-
tropical areas (16, 17, 29, 102, 103). Subcutaneous infection followed intramus-
cular injection in an otherwise healthy patient (107).
Description. Colonies are very fast-growing, gray to black, dense, with

aerial tufts of ropy mycelium. Conidia are produced from cylindrical phialides
within uni- or multiloculate, often hairy, black stromatic pycnidia. Conidiogenous
cells are intermixed with paraphyses (sterile hyphae) within the cavity. Conidia
are broadly ellipsoidal, initially hyaline, and single-celled, but at maturity are
two-celled, brown, and have longitudinal striations.
Comments. L. theobromae resembles Nattrassia mangiferae in geo-

graphic distribution, clinical presentation, and colonial morphology. Isolates do
not produce arthroconidia.
3. Nattrassia mangiferae (H. Sydow & P. Sydow) Sutton &

Dyko, Mycol Res 93:484, 1989 (Fig. 17)
Synanamorph: Scytalidium dimidiatum (Penz.) Sutton & Dyko, Mycol Res
93:484, 1989
Occurrence. Nattrassia mangiferae causes dermatomycoses of the nail,

toe webs, and glabrous skin, especially in immigrants from parts of Africa, India,
and the Caribbean (16–18, 29, 108, 109). Deep infections involve subcutaneous
or verrucose lesions on arm, foot, face, and finger, mycetoma, maxillary sinusitis,
endophthalmitis, fungemia, lesions of abdomen, and inguinal lymph node in pa-
tients with underlying immunosuppression, diabetes, chronic obstructive lung
disease, tuberculosis, arthritis, AIDS, or prior injury from handling wood (16–18,
86, 108, 110). Some individuals had concomitant onychomycosis. This species is
recognized and often identified under the name of the arthroconidial synanamorph
Scytalidium dimidiatum, which is the state found in primary subculture.
Figure 17 Nattrassia mangiferae. Synanamorph Scytalidium dimidiatum. (A) Arthro-

conidia. (B, C) Pycnidial conidia (bar ⫽ 20 µm).
666 Sigler
Description. Colonies are rapid-growing (filling petri dish in 7 days),

black, and ropy with deep aerial mycelium, producing diffusible black pigment,
sensitive to cycloheximide. Hyphae vary in width, ranging from 2.5 to 10 µm in
diameter, and are hyaline to subhyaline or dark brown, and often surrounded by
brown slime. Hyphae aggregate into strands and form loops. Very broad hyphae
do not fragment, but narrower hyphae form arthroconidia by schizolytic dehis-
cence. Arthroconidia are cylindrical, barrel-shaped to subglobose, subhyaline to
dark brown, nonseptate or one-septate, and measure 4 to 15 µm long by 2.5 to
7 µm wide. Morphological variants are recognized by growth rates, abundance
of arthroconidia, and production of pycnidia (108, 110). Fast-growing isolates
(form 1 or type A) are associated with individuals from subtropical areas of West
Africa, the Caribbean, or South America, and from infected plant hosts. Form 1
isolates can usually be induced to form flask-shaped to irregularly shaped pycnidia
or pycniostromata if grown on appropriate media (108, 110). Pycnidial conidia
are ellipsoidal and initially hyaline and may remain so for prolonged periods, but
eventually develop one—usually two—septa. The central cell is brown with the
end cells paler. Conidia measure 10 to 15 µm long by 4 to 5.5 µm wide. Slow-
growing isolates (form 3 or type B) are associated only with human hosts from
East Africa, the Indian subcontinent, and Southeast Asia, and it has been sug-
gested that this form represents an adaptation to anthropophilic transmission. Col-
onies are slower growing, gray rather than black, and aerial mycelium is scanty.
Hyphae are generally narrower, often form loops, and produce few or no arthro-
conidia. Pycnidia have not been obtained in isolates of this type. Intermediate
forms have also been noted, some of which will produce pycnidia (108).
Comments. Scytalidum hyalinum Campbell & Mulder was described for
fast-growing isolates that lack pigmentation. Colonies are yellowish-white, less
dense, and somewhat powdery. Arthroconidia are abundant, hyaline to subhya-
line, but no pycnidia are produced. This fungus is found exclusively from the
human host causing similar types of infections as those described for S. dimidia-
tum (17, 18, 108, 110). Moore’s (108) hypothesis that S. hyalinum could be a
white variant based on similarities in clinical presentation, immunological and
biochemical properties, and demonstration of mixed infections with S. dimidia-
tum, was confirmed by molecular typing of ribosomal genes (109). A PCR-RFLP
method to detect Scytalidium and dermatophytes in clinical samples has been
described (111). Scytalidium lignicola has not been reliably documented as an
agent of infection; cases referring to this species are based on misidentified S.
dimidiatum isolates (108, 110).
4. Phoma Species
Occurrence. Several Phoma species, including P. glomerata, P. hiber-
nica, P. minutella, P. eupyrena, P. minutispora, P. sorghina, and Phoma species,
have been reported mainly from subcutaneous infection (17, 103, 104), but re-
ports concerning P. minutispora and P. sorghina as well as P. cruris-hominis

and P. oculo-hominis are doubtful (104).
a. P. glomerata (Corda) Wollenw. & Hochapf. Z Parasitkde 8:592, 1936
Description. Colonies often appear slimy and pale to reddish-orange, be-
coming greenish-gray to dark brown to black in hyphal areas. Pycnidia are im-
mersed or superficial, globose or subglobose, and more pigmented around the
ostiole. Conidia are borne from minute phialides that line the inner cavity and
are unicellular, occasionally two-celled, ellipsoidal, hyaline, and measure 5 to 9
µm long by 2.5 to 3 µm wide. Chains of brown dictyochlamydospores (brown,
swollen structures having longitudinal and transverse septa) are common in the
hyphae.
Comment. There are a large number of species of Phoma, and a number
have been well characterized in culture. The dictyochlamydospores make P. glo-
merata one of the easier species to identify. These may appear superficially simi-
lar to the dictyoconidia of Alternaria species.
5. Pleurophomopsis lignicola Petrak, Ann Mycol 22:165, 1924

Occurrence. Subcutaneous infection and maxillary sinusitis have been
described (17, 103, 104).
Description. Colonies are light yellowish to grayish-brown and floccose.
Pycnidia are immersed or superficial, reddish-brown, subglobose to flask-shaped,
mostly uniloculate, and ostiolate. Conidiophores have one to two septa. Conidia
are hyaline, cylindrical, phialidic, and acropleurogenous (i.e., formed at the ends
and on the side with a lateral branch forming immediately below the terminal
septum) and measure 2.5 to 3 µm long by 1.5 µm wide (104).
Microsphaeropsis olivacea, implicated in dermatomycosis (17), is similar to
Phoma species in forming ostiolate pycnidia lined with minute phialides, but
conidia are brown rather than hyaline. Pleurophoma species, rare agents of subcu-
taneous and systemic infection, differ in having septate, filiform conidiophores
from which conidia develop at the tip or from small lateral openings below a
septum (17, 100, 103). Species of Pyrenochaeta have a similar conidiogenesis
to Pleurophoma species, but pycnidia are setose. Pyrenochaeta mackinnonii and
P. romeroi are often listed as agents of mycetoma, but the former species is
known only from the original description and the ex-type culture is sterile. Iso-
lates identified as P. romeroi are suspected to represent a Phoma species, possibly
P. leveillei (17). Phomopsis species are distinguished by forming conidia of two
types; alpha conidia are shorter and broader, ellipsoid to fusiform, while beta
668 Sigler
conidia are longer and narrow, filiform, straight or slightly bent or hooked. An
undetermined Phomopsis species was found to cause osteomyelitis (103, 112).
V. BASIDIOMYCETES
A. Species of Medical Relevance
1. Schizophyllum commune Fr., Syst Mycol 1:330, 1821
(Schizophyllaceae, Aphyllophorales)
Occurrence. S. commune has increasingly been reported as the cause of
chronic or allergic sinusitis, allergic bronchopulmonary mycosis, and other al-
lergy-related pulmonary diseases. Invasive infections of the brain, lung, and buc-
cal mucosa in immunosuppressed and immunocompetent individuals have also
been described (17, 18, 113–115). Sigler and Kennedy (18) suggested that a case
of sinusitis attributed to Myriodontium keratinophilum may refer to S. commune.
Description. Colonies are white, moderately fast-growing, dense, tough,
and cottony to woolly. They are thermotolerant with good growth at 37°C, toler-
ant of benomyl (2 µg/ml), and sensitive to cycloheximide. No anamorph is
formed. Isolates having clamp connections on the hyphae will normally produce
fruiting bodies (fan-shaped basidiomata with split gills) on appropriate media in
conditions of light. Many hyphae also bear small pegs (or spicules). Monokaryo-
tic isolates are flat and thinly cottony, have a pronounced unpleasant odor, and
lack clamps. Some isolates also lack spicules (115–116).
Comments. Clamped isolates with spicules can be identified as S. com-
mune, but clampless isolates lacking these structures may be difficult to identify
(116). Vegetative compatibility tests and comparison of ITS1 region sequences
have been used to identify atypical and aberrant isolates (113, 115). A clampless
monokaryon, if grown in paired culture with a compatible single basidiospore
isolate, will develop clamp connections at the interface of the paired mycelia.
2. Coprinus cinereus (Schaeffer: Fries) S.F. Gray, Nat Arr Brit

Plants p 634, 1821 (Coprinaceae, Agaricales)
Anamorph: Hormographiella aspergillata, Guarro, Gené & de Vroey, My-
cotaxon 45:182, 1992
Occurrence. Prosthetic valve endocarditis, fatal pulmonary infection in a
leukemic patient, and skin lesions have been attributed to C. cinereus or its ana-
morph, Hormographiella aspergillata (17, 18).
Description. Colonies are white to amber or tan, cottony, and dense. They
are thermotolerant, growing well at 37°C, and tolerant of benomyl. Clinical iso-
lates are normally encountered in the anamorphic state. Hyphae are simple sep-
tate, lacking clamps. Conidiophores bear several branches on the sides or at the
tip, and these branches divide schizolytically to form arthroconidia that remain
adherent at the ends of the conidiopores. Arthroconidia are hyaline, cylindrical,
thin-walled, and 3.5 to 6 µm long by 2 to 3 µm wide. Sclerotia may be formed
by some isolates.
Comments. The anamorph genus Hormographiella was described for

some clinical and environmental isolates that were thought to have basidiomyce-
tous affinity. Study of additional clinical isolates yielded some that formed Copri-
nus-type basidiocarps in culture. Compatibility tests and molecular analyses veri-
fied relationship between H. aspergillata and Coprinus cinereus and confirmed
that H. verticillata in a distinct species with an undermined Coprinus teleomorph
(117, 118).
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12
Molecular Methods to Identify
Pathogenic Fungi
Thomas G. Mitchell
Jianping Xu
McMaster University, Hamilton, Ontario, Canada
I. INTRODUCTION
Over the past two decades, the number of immunocompromised patients has con-
tinued to rise globally, promoting a dramatic increase in the incidence and variety
of fungal infections (1–3). As a result of this mycological crisis, there is a pressing
need to improve the accuracy and speed of the diagnosis, to identify the sources
of individual cases and outbreaks, and to understand the genetics and distribution
of populations of pathogenic fungi. As described in Part 1 of this book, the identi-
fication of mycotic species and strains is often problematic. Established methods
of classification rely on phenotypic differences in morphology and physiology,
and when possible, mating. For many taxa, definitive phenotypic features are
difficult to observe or are highly variable. Hence there is considerable scientific
and clinical interest in molecular approaches to the identification of species and
strains of pathogenic fungi. In recent years there has been substantial progress
in the development of innovative methods to analyze organisms at the genomic
level. In addition to the practical benefits, the application of molecular methods
to pathogenic fungi has reaped much new information about the epidemiology
and evolution of human mycoses. Consequently there are compelling reasons to
develop molecular methods to identify various taxa of pathogenic fungi.
Most medical fungi lack a sexual cycle, and for many pathogens the concept
of a species is poorly defined. Accordingly, there is a crucial need for rapid,
precise, and reproducible methods: (1) to identify fungal species accurately in
the clinical laboratory, (2) to determine the source of a mycotic infection, (3) to
677
678 Mitchell and Xu
resolve the status of problematic species, (4) to track the transmission of strains
involved in nosocomial mycoses, (5) to recognize strains with clinically impor-
tant phenotypes, such as specific virulence factors or resistance to antifungal
drugs, (6) to clarify the origin(s) of diversity and the population genetics of the
major pathogens, and (7) to provide or identify genotypes of typical strains for
use by basic scientists and by researchers in the pharmaceutical and diagnostics
fields.
In this chapter we will review molecular methods currently used for typing
species and strains of medically important fungi and discuss the selection of meth-
ods to resolve specific questions. The content will emphasize the concept of each
method, as well as its advantages and limitations. The literature in this field is
rapidly expanding, and we will not attempt to describe every application of each
method or the multiple studies applying various methods to the more common
medical fungi. References will be cited for broader issues, comprehensive re-
views, and detailed protocols.
II. MOLECULAR METHODS
The current revolution in molecular biology has provided techniques to identify

numerous taxa of medically important fungi, including pathogenic species, as
well as strains of a species. Many new methods exploit the tremendous variation
in the DNA of fungi. In this section we will introduce the more common typing
techniques and compare their value and drawbacks. The established methods for
typing medical fungi entail the comparison of allozymes, electrophoretic karyo-
types, hybridization of probes to DNA, PCR-based fingerprints, restriction frag-
ment length polymorphisms, and DNA sequencing. All these approaches are de-
signed to generate molecular markers to compare fungi.
Overall, molecular markers are definitive and more stable than phenotypic
observations. A molecular marker can be any detectable property that identifies
a specific region of the genome. There is no ideal molecular marker for every
organism or every purpose. Some markers may be better at discriminating indi-
vidual strains, separate species, or higher taxonomic groups. For some purposes,
it is important to identify markers in specific genes. In other situations, markers
in noncoding, usually anonymous portions of the genome are preferable because
they are assumed to be neutral or unaffected by selective pressure. Even with
the same organism, different markers will be used to address different questions.
A. Isozyme Electrophoresis
The electrophoretic migration of enzymes is among the most cost-effective meth-
ods to investigate genetic variation at the molecular level. The four common
methods of protein electrophoresis differ both in the nature of the supporting
Molecular Methods to Identify Fungi 679
medium or gel and whether they are run horizontally or vertically: starch (includ-
ing both horizontal and vertical systems), polyacrylamide (vertical), agarose (hor-
izontal), and cellulose acetate. These methods are compared and reviewed in
detail by Murphy et al. (4). The migration M of a protein is influenced by many
factors, including its net charge Q, its molecular size as measured by its radius
r, the strength of the electric field E, and the viscosity of the supporting gel V.
The relationship between M and other factors can be described as follows:
M ⫽ QE/4 π r 2 V
Under appropriate conditions, the rate of M increases with the net charge, which
is influenced by the pH of the buffering system and the strength of the electric
field, and M decreases as the molecular size of the protein and the viscosity of
the suspension medium are increased. Since not all proteins are globular, various
shapes may affect migration differently.
Most useful isozymes are functional enzymes that differ in amino acid se-
quence. After separation, the variant bands of an enzyme are recognized by add-
ing the appropriate substrate and a detection system. The substrate is often cou-
pled with a dye that is released upon enzymatic activity. The detection of specific
enzymatic activity in more than one band or electrophoretic mobility denotes an
enzyme with allelic variants, or isozymes.
The accurate application of isozyme data requires that the observed banding
patterns on gels are correctly interpreted. There are two commonly held assump-
tions. The first is that changes in the mobility of an enzyme in an electric field
reflect a change in its amino acid and thus encoding DNA sequence. Therefore,
if the banding patterns of two individuals differ, these differences are assumed
to be genetically based and heritable. The second assumption is that enzyme
expression is codominant; that is, every allele at a locus is expressed.
There are two general forms of protein data. Isozymes are functionally
similar forms of an enzymatic protein—including all its subunits—that may be
produced by different gene loci or by different alleles at the same locus. In con-
trast, allozymes are a subset of isozymes in which polypeptide variants of the
enzyme are formed by different allelic alternatives at the same gene locus. Allo-
zyme data are required for correct inferences about population structure. Isozyme
data are only useful under appropriate circumstances. The analysis of allelic status
is essential for complex isozyme patterns. Obtaining allozyme data from isozyme
data requires crosses and analyses of meiotic progeny. In asexual diploid fungi,
such as Candida albicans, it is impossible to infer allelic status correctly from
isozyme patterns that involve either multiple loci and/or enzymes with polymeric
structures.
Isozyme analyses have been successfully applied to studies of C. albicans
(5, 6), to other species of Candida (7, 8), and to Cryptococcus neoformans (9–
11).
680 Mitchell and Xu
B. Electrophoretic Karyotypes (EKs)

Variation in the number and size of fungal chromosomes can be detected by
electrophoresis under conditions that provide alternating fields of electric current,
referred to as pulsed field gel electrophoresis (PFGE) (12). Several instruments
and procedures have been developed for PFGE, and perhaps the most common
is the contour-clamped homogeneous electric field (CHEF). In these procedures,
intact chromosomes migrate through an agarose gel matrix under the influence
of the pulsed fields. Following electrophoresis and optimal separation of chromo-
somes, the gels can be stained with ethidium bromide and viewed under ultravio-
let light to analyze the patterns of chromosomal banding, or electrophoretic
karyotype (EK). This technique can potentially detect large deletions, insertions,
duplications, and translocations among chromosomes (13–15). Identifying genes
and chromosomes with these polymorphisms may require additional analyses,
such as digestion with endonucleases and analysis of restriction fragments and/
or blotting and probing the chromosomal gels (16, 17). A common method of
improving the resolution of the EK is to first digest the chromosomes with rare
cutting restriction endonucleases (e.g., the eight-base cutting enzyme, Sfi), and
then use PFGE or CHEF to separate these large restriction fragments (18, 19).
Depending upon the species, the EK method compares favorably with other
typing methods. It may be as good or better than other methods for discriminating
among species (20). Among the prominent medically important fungi, EKs have
been developed for Candida albicans (18, 21–24), other Candida species (25,
26), Cryptococcus neoformans (27–30), Aspergillus fumigatus (31), and others
(32–37).
There is evidence that EKs are often unstable in C. albicans (38–40) and
C. neoformans (41–43). They may be too variable and unstable to be useful in
species or strain delineation. Diverse EKs have been observed following asexual
propagation and subculturing within a single genotype. The loss of dispensable
chromosomes also causes variation (44). Because chromosomes must pair during
meiosis, it is often assumed that the EKs of sexual species should be less variable
than those of asexual ones, but the EKs of sexual species of fungi may indeed
vary in size and gene arrangement (45). Another drawback of EKs is the occa-
sional difficulty of scoring homologous chromosomes, which may migrate to-
gether and appear as one band. It is also difficult to quantify differences in EKs.
C. DNA–DNA Hybridization
Several increasingly sophisticated methods of molecular typing entail variations
of the hybridization of complementary strands of DNA. DNA–DNA hybridiza-
tion techniques offer a quantitative measure of the similarity between two or
among several individuals. This category of molecular typing methods began
with DNA reassociation kinetics. Currently, hybridization using oligonucleotide

probes and DNA microarrays are becoming powerful tools for assaying genetic
differences at defined genomic regions.
1. DNA Reassociation Kinetics

DNA hybridization takes advantage of the double-stranded nature of the DNA
molecule in which nucleotides on opposing strands are held together by hydrogen
bonds. Two hydrogen bonds are formed between adenine and thymine, and three
hydrogen bonds link guanine and cytosine. When double-stranded DNA is heated
to around 100°C, the hydrogen bonds between complementary base pairs are
broken and the opposing strands separate. During subsequent cooling of the solu-
tion, the complementary DNA strands will reanneal. If DNA from two different
species are combined, denatured, and then allowed to reassociate, the double-
stranded molecules that form between complementary strands from the two spe-
cies will contain base pair mismatches because of their evolutionary divergence
from a common ancestor (46). The conditions of reassociation must be standard-
ized because the amount of base pair mismatches that form in the hybrid mole-
cules is affected by the salt concentration, temperature, viscosity, and DNA frag-
ment size. Under highly stringent conditions of reassociation, which are generally
achieved by increasing the temperature and/or decreasing the salt concentration,
base pairing between DNA strands from different strains or species will only
occur between well-matched sequences. Under conditions of progressively lower
stringencies, more mismatches will be tolerated during reassociation.
The extent of mismatching determines the temperature at which these hy-
brid molecules melt when they are placed in a thermal gradient. The more mis-
matches, the lower the temperature at which the hybrid strands will separate.
The decrease in the melting temperature of a heteroduplex hybrid relative to a
homoduplex control provides an index of divergence between the DNAs under
consideration. Reassociation analyses have been used to investigate the relation-
ships among several closely related anamorphic species (46–50).
2. Oligonucleotide Hybridization
This technique utilizes known single nucleotide polymorphisms in a species. Oli-
gonucleotides of more than 20 bases are designed with known polymorphic sites
placed near the middle of the oligonucleotide, which can be end-labeled with a
radioactive tag or fluorescent dye. The labeled oligonucleotide probes are then
hybridized by conventional Southern hybridization to either total genomic DNA
or specific gene fragments amplified by the polymerase chain reaction (PCR).
The presence or absence of a hybridization signal for each probe can be scored
as alternative alleles at a specific site. The drawbacks of this technique are that
it requires two hybridizing procedures for every locus in diploid individuals, and
682 Mitchell and Xu
unknown mutations at any of the 20 or so nucleotides may cause the loss of a

hybridizing signal.
3. DNA Chip Technology

The new technology of DNA microarrays on chips represents a miniature but
mass version of individual oligonucleotide hybridization. Briefly, DNA chips are
glass surfaces to which arrays of specific DNA fragments have been attached
at discrete locations. These fragments serve as probes for hybridization. Under
conditions suitable for hybridization, the DNA spots on the chip are exposed to
a solution containing a complex sample of fluorescent-labeled DNA or RNA.
This technique has been established for only a few species; current use has con-
centrated on Saccharomyces cerevisiae (51). A typical array might consist of 20
complementary pairs of oligonucleotides (25-mers) for each gene. In addition,
there will be three permutations of each consensus 25-mer, each with a single
base change in the central nucleotide position. Thus many base pair substitutions
of the gene will be represented on the chip. A DNA sample of each isolate is
permitted to hybridize to the array. Comparing the hybridization intensities to the
consensus oligonucleotides and the one-base mutants provides internal controls in
the search for sequence polymorphisms. The amount of sequence divergence and
similarity among strains can be calculated.
Another broad application of DNA chip technology involves the analysis
of gene expression. At present this technology is limited to intraspecific compari-
sons. The high expense currently prohibits its extensive use.
D. PCR-Based Methods
The PCR technology has spawned many procedures for typing strains, some of
which have become established methods in systematics and strain typing. PCR
methods are easy to set up, rapid, and have the advantage of requiring only minute
amounts of starting material or template DNA. Although simple in concept, the
PCR entails unrivaled, often overlooked complexity. The source of this complex-
ity is inherent in the PCR itself; the products result from myriad ionic interactions,
kinetic constants, and enzymatic activities, which repeatedly affect the reactants
in a small volume over an extended time period. Some of the common PCR-
based strain typing techniques are described below.
1. Random Amplified Polymorphic DNA (RAPD)

In RAPD analysis, genomic or template DNA is primed at a low annealing tem-
perature (30–38°C) with a single short oligonucleotide (approximately 10 bases)
in the PCR. Multiple PCR products of different electrophoretic mobilities are
typically generated, and in comparing species or strains, each isolate will yield
Figure 1 Electrophoretic separation of RAPD fingerprints obtained by amplifying geno-

mic DNA from 23 strains of Cryptococcus neoformans with single primers: (A) OPA-03
(5′ AGTCAGCCAC 3′) or (B) OPA-17 (5′ GACCGCTTGT 3′). Lanes 1 and 25 are 100
bp and 1 kbp DNA ladders (GIBCO, BRL), respectively. Lanes 2 through 24 are RAPD
profiles of the following strains from the Duke Medical Mycology Research Laboratory:
CnA-1, CnA-2, CnA-3, CnA-4, CnA-5, CnA-6, CnA-7, CnA-8, CnB-1, CnD-1, CnD-2,
CnD-3, CnD-4, CnA-9, CnA-10, CnD-5, CnD-6, CnA-11, CnA-12, CnA-13, CnA-14,
CnA-15, and CnA-16, respectively. (Note: Cn denotes C. neoformans, and the letters indi-
cate the serotype, A, B, C, or D.)
several bands of different sizes. (See Fig. 1.) RAPD analysis detects variations
in the length between two primer binding sites or sequence length polymorphisms
in the fragments between PCR priming regions (52, 53). Nucleotide substitu-
tions in the region of PCR primer binding, particularly at the 3′ ends, can prevent
binding of the primer to the DNA template and subsequent PCR amplification,
and a band will be missing. Similarities in banding profiles among strains (i.e.,
the number and mobility, but not the density of the bands) can be calculated and
used to infer epidemiological relationships. When multiple primers are screened,
RAPD analysis is sensitive enough to detect variation among isolates that cannot
be observed by using other methods, although a combination of methods often
provides optimal discrimination (54–56).
Although technically fast and simple, there are some disadvantages to
RAPD. The major drawback is reproducibility. RAPD analysis can detect minute
684 Mitchell and Xu
variation among strains because, as noted above, even a single nucleotide substi-
tution in the priming region may permit or prevent the annealing and subsequent
production or absence of a characteristic band. Small differences in any aspect
of PCR conditions that affect binding of the primer will have the same effect;
consequently, RAPDs are sensitive to the vagaries of the testing procedure. This
problem can be minimized if strains under study are treated similarly. When
multiple strains are to be compared for distinct RAPD patterns, the same PCR
buffer, master mix, and thermal cycler should be used, and the strains being
compared should be amplified at the same time.
RAPDs can also be problematic because bands with the same electropho-
retic mobility may not share the same sequence. This problem may be common
for interspecific studies and is affected by the conditions of electrophoresis. With
the usual agarose gels (1–1.5%), it is difficult to distinguish RAPD bands with
size differences of less than 20 base pairs.
Another concern with the interpretation of RAPD profiles is the problem
of dominant and null alleles. In haploid organisms, both the dominant (presence)
and null (absence) alleles can be scored, but in diploid organisms, it is not possible
to distinguish genotypes that are homozygous for the dominant allele from those
that are heterozygous. Therefore, RAPD data are generally not ideal for infer-
ences of population genetic history (57).
Nevertheless, for comparing the similarities among strains and developing
fingerprints for molecular epidemiology, RAPD analyses have been widely ap-
plied to a number of medically important fungi (10, 56, 58–68).
2. Amplification of Microsatellite Repeats

This emerging technique exploits the hypervariability of DNA regions composed
of 10 or more tandem repeats of di-, tri-, or multiple nucleotides. This hypervaria-
bility can be caused by either strain slippage during DNA replication or unequal
crossing-over during meiosis. Useful microsatellites have been located by probing
a genomic library or searching databases of gene sequences (69, 70). PCR primers
flanking these repeat regions can be designed, and PCR products are typically run
on polyacrylamide gels to detect differences in a single repeat. This technique can
discern levels of variability as high or higher than PCR fingerprinting. Because
multiple alleles are typically found at a single locus, the relationships among al-
leles can be difficult to decipher, and alleles may not be identical by descent.
3. PCR Fingerprinting
This technique is similar to RAPD, except that the primers are longer (⬎15 bases)
and the annealing temperatures are more stringent. Most PCR fingerprinting
primers are designed from repetitive DNA sequences, microsatellites (as above),
or somewhat longer minisatellites (71, 72). Commonly used primers include
M13, which is derived from the core sequence of phage M13, T3B, which origi-
nates from internal sequences of tRNA genes, and TELO1, which is based on
fungal telomere repeat sequences. Because of more stringent reaction conditions,
PCR fingerprinting is generally more reproducible than RAPDs. Nonetheless, it
suffers the same problems of interpretation as RAPDs. However, under standard-
ized conditions, PCR fingerprinting has proven quite reliable for the identification
of species and strains (71, 73–75).
4. PCR-RFLP
With increasing knowledge of the genomics of human pathogenic fungi, the num-
ber of reported gene sequences is increasing. These gene or intergenic sequences
can be used to investigate the variability among strains and the history of popula-
tions of a species. One typical application is to design PCR primers to amplify
a particular stretch of DNA, and subsequently digest the amplicon with a battery
of four-cutter restriction enzymes to screen for variability. Variable restriction
sites can then be used to screen a larger sample of isolates. This approach was
used to develop multilocus genotypes of Candida albicans (43, 76). In a diploid
organism, codominant genetic information is obtained, and unlike RAPDs or PCR
fingerprinting, both alleles can be scored. This technique is highly reproducible.
5. SSCP and Heteroduplex

Single-strand confirmation polymorphism (SSCP) and heteroduplex are promis-
ing techniques that allow efficient detection of nucleotide substitutions in short
fragments (⬍500 bp) of DNA. SSCP analysis typically involves the amplification
of a discrete segment of genomic DNA, melting the PCR products, and analysis
of the single strands on a nondenaturing polyacrylamide gel (77). The DNA
strands are usually visualized by using radiolabeled DNA during PCR or by
silver-staining the DNA after gel electrophoresis. Polymorphisms in strand mo-
bility result from the effects of primary sequence differences on the folded struc-
ture of the single DNA strands; that is, the primary sequence differences alter
the intramolecular interactions that generate a three-dimensional folded structure.
The molecules may consequently migrate at different rates through the non-
denaturing polyacrylamide gel. Because these conformational variations may in
theory reflect a single base difference, they are subtle, and the success of any
particular SSCP assay depends highly on the particular DNA fragments being
investigated, including their sequence and length, and the optimization of the
experimental conditions to maximize the differential migration of the fragments.
A variety of methods have been invoked to improve the resolving power of SSCP,
including the addition of glycerol to the polyacrylamide gels, reducing the tem-
perature, and increasing the length of the gels or duration of electrophoresis.
Nevertheless, differentiation among polymorphic molecules on a polyacrylamide
686 Mitchell and Xu
matrix is not entirely predictable, and this method can produce false negative
patterns, ambiguous results, and experimental artifacts (77). SSCP works best
with fragments ⱕ300 bp. It has been used to develop markers for several patho-
genic fungi (78–83).
In contrast to SSCP, heteroduplex analysis is dependent on conformational
differences in double-stranded DNA. In this technique, equal amounts of two
PCR products (e.g., from wild-type and mutant DNA samples) are combined in
a nondenaturing buffer. The DNA is melted at 95°C and slowly cooled to room
temperature. During the cooling process, the complementary single strands from
the same sample reanneal to form double-stranded homoduplexes, and the non-
complementary single strands from different samples also reanneal to form
heteroduplex DNA. The mismatch in the heteroduplex DNA imparts a different
three-dimensional shape or flexibility compared to the homoduplex DNA, and
consequently the heteroduplex DNA will have less electrophoretic mobility than
the homoduplex DNA. The electrophoresis and detection methods for hetero-
duplex analysis are similar to those for SSCP. Heteroduplex analysis works well
for fragments ranging in length from 200 to 600 bp.
6. Amplified Fragment Length Polymorphism (AFLP)

The relatively recent development of the method of amplified fragment length
polymorphisms (AFLPs) has already exerted a significant impact as a genotyping
method. The determination of AFLPs is a powerful method for fingerprinting
genomic DNA, as well as generating a large number of dominant markers for
genotypic analysis (84, 85). AFLP technology combines the strategies of enzy-
matic digestion and PCR. With AFLP, after digestion with two endonucleases
(usually a frequent and a rare cutting enzyme), dsDNA adapters are ligated to
the ends of the DNA fragments to create template DNA for the PCR. AFLP
adapters consist of a core sequence and an enzyme-specific sequence. AFLP am-
plification primers consist of the core sequence, the enzyme-specific sequence,
and a selective extension of one to three nucleotides, which will amplify a subset
of the restriction fragments. AFLP usually involves two PCR steps: (1) in the
preamplification step unlabeled primers with a single selective nucleotide are
used; (2) after this preliminary PCR, the reaction mixtures are diluted tenfold
and used as templates for the second or selective PCR, which uses a longer exten-
sion and labeled primers.
AFLP has several powerful advantages over the other methods. Many more
fragments can be generated and analyzed. By varying the restriction enzymes
and the selective nucleotides, 30 to more than 200 fragments can be produced,
depending as well on the complexity of the genome. Different enzymes or exten-
sion nucleotides (or both) can be used to create new sets of markers. AFLP can
therefore provide an almost limitless set of genetic markers. In addition, the frag-
ments are stable and highly reproducible since they are amplified with two spe-
cific primers under stringent conditions. Like RAPD markers, AFLP markers are
amplified by using arbitrary sequences, but with greater reproducibility and fidel-
ity. An example of AFLP products is shown in Fig. 2.
In Table 1 comparisons are made of the steps involved with AFLP and two
other common approaches—the classic hybridization-based method to identify
RFLPs, and PCR-based fingerprinting techniques, which involve amplification
of particular DNA sequences using specific or arbitrary primers. Amplification
products are separated by electrophoresis and detected by staining the DNA or
by using radiolabeled primers and detected by exposure to X-ray film.
E. Restriction Fragment Length Polymorphism (RFLP)

Restriction endonucleases have been used to discriminate among species and
strains of fungi as well as other biological taxa. The most common approach is
to digest genomic DNA with a four-base or other frequently cutting enzyme and
examine the resulting bands on electrophoresis in agarose or polyacrylamide gels
(64, 86–91). Depending upon the size of the genome and the number of sites of
enzymatic cleavage, it may be possible to compare digests of genomic DNA
for different banding patterns, indicating DNA sequence polymorphisms. This
method of analysis can only detect differences in DNA molecules that are present
in high copy numbers, such as ribosomal or mitochondrial DNA. For genetic
elements that are low in number, it is almost impossible to observe restriction
site polymorphisms following a simple digestion and electrophoresis on agarose
or polyacrylamide gel.
Alternatively, an amplified gene or other PCR product can be digested and
analyzed (76), as described in section II.D.4 (page 685). For comparisons between
species, differences in the rDNA motif are often discernable by PCR-RFLP (92–
94). Because it is frequently difficult to determine accurately the migration of
bands (i.e., DNA fragment sizes), comparisons should be made on samples in
adjacent lanes of the same agarose gel with size gradients on both sides. Table
2 compares this classic method of DNA fingerprinting with PCR and AFLP fin-
gerprinting.
The most widely used RFLP method is a DNA–DNA hybridization tech-
nique that involves digesting genomic DNA with restriction endonuclease(s) fol-
lowed by electrophoretic separation of the DNA fragments. RFLPs are detected
by Southern hybridization with labeled probes targeted to single copy markers
or repetitive DNA. Several probes have been well characterized for C. albicans,
including Ca3, CARE2, 27A, MGL1, and the RPS family (95–100). These probes
may be as large as several kb. Other probes are specific for other species of
Candida (101–106), for A. fumigatus (107–109), and for Cryptococcus neo-
formans (110–113).
688 Mitchell and Xu
Figure 2 AFLP in Cryptococcus neoformans were generated by using the method of

Vos et al. (84) with fluorescent-labeled PCR primers. The gel images are analyzed by
GeneScan (Perkin-Elmer). Numbers on the right indicate molecular weight standards.
Thirty lanes containing AFLP bands from basidiospore progeny are loaded in the parallel
lanes. Arrows denote polymorphisms.
Table 1 Comparison of Steps in DNA Strain Typing by Hybridization, RAPD, and

AFLP
Conventional hybridization DNA fingerprinting

1. Extract DNA
2. Digest DNA with restriction enzyme(s)
3. Separate DNA fragments by agarose gel electrophoresis
4. Denature DNA
5. Blot single-stranded DNA to membrane
6. Hybridize with labeled oligonucleotide probe
7. Wash and detect bands (e.g., autoradiography, fluorescence)
RAPD (PCR, AP-PCR, or DAF) fingerprinting
1. Extract DNA
2. Randomly PCR amplify with single oligonucleotide primer
3. Separate products by agarose gel electrophoresis
4. Photograph and analyze bands
AFLP fingerprinting
1. Extract DNA
2. Digest DNA with restriction enzyme(s)
3. Ligate dsDNA adapters (i.e., core sequence ⫹ restriction enzyme-specific
sequence) to ends of the DNA fragments
4. PCR amplify restriction fragments with primers consisting of adapter ⫹
enzyme-specific site
5. Dilute and PCR selectively with primers consisting of adapter ⫹ enzyme-
specific site ⫹ 3′ extension of 1-3 nucleotides, thereby amplifying only a
subset of the restriction fragments
6. Separate products by denaturing polyacrylamide gel electrophoresis and analyze
bands
RFLPs generated from total genomic digests alone or by hybridization with

repetitive elements may be difficult to interpret. As the number of bands in-
creases, accuracy may be compromised and affected by the conditions of elec-
trophoresis. These banding patterns are often used to recognize species and to
fingerprint strains, but it is difficult to use them to identify the allelic status of
individual loci. Conversely, for RFLPs detected by hybridization with probes that
target single-copy DNA markers, the interpretation is uncomplicated and the data
can be used for multiple purposes. For example, single-copy RFLPs are quite
useful for population genetic analyses of diploid fungi (76).
F. DNA Sequencing
The most exacting and laborious method to catalog differences at the DNA level
is directly sequencing cloned genes or PCR products. This approach provides the
690
Table 2 Comparison of Methods Currently Used to Study Genetic Variation in Fungal Populations
Techniques → EK DNA–DNA DNA probe RAPD/AP-PCR DNA
↓ Considerations MLEE (PFGE/CHEF) hybridization hybridization PCR fingerprint AFLP SSCP RFLP sequencing
Typical applications
Population structure Yes No Usually yes Yes Yes or no Yes Yes Yes Yes
Identify taxa No Yes or no Yes Yes Yes Yes Yes Yes Yes
Phylogenetic analysis Yes or no No No Yes or no Yes or no Yes Yes Yes Yes
Practical factors
Pure cultures required Yes Yes Yes Yes or no Yes Yes No Yes or no Not with
PCR
Sample preparation Minimal Medium to Medium to Medium Minimal Minimal Minimal Medium Maximal
high high
Reproducibility Good Good Very good Good Good to poor Very good Very good Very good Best
Cost Least Moderate Expensive Moderate Low to Moderate Expensive Low to Most
expensive moderate moderate expensive
Turnaround time Moderate Slow Slow Slow Rapid Moderate Slow Moderate Slowest
Analytical factors
Sensitivity to detect Low (but often Low to high High Moderate High Very high Extremely Moderate Highest
polymorphism adequate) high
Utility of genetic mark- Codominant Chromosome Codominant Usually Dominant Codominant Codominant Usually Codominant
ers (dominant or co- markers codominant codominant
dominant) codominant
Note: MLEE, multilocus enzyme electrophoresis; RFLP, restriction fragment length polymorphism; EK, electrophoretic karyotype; PFGE, pulsed field gel
electrophoresis; CHEF, contour-clamped homogeneous electric field; RAPD, random amplified polymorphic DNA; AP-PCR, arbitrarily primed-PCR; SSCP,
single strand conformational polymorphism.
Mitchell and Xu
most accurate data for phylogenetic analyses. As with other procedures, several
investigations have analyzed medical fungi by direct DNA sequencing (80, 114–
123). For phylogenetic analyses, the most common regions to be sequenced and
compared are portions of the ribosomal DNA cluster, including the internal tran-
scribed spacers (ITS1 and ITS2), the intergenic spacer (IGS), and the 18S, 5.8S,
and 28S rDNA. These regions are highly conserved within species and variable
among species.
At the species level, ribosomal RNA genes are under strong evolutionary
constraints. Conversely, stable mutations are more common in noncoding se-
quences, and third-base substitutions are common in protein-encoding genes.
Consequently, for comparisons among strains of a species, sequences of protein-
encoding genes or nonfunctional DNA are usually more informative than rDNA.
For example, nucleotide substitutions were found among strains of Coccidioides
immitis in genes encoding five proteins: chitin synthase, chitinase, orotidine mo-
nophosphate decarboxylase, serine proteinase, and a T-cell reactive protein simi-
lar to mammalian dioxygenase (124). Phylogenetic analysis of these variations
identified geographically isolated populations of C. immitis. In the ensuing years,
more studies will employ this type of gene genealogical analysis.
Analyses of this type will also be facilitated by whole genome sequence
projects. The genome of S. cerevisiae has been fully sequenced, and the genomes
of both Candida albicans and Cryptococcus neoformans are currently being se-
quenced. These data will permit direct comparisons of specific sequences among
isolates within these species. Future evolutionary studies of these and other fungal
pathogens will feature comparative genomics across and within taxa.
III. NO UNIVERSAL MOLECULAR METHOD SUITS

EVERY PURPOSE
There is not an ideal molecular method for every application, nor is there a best
or worst method of molecular typing. Different typing methods are appropriate
for different epidemiological or evolutionary studies in different species. Table
2 summarizes the advantages and limitations of the major molecular techniques.
In the remainder of this section we will attempt to recommend appropriate molec-
ular methods to address specific questions. However, as indicated below, aside
from the technique, the availability of appropriate control samples may be more
crucial than the selection of a typing method.
A. Species Identification
Most pathogenic fungi can be identified by conventional laboratory methods,
which are based primarily on morphology and physiological tests for molds and
692 Mitchell and Xu
yeasts, respectively. However, the interpretation of these tests may be subjective,

requiring a skilled mycologist, and some isolates yield variable or atypical results.
Phenotypic variation is common among and within fungal species. As the panoply
of pathogenic fungi continues to expand, there is a growing need to develop a
more efficient, accurate, and rapid approach to identification.
To develop a DNA-based protocol to identify pathogenic fungi, it is feasible
to consider signature sequences in the 28S or large nuclear subunit of rDNA.
Portions of the 28S rDNA sequences of most human pathogenic yeasts are cur-
rently available in Genbank. In addition, many of the molecular methods de-
scribed in Sec. II are amenable to species identification, and many have been
applied to this purpose. Indeed, a variety of DNA-based methods for species
identification have been reported, and the results are often reliable, reproducible,
and easy to interpret. However, no standard database or protocols have been
established for these methods, and it is difficult to compare the results from differ-
ent laboratories.
B. Molecular Epidemiology
Besides species identification, molecular methods can help determine the origin
of a clinical isolate. Although a few human fungal pathogens are geographically
restricted, such as Coccidioides immitis, most are highly prevalent. They exist
either as members of the normal flora, such as species of Candida, or they are
ubiquitous in nature, such as Cryptococcus neoformans and A. fumigatus.
To determine whether the source of a case or outbreak of nosocomial can-
didiasis can be attributed to a commensal isolate, the local yeast microflora should
be sampled. Environmental isolates might be obtained from a variety of clinical
settings and health care workers. Control samples could be isolated from vicinal
patients, similar body sites, and patients with similar risk factors. After the appro-
priate isolates and case histories are obtained, a variety of molecular techniques
may be employed to determine the similarity of the isolates. For such studies,
DNA sequencing of specific genes may have little value. The most useful meth-
ods are those that detect numerous polymorphisms throughout the genome and
are highly discriminatory, such as AFLP, RAPD, PCR fingerprinting, and probe
hybridization methods. Other markers may also be used, especially those that
detect polymorphisms that can be unambiguously scored and quantified. Obvi-
ously, molecular markers that do not detect intraspecies variation are not helpful,
although such invariable species markers have great value for species identifica-
tion. When the data are collected, appropriate statistical tests should be applied
to determine the significance or statistical support for similarity clusters. The
subsequent detection of significant clusters can be used to infer the source of the
strain responsible for an infection(s). However, because similarity among strains
does not prove identity, strain typing can be used with more confidence to exclude
certain sources or strains from involvement.
Determining the environmental source of an infection can be more prob-
lematic. This is because the extent of genetic variation among environmental
populations of pathogenic fungi is usually unknown. As described above, appro-
priate samples from nature can be collected from the suspected sources of the
pathogen and critically evaluated. For comparison, additional control isolates
should be obtained from similar environments.
C. Antibiotic Resistance
Methods similar to those for determining the source of an infection (see Section
III.B) can be used to investigate the origin of a strain with antibiotic resistance.
Controls include isolates from different samples, for which the minimal inhibitory
concentration to an antifungal agent must also be determined. In general, if mo-
lecular typing determines that several drug-resistant strains are dissimilar, resis-
tance can be assumed to have arisen independently in each strain. Alternatively,
a significant clustering of the resistant strains suggests a clonal origin of the
resistant genotype and spread of the resistant genotype among patients (125–
128).
D. Genetic Diversity and Population Structure

To investigate population structure, it is necessary to have markers that can be
interpreted genetically and population samples of sufficient sizes. The selection
of markers is influenced by the ploidy of the species and the extent of existing
genetic variation in the population. For haploid species, dominant-recessive
marker systems can be used, such as AFLP or RAPD. Codominant markers are
preferable because multiple alleles can be detected at the same time. For diploid
species, only codominant genetic markers provide enough information to infer
the mode of reproduction in nature and genetic differences among populations.
The analytical methods for understanding the genetic structure of fungal popula-
tions are discussed in detail in Chap. 13.
IV. FUTURE CONSIDERATIONS
With the completion of the sequencing of the genome of S. cerevisiae, near com-
pletion of the Candida albicans genome, initiation of the sequencing of the ge-
nome of Cryptococcus neoformans, and sequences of other microorganisms in
public databases, abundant genomic information is now available to design prim-
694 Mitchell and Xu
ers to develop systems for the identification of fungal species. The sequence
databases can also be used to generate gene-specific products with which to fur-
ther compare strains within a species. Developing a set of genetic markers from
genes with known functions could also facilitate the analysis of interspecific pop-
ulation genetics. Subsequent standardization will promote the exchange of ge-
netic information among laboratories and improve the usefulness of studies of
molecular epidemiology.
In this era of fungal molecular biology and genetics, a wealth of genotyping
methods is available. Determining the origin and spread of fungal pathogens re-
quires thoughtful selection of control samples and appropriate typing methods.
Molecular methods provide new tools, not necessarily the solution, to epidemio-
logical questions.
ACKNOWLEDGMENTS
Support from Public Health Service grants AI 25783, AI 28836, and AI 44975 is
greatly appreciated. The authors are members of the Duke University Mycology
Research Unit.
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13
Population Genetic Analyses
of Medically Important Fungi
Jianping Xu
McMaster University, Hamilton, Ontario, Canada
Thomas G. Mitchell
I. INTRODUCTION
In the last decade there has been a vast accumulation of information on the popu-
lation structure and epidemiology of fungi pathogenic to human and other ani-
mals. This increase is due both to the rising incidence of mycotic diseases and
to the increasing availability of strain-typing techniques. As reviewed in Chap.
12, there are numerous applications of these methods to analyzing the similarity
among individual strains (1–10). Equally important are studies of the population
structure of medically important fungi. Understanding the population dynamics
of a pathogenic fungus will clarify epidemiological trends and assist researchers
in the selection of appropriate strains in the quest for virulence factors and target
molecules for novel antifungal drugs, vaccines, or diagnostic tests.
In this chapter we will review approaches based on population genetic anal-
yses to understand the patterns and mechanisms of genetic variation. Rather than
exhaustively reviewing the population genetic studies of all medically important
fungi, in this chapter we will introduce basic concepts, issues, and rationales for
population genetic studies of medically important fungi. We will also review
the analytical methods, present examples of their application, and indicate their
limitations in addressing population genetic questions.
II. WHAT IS A POPULATION?
There are several overlapping definitions of ‘‘population’’: a population can be

(1) the organisms that inhabit a particular locality, (2) a group of interbreeding
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704 Xu and Mitchell
organisms that represent the level of organization at which speciation begins, or

(3) a group of individual persons, objects, or items from which samples are taken
for statistical measurement. The first definition is based on geographic locations,
although the size and boundaries of individual populations can vary widely and
are often arbitrary. This definition fits most population genetic studies of fungi
and other organisms. The second definition is based on genetics and is the most
restrictive. By this definition, a population refers to groups of individuals that
are genetically isolated but still capable of interbreeding with individuals of other
populations of the same species. For many species, including human pathogenic
fungi, it is usually difficult to establish the precise mating boundaries for groups
of individuals. In species of plants and animals, both geographical and ecological
factors contribute to determining the population breeding boundaries (11). An
obvious problem with this definition is that many species of pathogenic fungi
have not been observed to undergo sexual mating and meiosis.
The third definition is the most versatile. It allows multidimensional analy-
ses of the distribution of genetic variation within a species. This is an operational
definition of a population. For example, a population of Candida albicans can
be a collection of individual strains from a particular locality. The locality may
be defined as a geographical entity (e.g., a continent, country, state or province,
city, hospital, or ward) or ecological niche (e.g., isolates from patients with cer-
tain medical conditions, specific body sites, or different body sites of the same
host). A population of C. albicans isolates could be further circumscribed ac-
cording to the vital statistics of their hosts (sex, age, race, and ethnic background).
The only limitation on the number of delineating criteria is that the sample
size of the smallest category must be sufficiently large for statistical analysis.
The sample size that is sufficient to detect differences among samples depends
on the patterns of genetic variation of the population. The smaller the actual
difference in allele frequencies, the larger the sample sizes required for their
reliable detection. For populations with small numbers of members, the sample
size N needed to detect a given level of differentiation at a diploid locus among
populations at least 50% of the time (i.e., a power of 0.5) with a statistical signifi-
cance of 0.05 can be approximated as
N ⫽ 1/2 FST and FST ⫽ 1/2 N
where FST represents the proportion of the total genetic variation explained by
the difference between samples. For example, to detect an FST value of 0.05,
samples of just 10 diploids per locality may be adequate (or 20 strains per locality
for haploids) (12). A more detailed explanation and calculations are presented
in Sec. V. This definition of a population is commonly applied to study the epide-
miology and prevention of infectious diseases and to identify risk factors among
humans and potential virulence factors in pathogens.
Population Genetic Analyses 705
III. GENETIC MARKERS
A genetic marker can be defined as an allele. For the species under investigation,
the types of markers amenable to population genetic-based analysis depend on
the ploidy and the mating system. Genetic markers must be unambiguously inter-
pretable as alleles of a specific locus, and all alleles at each locus must be scorable
for all isolates. In haploid species, most markers can be easily analyzed by popu-
lation genetic approaches. Since there is only one set of genetic material (1N)
for each strain, all markers can be scored for each strain. However, in diploid
species (2N), determining the number of loci and the number of alleles per locus
for many fingerprinting-based markers can be tedious and is often prone to error.
In a species with a tractable mating system, strains can be crossed and the meiotic
progenies can be analyzed to detemine the number of loci and the number of
alleles per locus for a marker system. With diploid species lacking in sexual
mating and/or meiosis, the interpretation of markers is highly problematic. Gener-
ally speaking, codominant, single-copy genetic markers are best suited for allelic
assignments in strains of diploid species (13). These markers include allozymes
(see Chap. 12), single locus RFLP, and DNA sequence-based single-nucleotide
polymorphisms.
Some markers are better at addressing certain questions. Selectively neu-
tral, codominant markers are best suited to investigate recombination and gene
flow between populations. Loci that are under selective pressure will have a
higher probability of convergent and sometimes diversifying evolution than neu-
tral loci. For example, genes involved in drug resistance and in response to host
defenses are under constant selective pressure in clinical settings, and these genes
may not be well suited for examining recombination and genetic differentiation
in natural populations.
IV. ANALYSIS OF VARIATION WITHIN A POPULATION

A. Issues and Rationales
Many simple measures have been used to describe the genetic variation within
a population. Among others, they include: (1) observed heterozygosity (11), (2)
gene diversity (11), and (3) observed genotypic diversity (13,14). The observed
heterozygosity Ho represents the percentage of observed heterozygotes at each
locus, applicable only in diploids. Gene diversity is defined as the expected heter-
ozygosity He as follows:
He ⫽ 1 ⫺ ∑p 2i
where pi is the frequency of the ith allele at a locus. Both the observed heterozy-
gosity and the gene diversity (i.e., both Ho and He) are individual locus-based
706 Xu and Mitchell
measures of genetic variation for population samples. The mean observed hetero-
zygosity or gene diversity of a sample is estimated as the arithmetic mean of all
loci sampled.
A common measure of multilocus population genetic variation is the ob-
served genotypic diversity (G0): G0 ⫽ 1/∑p2i, where pi is the frequency of a partic-
ular multilocus genotype. G0 ranges from 1 to N, where N is the sample size.
This measure was originally proposed as an overall index of within-population
genetic diversity (12). Another potentially useful measure of the distribution of
genotypes is the probability that random pairs of isolates will have different
multilocus genotypes. This probability is calculated as 1 ⫺ ∑p2i, where pi is the
frequency of a particular multilocus genotype.
Examining the patterns of genetic variation in natural populations, a major
question about within-population genetic variation in fungi (and other pathogenic
microbes) is the contribution to this variation, if any, of recombination.
Since all microbes are known to reproduce asexually via mitosis, it is there-
fore expected that most microbial species would show some evidence of clonality
in nature. One focus of fungal population genetic studies has been whether recom-
bination plays any role in generating genetic variation in natural populations.
The amount of recombination that occurs in natural populations of microor-
ganisms effects their evolution. Sexual reproduction and recombination pose an
evolutionary paradox. Whereas an organism that reproduces asexually passes on
all its genes to each of its progeny, one that reproduces sexually passes on only
half of its genes to each progeny. Under uniform conditions, natural selection
therefore favors the organism that reproduces asexually because with an equal
number of progeny, the asexual individual has double the fitness of the sexually
reproducing one.
Sexual reproduction affords microorganisms two possible advantages: pan-
mixia and DNA repair (15, 16). The panmictic argument suggests that without
the mixing of genes generated by sexual recombination, adaptive evolution is
limited to the accumulation of favorable mutations that occur successively in
each independently evolving lineage. Sexual recombination allows favorable mu-
tations that arise in separate lineages to become combined in the same individual,
providing an advantage in the adaptation to different environments. The repair
argument points out that the two haplotypes associated wth diploidy during sex
provide an error-correction mechanism for repairing genetic damage. Genetic
damage can occur spontaneously and continuously during replication and possi-
bly transcription. The intact DNA of one haplotype can serve as a template for
correcting the damaged DNA in the other haplotype. Moreover, deleterious muta-
tions in one haplotype can be overcome by compensatory dominant mutations
in diploids. Whether one or both of these purported advantages of sexual recombi-
antion accounts for the origin and maintenance of sexual reproduction is subject
to considerable debate and investigation (15).
A second reason to assess the role of recombination in natural populations

relates to issues of sampling, research, and treatment strategies. If recombination
occurs in natural populations, there may be significant ramifications for the evolu-
tion and dissemination of genes related to antibiotic resistance, pathogenicity,
and host specificity. A recombinant population structure implies that the selected
properties reflect distinct genes or nonrecombining genetic elements. (Discreet
genes are usually assumed to be nonrecombining units, even though intragenic
recombination has been demonstrated in all organisms that were critically exam-
ined.) Conversely, a clonal population structure implies that the selected units are
clones or clonal lineages. To study medically relevant traits in a clonal population,
representatives of every clonal lineage should be sampled. However, in a recom-
bining population, it would be more profitable to focus primarily on individual
genes.
To determine whether a microbial population structure is predominantly
clonal or recombining, population microbiologists employ genetic tests. The next
section will discuss the tests used to dissect the genetic structure within a popula-
tion. These tests differ somewhat for haploid and diploid species.
Many of the methods used in the analysis of population genetic structure
require rigorous statistical measurements. These statistical tests, such as those
used to test null hypotheses, are selected to address specific questions and evalu-
ated according to their appropriateness and accuracy. Statistical tests should be
examined for their propensity to be erroneous. Statistical tests are prone to two
distinct and opposing types of errors, known as type I or type II errors. Type I
errors occur if a test is too rigorous for the data, perhaps by not accounting for
errors due to random sampling, which results in rejection of the hypothesis, even
when it is true. Conversely, with a type II error, the statistical test permits exces-
sive leeway in the data, and the hypothesis will seldom be rejected, even when
it is false (11). The balance between type I and type II errors is such that reducing
the probability of one type will increase the probability of the other. For null
hypotheses, the 5% level of statistical significance is applied, indicating there is
a 95% probability that the hypothesis is true a 5% chance that a true hypothesis
has been rejected (type I error). The chance of a type II error, failure to reject a
false hypothesis, will vary.
B. Analysis of Clonality and Recombination in Haploids

Since a haploid genome contains only one set of chromosomes, each isolate has
only one allele for each locus. Consequently, tests of recombination in natural
haploid populations involve the examination of the allelic associations between
different loci.
For the purposes of population genetics, a genetic locus may be defined
as: (1) a single polymorphic nucleotide site, (2) a polymorphic restriction endonu-
708 Xu and Mitchell
clease recognition site (four to six base pairs), (3) a single nucleotide insertion
or deletion, (4) any nucelotide sequence difference(s) within a region of continu-
ous DNA of variable length, possibly constituting a distinct allele, or (5) an enzy-
matic staining profile. Because of the large variation in the size of the DNA
segment or gene being recognized as a locus, analytical methods may be different.
All analytical methods rely on two basic assumptions: (1) once a locus is defined,
recombination within the locus is assumed to occur very rarely, therefore negligi-
bly or not at all, and (2) each distinct allele is the result of a unique mutational
event that occurred only once in the population. Within these assumptions, indis-
tinguishable alleles are considered to be ‘‘identical by descent.’’
In tests for the occurrence of recombination in natural populations, there are
two distinct questions. First, is the population panmictic? A panmictic population
structure implies that alleles at all loci are randomly associated with each other.
If the hypothesis of panmixia is statistically rejected and a clonal population
structure is assumed, the second question arises: Can all or part of the genetic
variation in natural populations be attributed to recombination? Since all medical
fungi are capable of reproducing asexually through mitosis, a clonal component
in populations is expected. A widely used approach is to analyze representatives
of each different multilocus genotype to distinguish between the null hypothesis
of recombination and the alternative hypothesis of clonality.
Often, a small number of genotypes are dominant or overrepresented
within a population. It is common in these analyses to include the genotypes of
all isolates in the population as well as only the unique genotypes within the
population. This smaller group is the ‘‘clone-corrected’’ sample. The rejection
of panmixia for the total sample but acceptance of panmixia for the clone-cor-
rected sample is usually regarded as evidence for clonal expansion with evidence
of random mating in the genetic structure. There are several caveats related to the
truncation of a sample by clone correction before testing. Even though generally
assumed, it is usually not confirmed that identical multilocus genotypes are actu-
ally clonal in origin. The justification for clonal-correlation of the sample should
be based on biological and ecological considerations. The decrease in sample
size may also decrease the power of the statistical test for rejection of the null
hypothesis of recombination, thus increasing the possibility of type II error (11,
17).
Tests for whether or not the haploid population is panmictic involve com-
paring observed allelic associations with those derived under the null hypothesis
of random mating. The two most widely used population genetic tests for haploid
genomes are tests for allelic association (linkage equilibrium) between pairs of
loci, and the overall index of association (IA) involving alleles at all loci. A third
test compares the observed genotypic diversity with that expected under the null
hypothesis of random mating (14). A fourth test uses phylogenetic analysis of
DNA sequence-based characters (18,19). In this test, incongruences between dif-

ferent gene genealogies for a set of strains are considered to be consistent with
hypothesis of recombination.
1. Linkage Disequilibrium
Linkage disequilibrium (gametic phase disequilibrium or gametic disequilibrium)
is a measure of the association between alleles at pairs of loci (11). Random
association between alleles at different loci in a population is considered to be
an indication of recombination between these two loci. The test for random asso-
ciation can be described as follows. If alleles at two loci, A and B, segregate
independently, then the expected frequency of the genotype AiBi is simply the
product of the frequencies of the two alleles Ai and Bi. A chi-square test or
Fisher’s exact test can be performed to determine whether or not the observed
genotypic counts are significantly different from the expected counts (11,17). If
the observed and expected counts are not significantly different, then the popula-
tion under study is assumed to have a recombining structure. Conversely, if the
observed and expected counts are significantly different, the population is as-
sumed to have a clonal structure as inferred by this pair of loci. With balanced
gene frequencies, in a completely panmictic population less than 5% of locus
pairs are expected to have genotypic counts significantly different from those
expected.
A key factor in this test is the independence of the loci. If the paired loci
are linked, they are not totally independent. The degree of linkage between loci
affects the association of alleles at these loci. However, it has generally been
assumed and has been mathematically proven that if the loci under study are
selectively neutral, any positive and/or negative allelic association between loci
will be broken down rapidly if sexual mating and meiosis are frequent (11). If
an organism reproduces clonally (through either mitotic division or homothallic
mating), the entire genome is effectively linked since there is no segregation and
reassortment of alleles. Both linkage and clonal reproduction can cause deviations
from the expected (random) genotypic frequencies for any pairs of loci. The
degree of deviations (D) or nonrandom association between two loci, each with
two alleles, A1 and A2, and B1 and B2, is computed as: D ⫽ pA1B1pA2B2 ⫺
pA1B2pA2B1, where pA1B1 is the observed frequency of genotype A1B1, and
so on.
This approach has the drawback that many different tests are required and
the results are not easily interpreted. If there are n polymorphic loci in a sample,
the number of tests between possible pairs of loci are n(n ⫺ 1)/2. Even if a
population is panmictic, some tests are likely to show significant deviation from
random mating by chance. Conversely, even in a strictly clonal population, some
710 Xu and Mitchell
tests might still indicate random association, especially when the sample size is
small and allelic frequencies are skewed. To avoid some of the problems in the
test for linkage disequilibrium, an index was introduced to measure the overall
allelic association in a sample, described below.
2. Index of Association (IA)

To assess overall allelic associations in haploid organisms, an index of association
IA has been used widely in fungal population genetic analysis. This index was
first used by Brown et al. (20) to measure population structure in Hordeum spon-
taneum, and it was used later to demonstrate nonrandom association of alleles
in Escherichia coli (21) and other bacteria (22). IA is a generalized measure of
linkage disequilibrium and has an expected value of zero if there is no association
between loci. The calculation of IA is derived as follows:
Suppose that M loci have been analyzed in N individuals. Let p ij represent
the frequency of the ith alleles at the jth locus. Then the gene diversity h j
⫽ 1 ⫺ ∑p 2ij, which is the probability that two individuals are different at the
jth locus. Let K represent the number of loci having different alleles between
two different individuals. The observed variance of K, or Vo, can be calcu-
lated based on the distributions of K. Since there are N(N ⫺ 1)/2 possible
pairs of individuals, the mean difference between any two individuals, K′,
is ∑ h j. The expected variance of K is Ve ⫽ ∑h j (1 ⫺ h j ). The index of
association is IA ⫽ Vo/Ve ⫺ 1.
There are two ways to test whether or not IA is significantly different from
zero—that is, the null hypothesis of random association of alleles at different
loci. The first test assumes that the sampling distribution of the error variance
of IA, which is calculated as
Var(Ve) ⫽ [ ∑h i ⫺ 7∑h 2i ⫹ 12 ∑ h 3i ⫺ 6∑ h 4i ⫹ 2(∑ h i ⫺ h 2i )2]/N
approximates normality, then the upper 95% confidence limit for Var(Ve) is
L ⫽ ∑ h j ⫺ ∑ h 2j ⫹ 2[Var(Ve)]1/2
If Vo does not exceed L, then the null hypothesis of random association of alleles
at all loci is not rejected. The population under study is therefore concluded to
have a population structure not significantly different from panmixia. If Vo is
greater than L, the population is assumed to have a significant clonal reproduction
component.
The second statistical test of IA is to use a randomization approach in which
the null distribution of Vo is generated by randomly permuting the alleles among
all individuals within each locus and calculating Vo many times. The Vo from
the observed sample is then compared to the null distribution from permuted
samples to determine whether or not there is a significant difference. If the ob-

served variance Vo is ⬎(1 ⫺ α) of the Vo from randomized data sets, where α
is the acceptable type I error rate (0.05 or 0.01), then the sample deviates signifi-
cantly from random mating. Since the sampling distribution of Vo is not known
to be normal, randomization tests for significance are preferable, especially when
sample sizes are relatively small.
3. Phylogenetic Methods
As shown above, tests for both linkage disequilibrium and asosciation index were
against the null nypothesis of random mating. However, testing against a null
hypothesis of random mating can create a type II error, the probability of ac-
cepting a false hypothesis, especially when the sample size is small and the allele
frequencies are skewed. Furthermore, it is often difficult to determine whether
or not recombination contributes at all to variation in a population determined
to have a predominantly clonal structure.
To overcome these problems associated with testing against the null hy-
pothesis of panmixia, phylogenetic analysis offers tests against the alternative
hypothesis of strict clonality. There are two types of phylogenetic tests for clon-
ality and recombination. The first uses population genetic data of allelic informa-
tion at individual loci for each strain. The premise is that if populations are clonal,
the ancestry of isolates will be represented by a phylogenetic tree of good fit.
However, for recombining populations there will be little or no phylogenetic
consistency because different loci reflect different patterns of descent among indi-
viduals. Furthermore, the length of the tree can be compared with the minimum
length expected if the population were strictly clonal (2).
The second phylogenetic test uses gene sequences from several genes. By
comparing phylogenetic trees built for different genes, clonality can be distin-
guished from recombination. There is strong evidence for clonality if the trees
for different genes are concordant, but if the trees are in conflict, recombination
is a likely explanation. The partition homogeneity test (PHT) was developed to
assess gene genealogy congruences (23). For congruent gene trees, the sum of
the lengths of the most parsimonious tree for each gene should not change sig-
nificantly if the polymorphic nucleotides in each gene are swapped among genes.
In contrast, for incongruent gene trees, the sum of the gene trees for the observed
data should be shorter than the sum for gene trees made after polymorphic nucleo-
tides have been swapped among genes. This is true because recombination is
assumed to be correlated with linkage relationships in the genome. Intragenic
recombination, if it exists at all, would be much rarer than intergenic recombina-
tion. The statistical significance of this test is established by making many resam-
pled data sets and comparing the observed sum of length to the distribution of
1000 or more resampled data sets.
712 Xu and Mitchell
4. Examples
Although most human fungal pathogenic species are haploid, few have been criti-
cally examined for the patterns of genetic variation in natural populations. Four
species of medically important fungi have been examined for the roles of clonality
and recombination in natural populations—Coccidioides immitis, Histoplasma
capsulatum, Cryptococcus neoformans, and Aspergillus flavus.
Three populations of C. immitis were analyzed with the same set of genetic
markers by Burt et al. (2,24). All the populations—one from Tucson, Arizona,
one from Bakersfield, California, and the third from San Antonio, Texas—had
genetic structures not significantly different from panmixia. The allelic associa-
tions between many pairs of loci were in linkage equilibrium (24; J. Taylor,
personal communication). The overall association index IA for the Tucson popula-
tion was 0.041, not significantly different from zero (2).
Thirty isolates of H. capsulatum from Indianapolis, Indiana, were examined
for associations of alleles at 11 different loci (25). Every isolate had a unique
multilocus genotype. Alleles at many pairs of loci were not significantly associ-
ated with each other, and with an IA close to zero (0.0428), this sample of H.
capsulatum was concluded to have a recombining population structure (25).
Cryptococcus neoformans is somewhat different from the above two spe-
cies. The current taxonomy classifies this biological species into two varieties,
C. neoformans var. neoformans and C. neoformans var. gattii. Each variety has
two predominant serotypes, serotypes A, D (or AD) in C. neoformans var. neo-
formans and serotypes B and C in C. neoformans var. gattii. Despite cross-hybrid-
ization among all serotypes in the laboratory (26,27), various strain-typing studies
have revealed clear differences between the two varieties (1,28–33). Strains
within each variety can also be readily sorted according to their serotypes
(28,30,32,34). It has been suggested that different serotypes might correspond to
different cryptic species reproductively isolated for a significant amount of time
(35). When populations of C. neoformans were analyzed through multilocus en-
zyme electrophoresis, abundant evidence supported a predominantly clonal ge-
netic structure (28). Significant clonal components were still observed when only
representatives of unique multilocus genotypes were analyzed separately for indi-
vidual serotypes (36). While the null hypothesis of panmixia is rejected for iso-
lates of C. neoformans from natural and clinical sources, it is not known whether
sexual recombination contributed to the patterns of genetic variation in this spe-
cies. As with C. neoformans, the genetic structure of Aspergillus fumigatus is
clonal (37).
The use of phylogenetic methods for detecting recombination in medical
fungi was best illustrated with Aspergillus flavus. Analyzing the sequences of five
genes, Geiser et al. (38) demonstrated recombination in one of the two genetically
isolated groups of A. flavus. The PHT was highly significant (p ⬍ 0.0001) for
incongruence among five gene genealogies, consistent with recombination (38).
C. Analysis of Clonality and Recombination

in Diploid Species
1. Hardy–Weinberg Equilibrium Test (Association of Alleles
Within a Locus)
Since each diploid strain has two alleles at each locus, tests for recombination
in diploid organisms are somewhat different from haploid species. In diploid
species, the association of alleles within a locus is quite often used as a measure
of recombination. A chi-square goodness of fit test can be performed to compare
the observed and expected genotypic counts at each locus and summed across loci
(11,39). This computation is the test for Hardy–Weinberg equilibrium (HWE). To
illustrate, for a single locus A with two alleles, A1 and A2, there are three possible
genotypes at this locus, A1A1, A1A2, and A2A2. The expected Hardy–Weinberg
frequencies of genotypes A1A1, A1A2, and A2A2 are p2, 2pq, and q2, respec-
tively, where p is the frequency of allele A1 and q is the frequency of allele A2,
and A1 ⫹ A2 ⫽ 1. If observed counts of genotypes are not significantly different
from expected counts, then the population is in HWE and assumed to have a
recombining structure. However, Hardy–Weinberg frequencies are valid only
when populations comply with several common but crucial assumptions: (1) the
population size is large, (2) there is no selective pressure on the markers being
analyzed, (3) migration and mutation are negligible, and (4) the population under-
goes random mating (11).
It is imprudent to infer population structure from HWE tests alone. First,
violation of any of the first three assumptions above may cause significant devia-
tion from the expected frequencies, even if the population is randomly mating.
Second, failure to reject the null hypothesis of HWE does not guarantee that the
population is indeed randomly mating (type II error). Third, different populations
of the same species may have different population structures (24).
2. Composite Genotypic Disequilibrium

The ‘‘exact test’’ for allelic association among loci was developed by Zaykin et
al. (40). In this test, the probability of the set of multilocus genotypes in a sample,
based on allelic counts, is calculated from the multinomial theory under the hy-
pothesis of no association. Alleles are then permuted and the conditional probabil-
ity is calculated for the permuted genotypic array. The proportion of arrays no
more probable than the original sample provides the significance level of the test.
The exact test is not restricted by the number of loci. It also allows the calculation
714 Xu and Mitchell
of the probability of multilocus genotypes conditioned on genotypic counts at

individual loci. Separating the allelic association test within a locus from geno-
typic association among loci can be very useful for diploid species, such as Can-
dida albicans, because deleterious recessive mutations may be common and many
loci with genotypic counts deviated significantly from Hardy–Weinberg expecta-
tions (11).
3. Examples
Pujol et al. (41) used 21 isozyme loci to characterize the genotypes of 55 strains
of C. albicans isolated from patients infected with the human immunodeficiency
virus (HIV). Thirteen of the 21 enzymatic loci were polymorphic among these
strains. Six of the 13 polymorphic loci deviated significantly from HWE. The
expected counts for some of the multilocus genotypes were also much lower than
observed counts. They concluded that C. albicans has a clonal population struc-
ture (41).
Gräser et al. (42) used 12 nucleotide-based markers to characterize a mixed
sample of C. albicans isolated from Durham, North Carolina. Among the 52
strains analyzed, 27 unique multilocus genotypes were detected. Similar to the
findings of Pujol et al. (41), about half of the markers showed significant deviation
from HWE (42). However, when they calculated the associations of alleles at
different loci, ⬎70% of the pairwise loci comparisons were not significantly dif-
ferent from random association. They concluded that the population structure of
C. albicans included both clonal and recombinant components. Similar patterns
have been confirmed among populations of C. albicans from different groups of
hosts, including HIV-infected patients, non-HIV patients, and healthy persons
(43).
D. Epidemic Clones: Calculations and Implications

Analyzing the genetic structure of populations to determine the extent of clonality
versus recombination is only the first step, albeit a critical one, toward under-
standing population history and the evolution of pathogenicity. The subsequent
questions concern the reason(s) for a particular genetic structure for certain popu-
lations. Approaches to address this question are beginning to emerge, but defini-
tive answers are lacking. Many factors can contribute to the genetic structure of
a population, including the history of the population, the mutation rate, the muta-
tion spectrum (i.e., the distribution of mutations that are lethal, deleterious, neu-
tral, and advantageous), genetic drift, population size, mating system (or lack of),
and the selection pressure from the environment. It is often difficult to examine at
the same time the relative contributions of these factors to the genetic structure
of natural populations.
One approach is to investigate whether and why certain genotypes are more
prevalent than expected. This information can be obtained by inspecting the rela-
tive frequencies of different multilocus genotypes or by calculating the expected
frequencies of individual genotypes based on allelic frequencies and under the
hypothesis of random mating (i.e., random associations of alleles within and
among loci). When predominant genotypes are identified, the medically important
traits of those isolates can be compared with less common genotypes in the popu-
lation to explore possible associations between the genotypes and relevant pheno-
types.
In a recent analysis of different samples of C. albicans by codominant,
single-copy PCR-RFLP markers, one multilocus genotype predominated in all
four samples (43,44). One sample was obtained from HIV-infected patients in
Vitória, Brazil, and three from the United States (Durham, North Carolina). The
predominant genotype was much more prevalent in all four samples than would
be expected under random mating. This genotype might be selectively more ad-
vantageous than other genotypes of C. albicans in human populations. The patho-
genicity, transmission, and antifungal drug susceptibility profiles of isolates with
this genotype warrant further investigation.
V. ANALYSIS OF GENETIC VARIATION

BETWEEN POPULATIONS
A. Issues and Analytical Methods
Depending on the questions of interest, there are multiple ways to compare ge-
netic variations among populations. The first general approach could be to com-
pare the genetic parameters calculated for each population. These parameters
include gene diversity, heterozygosity, genotypic diversity, relative percentage
of polymorphic loci, and number of alleles per locus. The extent of recombination
and clonality might differ among natural populations of the same species because
population histories, mutation rates, environmental conditions, and selection pres-
sure can vary among populations. Any associations between specific environmen-
tal factors and genetic elements can be determined through general statistical
analysis.
The second approach is the more traditional population genetic analysis.
Calculations can determine the extent of population subdivision, genetic differen-
tiation, and gene flow among populations based on differences in the gene and
genotype frequency and measures of inbreeding coefficients.
Population subdivision involves an inbreedinglike effect within a popula-
tion and a lack of interbreeding among strains between populations (11, 39).
This effect can be assessed from the decrease in the proportion of heterozygous
genotypes. A subdivided population of a diploid organism has three distinct levels
716 Xu and Mitchell
of complexity: individual organisms I, subpopulations S, and the total population

T. Let
HI ⫽ the observed heterozygosity of an individual averaged over

subpopulations
Hs ⫽ the expected heterozygosity of an individual in an equivalent
random mating subpopulation
Hsa ⫽ the average of Hs taken over all subpopulations
HT ⫽ the expected heterozygosity of an individual in an equivalent
random mating total population
The effects of population subdivision are measured by a quantity called
the fixation index (symbolized FsT), which is the reduction in heterozygosity of
a subpopulation due to random genetic drift (45).
FsT ⫽ (HT ⫺ Hsa)/HT
FsT is always ⱖ0, because the Wahlund effect (11) assures that HT ⬎ Hsa. If all
subpopulations are in HWE with the same allele frequencies, then FsT ⫽ 0.
In haploid organisms, all the calculations for FsT are similar to those for
diploid organisms. However, the symbols have different meanings. The expected
heterozygosity Hs of an individual in subpopulations of diploids is the same as
the gene diversity averaged among subpopulations in haploids, and the HT of an
individual in the total population of diploids is the same as gene diversity in the
total population of haploids.
The calculations of gene flow based on FsT are different for haploid and
diploid organisms because each migrant individual carries two alleles per locus
in diploid species but only one allele per locus in haploids. The number of mi-
grants per generation Nm equals (1 ⫺ FsT)/4 FsT for diploids (46) but (1 ⫺ FsT)/
2 FsT for haploids. N is the number of individuals in a subpopulation, and m is
the migration rate between pairs of populations. The estimation of gene flow
based on FsT is conditional on many assumptions (46). These assumptions include
low mutation rates, stable environments, no selection on the marker(s) under
investigation, large population sizes, and genetic equilibrium within each popula-
tion. If these assumptions are violated, Nm cannot be used as a parameter to
estimate the gene flow between populations.
The statistical test of whether or not pairs of populations are significantly
different is usually achieved by a direct comparison of gene frequencies (47).
This is done by using the chi-square contingency table tests. Alternatively, Fish-
er’s exact test is used when the sample sizes are small or when the lowest ex-
pected allele count is smaller than five. These tests are described in general biosta-
tistical and epidemiological texts (e.g., 17).
Population genetic relationships are also frequently described in un-

weighted pair-group method using arithmetic means (UPGMA) phenograms (11).
These phenograms are based on population genetic distances. There are several
measures of genetic distances, all based on population gene frequencies and in-
breeding coefficients. The most widely used is Nei’s genetic distance D (48).
The basis of the measure is a so-called normalized identity, which expresses the
probability that a randomly chosen allele from each of two different populations
will be identical, relative to the probability that the two randomly chosen alleles
from the same population will be identical. (‘‘Identical’’ in this context means
indistinguishable, not ‘‘identical by descent.’’) The calculations can be illustrated
as follows:
Suppose there are two hypothetical populations, A and B. At locus X, the
frequency of allele I in population A is pi and in poulation B is qi. JAA is
defined as the probability that two alleles chosen at random from population
A are identical, then,
JAA ⫽ ∑ p 2i
This is because with random mating, JAA equals the homozygosity in popula-
tion A. Likewise, JBB is defined as the probability that the two alleles chosen
at random from population B are identical.
JBB ⫽ ∑p 2i
JAB is the probability that two alleles are identical when one allele is chosen
from population A and the other is chosen from population B, or
JAB ⫽ ∑p i qi
Nei defines the normalized identity, I, for this gene as
I ⫽ JAB /(JAA JBB)1/2

and the standard genetic distance, D, as
D ⫽ ⫺ln(I)
D varies from 0 to ∞. This measure of genetic distance is more reliable

when data from many genes are available, because with many genes the quantities
JAA, JBB, and JAB, are the arithmetic means of the individual values. Using the
average of JAA, JBB, and JAB, the same formulas for I and D apply. If two popula-
tions are identical, then JAA ⫽ JBB ⫽ JAB, and the normalized identity is 1. Lack
of genetic divergence between populations results in a distance D ⫽ 0. Since the
statistic is calculated from samples from populations, the exact value of D be-
tween populations can vary from one sample to another. The degree to which
different samples produce different D values depends on the amount of genetic
divergence, the genes assayed, and the size of the samples.
718 Xu and Mitchell
After pairwise population genetic distances are calculated, population simi-

larities can be described on a UPGMA phenogram (11,17). Genetic distances
are often compared to geographic distances. Assuming roughly constant rate of
migration, one might expect that pairs of populations that are situated farther apart
geographically would show greater genetic distance. This principle is known as
isolation by distance (49).
Figure 1 UPGMA phenograms describing genetic similarity among four samples of

Candida albicans (44). The distance measure is based on Reynolds’ genetic distance (39).
These two phenograms are based on two different sample types: (A) the total samples;
(B), the clone-corrected samples in which only one representative of each multilocus geno-
type from each population is used for analysis. Bootstrap values from 1000 replicates are
given.
B. Example
At least two studies have estimated the genetic differentiation and gene flow
between geographic populations of human pathogenic fungi. In the analysis of
three geographic samples of the haploid Coccidioides immitis from Bakersfield,
Tucson, and San Antonio, the overall FST between pairs of populations ranged
from 0.198 to 0.944 (24). The overall allele frequencies were significantly differ-
ent between all pairs of populations. Taken together, the results suggest signifi-
cant genetic differentiation between geographic populations of C. immitis (24).
Conversely, the geographically separated populations of the diploid Can-
dida albicans from HIV-infected patients in Vitória, Brazil, and Durham, North
Carolina, were genetically indistinguishable (44). The overall FST estimated from
16 loci between these two samples were 0.017 and 0.002 for the total samples
and the clone-corrected samples, respectively. The number of migrants estimated
from FST values ranges from 6.89 to ∞ per generation between the two geographic
populations. Both samples shared the same most common multilocus genotype
for the 16 loci examined (44). Furthermore, these two samples were genetically
more similar to each other than either was to the sample from healthy persons
in Durham. (See Fig. 1.)
VI. CONCLUSIONS
Although the study of population genetic structure of medically important fungi

is still embryonic, clear trends are emerging. Different species and different popu-
lations of the same species may have very different genetic structures. Under-
standing the evolutionary and ecological bases for these differences should con-
tribute to the understanding of fungal pathogenicity and epidemiology. This
knowledge can be expected to influence the design of effective strategies for the
treatment and prevention of fungal infections.
ACKNOWLEDGMENTS
Support from Public Health Service grants AI 25783, AI 28836, and AI 44975 is
greatly appreciated. The authors are members of the Duke University Mycology
Research Unit.
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14
Genetic Instability of
Candida albicans
Elena Rustchenko and Fred Sherman

University of Rochester Medical School, Rochester, New York, U.S.A.
I. INTRODUCTION
Candida albicans is a polymorphic, commensal, and opportunistic pathogenic

fungus, often incorrectly referred to as ‘‘dimorphic,’’ with a remarkable degree
of both genetic and phenotypic variability. Under normal circumstances Candida
survives on the mucous surfaces of a host in different niches, which include the
gastrointestinal tract as well as the genitalia of mammals. The mucus is believed
to be a source of a variety of aminosugars, which can be utilized as energy sources
(1). As at least eight different mucinlike genes are differently expressed in differ-
ent body niches (2), it is reasonable to suggest that the content of mucus, as well
as the content of body fluids, vary between different regions of the body, between
different individuals within the same species depending on the individual’s me-
tabolism and diet, and between different mammalian species. In this respect,
Singh and Datta (3) demonstrated that, in contrast to C. albicans, the nonpatho-
genic species of the genus Saccharomyces cannot utilize the aminosugar N-ace-
tylglucosamine, which suggests that the aminosugar metabolic pathway is impor-
tant in pathogenesis or at least in survival in a mammalian host. Because of the
above-mentioned factors, environmental diversity requires phenotypic plasticity
in the form of tolerance to different pH, food supply, and coexistence with other
natural microflora. One example of the means of C. albicans to cope with a
diversity of niches is the induced expression of catabolic enzymes on the path-
way of N-acetylglucosamine (4). Another example is transcriptional induction of
PHR1 and PHR2 genes, which respond to certain ranges of pH (5–7). Still other
723
724 Rustchenko and Sherman
potential mechanisms involve genes, which sense the environment, and which
are also implicated in morphogenesis. (See, e.g., Refs. 8–25.)
The subject of this chapter will be genetic instability based on mutational
events, in contrast to both the transcriptional induction or repression implicated
in the above-mentioned forms of regulation and the control of such transitory
cellular stages as budding, pseudohyphae, hyphae, chlamydospores, and germ
tubes.
The sites of occurrence and mechanisms of genetic instability in the host
are still not understood, although they obviously play the major role in the forma-
tion of new phenotypes. As emphasized in this review, chromosomal alteration
is the main cause of this instability, and we have considered two major aspects
of this phenomenon. One is chromosomal instability, which occurs spontaneously
(i.e., without an obvious selective condition), resulting in preadaptation of a por-
tion of the population (26). Another aspect is chromosomal instability occurring
in response to the changing environment (27). So far, neither of these mechanisms
has been addressed in animal models.
The mechanism of C. albicans pathogenicity is currently a popular subject
of study. As summarized by Odds (28, 29), the traditional understanding of Can-
dida virulence has been derived largely from studies in which each of the attri-
butes reported to contribute to pathogenicity, such as adhesion, hyphae formation,
and proteinase secretion, was assessed one at a time in vitro or in attenuated
variants. Some findings have demonstrated that the Candida–host interplay is
subtler than previously appreciated. Odds also emphasized that a panel of special-
ized virulence attributes may play a role at each stage of the infectious process.
Cutler (30) also made a special point in his thorough review on putative virulence
factors that no single factor accounts for virulence and that some, but not all,
virulence factors may be important at specific tissue sites. Similarly, Hube (31),
in his review on SAP genes and Sap isoenzymes, emphasized that C. albicans
pathogenesis is multifactorial. In fact, Odds et al. (32) recently concluded that
the pathological factors that ultimately led to the death of animals infected with
certain strains of C. albicans are unknown, but they were, for example, clearly
not absolutely dependent on the capacity of the infecting strain to form hyphae.
Although several genes have been suggested to encode virulence factors, there
has been a conceptual problem with defining pathogenicity genes. For example,
ura3/ura3 mutants are not pathogenic, and neither are most, if not all, mutants
defective in genes affecting growth (33). In this regard, strains with diminished
growth are rapidly cleared from the body. Although the pathogenicity of fungi
in general is poorly understood, there is a growing body of evidence that genes
expressed, for example, by phytopathogenes during starvation for either nitrogen
or carbon are also expressed during infection in plants (34–36). Some, but not
all, positive regulators of metabolic genes of phytopathogens were shown to be
directly implicated in pathogenicity (37).
Genetic Instability of Candida albicans 725
A striking example of importance of secondary carbon utilization for C.

albicans pathogenicity was recently published by Lorenz and Fink (38). After
ingestion in vitro of C. albicans cells by cultured mammalian macrophages, two
principal enzymes of the glyoxylate cycle that permits utilization of two-carbon
compounds, isocitrate lyase and malate synthase, were upgraded in the surviving
cells. The mutants lacking isocitrate lyase were markedly less virulent in mice
than the parental strain. The authors speculated that glucose-deficient environ-
ment of phagolysosome—which, however, is rich in fatty acids and their break-
down products (e.g., acetyl-CoA)—provides alternative nutrients, which can be
utilized for the synthesis of glucose through glyoxylate cycle. Some products,
such as acetyl-CoA, can be utilized only through glyoxylate cycle. The authors
concluded that isocitrate lyase is essential for full virulence, and that glyoxylate
pathway is necessary, but not sufficient for the virulence.
Because under regular circumstances the major factor for Candida infec-
tivity is undoubtedly the condition of the host, the ability of C. albicans to adjust
and to survive in a healthy host is as important as the action of so-called patho-
genic factors. In fact, C. albicans phenotypic diversity, with the rapid ability
to alter phenotypes, was recognized a long time ago as an important putative
contributing factor for virulence (39; reviewed in Refs. 28,30). The variable phe-
notypes represent a wide spectrum of the morphological, physiological, and bio-
chemical properties. Some important phenotypes include the resistance to antibi-
otic (40,41,42), the radiation sensitivity (43), the ability to assimilate different
nutrients (38,44), and the virulence in animal model (these and other phenotypes
are also reviewed in Refs. 26, 28, 45). We recently showed that adaptation to new
environment is achieved through a mechanism of adaptive mutagenesis, which
provides a dramatic increase in several order of magnitudes in “fit” mutants (re-
viewed herein). This study can be viewed as a model of an opportunistic pathogen
strategy to adapt to different niches in host.
Since the first report in 1990 of a large repertoire of frequent spontaneous
chromosomal alterations within the same population of Candida cells, two
hypotheses were introduced: (1) chromosomal instability is a means to gain ge-
netic variability in the absence of the more conventional way by meiotic segrega-
tion (46); and (2) chromosomal instability observed within the same population of
cells under laboratory conditions is a source of genetic and phenotypic variability
among isolates in nature (47). During the course of the last decade, we presented
evidence supporting these hypotheses (reviewed in Refs. 26, 48). A causal rela-
tionship between specific and different chromosomal alteration and different
stressfull environments, i.e., presence of secondary nutrient, antibiotic or other
toxine, was established (27,44,40; M. Wellington and E. Rustchenko, unpub-
lished data). Most important, we identified chromosome copy number, as pro-
vided by the loss or gain of a chromosome, as a novel means for regulating gene
expression in microbes. Because this novel regulation controls in C. albicans
important functions, we believe it may be a general regulatory mechanism in C.

albicans (44). For the sake of clarity, we will distinguish between natural variabil-
ity among independent strains and spontaneous instability observed within a par-
ticular strain. The genetic variability with emphasis on chromosomal instability
will be the main subject of this chapter.
II. MATING
The hypothesis of high-frequency chromosomal rearrangements that lead to ge-

netic and subsequently to phenotypic diversity in C. albicans was proposed 10
years ago (46) as a substitute for mating and meiosis. Recent experiments with
specially manipulated diploid strains demonstrated that mating could occur, and
may therefore function as a means of introducing genetic diversity in C. albicans.
With the demonstration that mating can be induced, we must now compare the
extent to which chromosomal instability and mating is used to generate diversity.
Mating was achieved by two different approaches. Johnson’s group (49)
analyzed the structures of a mating-type-like (MTL) locus, identified MTLa1,
MTLα1, and MTLα2 alleles similar to the Saccharomyces cerevisiae MAT genes,
and constructed hemizygous derivatives of the strain with either the MTLa1 or
the MTLα1 and MTLα2 alleles disrupted (50). On the other hand, Magee’s group
(51) eliminated either one of an opposite type of alleles, a or α, by passing an
original heterozygous C. albicans strain through L-sorbose plates. This treatment,
based on the study in C. albicans of the chromosome copy number regulating
utilization of L-sorbose (44), causes the loss of either one of two chromosome
5 homologues, carrying the opposite MTL allele in an otherwise diploid cell. The
loss of one homologue, however, can be compensated for by the duplication of
the remaining homologue, leading to homozygosity of the MTL allele, which
occurs after growth of cells on a rich medium. The frequency of duplication is
approximately 10⫺3 in strain 3153A, but varies with different strains (44). The
genetically manipulated strains of Hull et al. (50) did not mate on a plate under
the initial conditions tested, but mating occurred when the mixture of cells was
passed through a mouse by injection in the tail vein followed by the removal of
animals’ kidneys, which were homogenized and spread on a plate with selective
medium. A total of 12 out of 16 products contained a combination of two alterna-
tive functional alleles, a and α, and the two alternative disrupted alleles a and
α present in diploid parents. These results are consistent with mating of the two
parental diploid strains. The four other products lacked one MTL allele, which
could have occurred by the loss of one homologue of chromosome 5 or by the
homozygosis of the MTL locus after mating. The frequency of mating varied and
was not determined, but appeared to be low (A. D. Johnson, unpublished results).
On the other hand, Magee and Magee (51) achieved mating by mixing post-L-
sorbose cultures containing the opposite MTL locus directly on plates. The fre-
quency of the event was not estimated, although it was observed that longer
incubation (up to 8 days) and lower temperature increased mating, the latter being
consistent with the property of chromosome 5 to increase its instability about
one order of magnitude at room temperature (E. Rustchenko, unpublished data).
Southern blot analysis showed the presence of both a and α alleles in the products
of mating. Earlier we suggested that the unique karyotype of each C. albicans
strain creates a unique genomic condition (47; also reviewed herein), thus defin-
ing the phenotypic differences between strains. The expression of the genes im-
portant for mating may significantly differ in the different Candida strains, thus
diminishing the efficiency of mating. Consistent with this view, many genes have
been identified previously in C. albicans that are homologous to S. cerevisiae
genes encoding components of the mating pheromone response and meiosis path-
ways (e.g., GPA1, STE12, DMC1) (reviewed in Ref. 49). While some of the
sexual cycle homologues have been found to be expressed and functional, how-
ever, the expression of others has not been detected under laboratory conditions.
In this respect, it is important to note that Magee and Magee obtained mating
with Sou⫹ cultures from different strains, but not from the same one, with only
one tested strain being an exception.
In S. cerevisiae mating occurs with high frequencies between a and α
strains independently of the ploidy of cells. Hull et al. (50) suggested an explana-
tion of why Candida mating appears to occur only rarely, if at all, in nature. The
authors suggested that mating requires a and α strains that arise by homozygosis
of the MTL allele or by chromosome loss. The appropriate pairs of a and α cells
may simply arise only rarely in the same host. As suggested above, the ‘‘weak-
ness’’ of genes involved in mating, which requires the combination of different
genetic backgrounds, may add to the rarity of the appropriate strains, additionally
limiting mating. About 5–7% of C. albicans strains are homozygous for the MTL
allele (B. B. Magee, personal communication), which poses one more obstacle
to mating.
The current research focus of Johnson’s group is determining the role of
the MTL locus in the life cycle of C. albicans. An unusual aspect of MTL is that
in addition to three regulatory proteins (MTLa1p, MTLα1p, and MTLα2p), the
MTL locus contains three additional gene pairs never seen before in fungal mat-
ing-type loci: poly (A) polymerases, oxysterol binding proteins, and phosphatidyl
inositol kinases (49). This complexity implies some unknown functions of the
MTL locus. Recent comparison of the X-ray survival curves of the parental strain
3153A heterozygous by MTL and sorbose-positive derivatives of this strain,
which were represented by mix populations of hemizygous and homozygous by
MTL cells, showed that contrary to the expectation and by the analogy with S.
cerevisiae, the heterozygous parental strain did not have higher resistance to ion-
izing radiation (43). This result indicates that the normal heterozygous strains
may not have the properties of heterozygous diploid strains of S. cerevisiae de-
pendent on the dimer protein MATa1p-MATα1p, a product of the MAT locus
that induces the repair genes belonging to the RAD52 epistatic group (52). The
further analysis of the MTL locus will elucidate its presumably different functions
in C. albicans.
One observation of the mating products is relevant to studies of chromo-
somal instability. Hull et al. (50) established the absence of one allele of MTL
in some mating products, suggesting the loss of one copy of chromosome 5.
Similarly, Magee and Magee reported that not all the mating products contained
a full tetraploid DNA content, as established by fluorescence-activated cell sorter,
and also that some markers were lost. It remains to be seen whether or not specific
chromosomes alternate, as well as whether or not mating depends on these chro-
mosomes’ alternation.
Overall, mating does not seem to be a prevailing means for genetic variabil-
ity. As discussed herein, chromosomal instability was shown to be responsible
for the formation of such phenotypes as utilization of various nutrients or primary
resistance to drugs. We currently believe that chromosomal instability is the ma-
jor cause of phenotypic diversity in populations of C. albicans, although mating
may be responsible to a minor degree.
III. VARIABILITY AND INSTABILITY OF DIFFERENT

ASPECTS OF THE C. ALBICANS GENOME
The first reports on genomic variability among Candida natural isolates included
several aspects of the genome. Mitochondrial DNA was analyzed and reviewed
by Riggsby (53). Restriction-site polymorphism was uncovered as variations be-
tween homologous chromosomes in different C. albicans strains with the hybrid-
ization probes containing the markers ADE2, URA3, and possibly LEU2 (54, 55).
Differences were even observed between alleles at the URA3 locus in certain
strains. At the same time, the first separations of electrophoretic karyotypes were
providing an insight into a natural chromosomal variability (56–58). Subse-
quently, other aspects of the genome were shown to be variable, including, for
example, the number of rDNA, telomeres, and dispersed repetitive sequences,
thus providing a potential to control some still unknown phenotypes. The possible
mechanisms of genomic variability including for example, unequal crossing-over
or mitotic recombination, have been reviewed by Wickes and Petter (59). We
wish to emphasize that during the last decade most of the understanding of the
phenotypic diversity came from studies of chromosomal instability within the
same population of cells, which will be discussed in a special section in this
chapter. (See section on chromosomal instability.) Recently evidence was ob-
tained that C. albicans genetic instability, which results in chromosome copy
number differences, controls such important functions as utilization of secondary

carbon sources and resistance to the drug fluconazole, as well as a drastic increase
in the frequency of specific mutation under adverse condition.
A. Chromosomes, Ploidy, and Cell DNA Content

Initially the combined use of several methods, such as the measurement of the
total DNA content of a cell, reassociation kinetics, and UV survival curves, pro-
vided evidence for the basic diploid nature of C. albicans. This approach was
important before the introduction of the pulsed field gel electrophoresis (PFGE)
technique, a method that allowed the visualization of native chromosomes in the
form of ethidium bromide-stained bands on a gel, with each band representing
the bulk of a particular chromosome from a population of cells. The improvement
of the quality of separation permitted a comparative analysis of chromosomal
size and copy number, which was apparently a more discriminative approach than
measurement of DNA content. In some situations, as for example the hypothetical
ploidy shift, the electrophoretic karyotype may not be helpful in detecting the
chromosome copy number, and the determination of the DNA content has to
complement the analysis of chromosomal pattern. Another advantage of the use
of the chromosomal separation was the opportunity to apply Southern blot analy-
sis to analyze the differences in the distribution of genes over chromosomes.
In the early 1990s a substantial amount of results accumulated showing
that the majority of C. albicans strains contain a haploid number of eight chromo-
somes. This genomic organization is not rigid, however. Rearrangements of link-
age groups, aneuploidy, and multinuclei conditions were documented. The con-
tinuous improvement of the PFGE technique was instrumental in establishing
the haploid number of chromosomes and natural chromosomal variability among
laboratory strains, as well as spontaneous chromosomal instability of the same
strain.
Many earlier versions of pulse electrophoresis, as for example, PFG (pulsed
field gradient), FIGE (field inversion gel electrophoresis), Pulsarphor, TAFE
(transverse alternating field electrophoresis), or RGE (rotating gel electrophore-
sis), which have been reviewed in Refs. 60–62, are rarely used. A commercial
version of the CHEF (contour clamped homogenous electric field) (63), denoted
CHEF-DR and provided by Bio-Rad Laboratories (Hercules, California) is cur-
rently popular. The advantages of the CHEF-type system are the straight lanes
and the large number of samples that can be tested with one gel. One of the early
systems with a nonhomogeneous field, however, OFAGE (orthogonal field gel
electrophoresis) (64), produces sharper bands. It is worthy to note that the reliable
visual comparison of the amounts of DNA in bands, as allowed by the OFAGE
system, proved to be essential for the analysis of changes in chromosome number.
(See, e.g., Refs. 40, 44.) The present limits of resolution in OFAGE are approxi-
mately 600 bp (65), and probably can be further extended if needed, as it is a

matter of the combination of different parameters.
The chromosomes of C. albicans were denoted by at least five numbering
systems, as reviewed by Pla et al. (66). These systems differed in the use of either
roman numerals or arabic numbers, or letters, as well as in how the chromosomes
are numbered, either from the smallest to the largest or vice versa. We will use the
currently accepted nomenclature, in which the penultimate largest to the smallest
chromosome is designated by arabic numbers 1–7, whereas the largest chromo-
some, containing the ribosomal DNA cluster, is designated R. The following are
the equivalencies of one of the earlier and current nomenclatures:
Chromosome VIII VII VI V IV III II I

R 1 2 3 4 5 6 7
1. Natural Isolates and Laboratory Strains

Total DNA Content per Cell and Ploidy. The convincing evidence for the
diploid nature of C. albicans came from the sequence complexity, which was
measured as the reassociation kinetics of the total DNA. These included three
independent methods, the susceptibility of single-stranded DNA to S 1 nuclease
digestion, DNA separations with hydroxyapatite chromatography, and optical hy-
pochromicity (67). It was concluded that the unrepeated genome size is approxi-
mately 18 fg, which was approximately one-half of the cell DNA content (53).
The measurements of total DNA per cell using the standard diphenylamine
method supported this conclusion. Riggsby (53) comprehensively reviewed the
results at that time. These early estimates were corroborated by Rustchenko-
Bulgac et al. (46) using the diphenylamine method, and by Suzuki et al. (68)
using diaminobenzoic acid. The size of the genome was subsequently estimated
to be 16 to 17 Mbp by physical mapping (69). A definitive evaluation of the DNA
content will come from the entire genomic DNA sequence, which is essentially
completed and only recently available on the Web (70). At the time of this writ-
ing, it is estimated that C. albicans contains approximately 6,500 genes (S.
Scherer, personal communication).
The first indication that C. albicans contains a haploid number of eight
chromosomes came from Southern blot analysis of then still limited chromosomal
separations. One difficulty that had to be overcome was frequent deviation in
size of two homologous chromosomes, which appeared as two different bands
on the pulsed field electrophoresis gels. Some workers purposely chose strains
that lacked deviated homologues for use in representative studies, even though
such strains were exceptional. Using three laboratory strains and an elaborate
collection of markers, Lasker et al. (71) suggested that the haploid number of
Figure 1 The precise separation of the chromosomes of two C. albicans laboratory

strains, 3153A and SGY-243. Orthogonal-field alternating gel electrophoresis (OFAGE)
conditions were chosen to accentuate the separations of either: (a) only the bottom, B,
group; or (b) the bottom, B, and middle, M, groups of chromosomes. These conditions
of separation did not resolve the top group, T, of large chromosomes, which can be seen
schematically in Fig. 2. When two groups, B and M, were separated on the same gel,
there was less resolution in the B group, and as a result, some closely running bands in
strains SGY-243 co-migrated. Source: Adapted from Ref. 40.
chromosomes is eight. Two other reports, in which chromosomal separations

of the laboratory strains were clearer but the number of markers was limited,
corroborated the haploid number of eight chromosomes (46, 72). In one of these
reports, in which a strain with five pairs of deviated homologues, as well as its
mutants with spontaneously altered chromosome copy number and other alter-
ations were used, the authors demonstrated the variety of ways by which a diploid
parental strain could give rise to high frequencies of aneuploidy by either a single
or multiple changes in the chromosome copy number (46). This and still other
publications, in which more laboratory strains with improved chromosomal sepa-
rations were used, contributed further evidence for the predominantly diploid
state of the laboratory strains with some strains being aneuploid, as exemplified
in Fig. 1 and summarized in Fig. 2 (40, 44, 47, 48). As discussed below, the
discrepancy between relatively frequent spontaneous aneuploidy within the same
strain, and rare cases of the aneuploidy among natural isolates, remains to be
clarified.
Natural Chromosomal Polymorphism. Although the early PFGE-separat-
ing techniques did not fully resolve C. albicans electrophoretic karyotype, the
differences in chromosomal patterns among the strains were convincing. Two
groups, each using as an example five different laboratory strains, first pointed
Figure 2 Schematic representation of the chromosomal patterns of some popular C.

albicans laboratory strains. The following two chromosome numbering systems are pre-
sented: 1 to 7 and R; and I to VIII and homologues, a or b. Numeration presented on the
right refers solely to strain SGY-243. Dotted, thin, and thick lines correspond, respectively,
to one, two, and three or more chromosomes. The array of lines for chromosome R repre-
sents a cloud of inseparable weakly stained bands. Three groups of C. albicans chromo-
somes, bottom, B, middle, M, and top, T, can be precisely resolved singly or in various
combinations by several different electrophoresis conditions. (See, e.g., Fig. 1.) The sym-
bol 䊉 designates comigration of two to four homologues of two different chromosomes;
䉱 designates comigration of the following chromosomes: one homologue of 1, two homo-
logues of 2, and one homologue of the chimeric chromosome; and ❋ designates a chimeric
chromosome (see Ref. 47). The chromosomal assignment of SOU1 gene in strains 3153A
and CAF4-2 is indicated. The assignment of chromosomes to individual bands for refer-
ence electrokaryotype of 3153A and their approximate sizes, as well as the rationale for
identifying the eight pairs of chromosomes, have been previously published (46–48, 65).
The chromosomal assignment of FC18 and C9 with the 16 markers was previously pub-
lished (47). Chromosomes of strain SGY-243 were identified from the similarity of posi-
tions and intensity of bands to the reference karyotype of 3153A, as well as by assignment
of the seven markers (40). Chromosome copy number of strains SGY-243 and WO-1 was
corroborated by densitometry. The assignment of largely rearranged chromosomes of
strain WO-1 is not presented in this figure (see 47, 69), except for the R chromosome.
The karyotype of strain CA14, which is not shown here, was identical with CAF4-2, except
for chromosome R, which was represented by inseparable weak stained bands similar to
the chromosome R of strain SGY-243. The relatively precise estimate of the differences
between two homologous chromosomes is available for chromosome 7 in strain 1006, 20
kbp (134), in strain NUM114, 100 kbp (H. Chibana and P. T. Magee, unpublished results),
and chromosome 7 in strain CAF4-2, 10 kbp (Y.-K. Wang and E. Rustchenko, unpublished
results).
out the high natural chromosomal polymorphism of C. albicans (57, 58). Their
observation was supported by partial separation of four more individual chromo-
somal patterns of laboratory strains published by Suzuki et al. (73). At the same
time, Merz et al. (56) separated chromosomes of clinical isolates from 17 patients
and obtained 14 unique patterns. Undoubtedly a poor resolution of the long chro-
mosomes in the early separations was a limiting factor in establishing a unique
electrophoretic karyotype for each of the 17 strains. Lasker et al. (71) and Iwa-
guchi et al. (72) subsequently reported unique patterns of three and 27 more
laboratory strains, respectively, combining PFGE separation with the assignment
of the chromosomal markers. In a study of 100 clinical isolates by Monod et al.
(74), the chromosomal polymorphism was not emphasized, and was regarded as
a minor variation. Nevertheless, their work contributed to the understanding of
chromosomal variability. Asakura et al. (75) and Doi et al. (76) continued to
document natural chromosomal variability by providing statistics on chromo-
somal separations of approximately 160 clinical isolates and revealing that each
strain probably had a unique electrophoretic karyotype. At approximately the
same time, two independent groups addressed electrokaryotypic variations of the
laboratory strains. Rustchenko-Bulgac (47) analyzed four strains using improved
PFGE separation procedures and 16 chromosomal markers, and documented
varying sizes of the homologous chromosomes in different strains as well as
dramatic chromosomal differences in the popular laboratory strain WO-1. These
included multiple translocations, a large truncation of chromosome 5, and a re-
cently analyzed duplication of the longer homologue of chromosome R (D. H.
Huber and E. Rustchenko, unpublished data) (Fig. 2). In addition, Rustchenko-
Bulgac (47) found it difficult to explain the multiple comigrations of the non-
homologous chromosomes in this strain, which included combined groups of
linkages not present in the other strains, as well as common groups of linkages.
The SfiI map of WO-1 provided further details on the chromosome structures of
this strain (69).
Furthermore, Rustchenko-Bulgac (47) unexpectedly found that in spite of
the high karyotypic variability under the condition of lack of meiosis, the chromo-
somal patterns of different strains retained the same pivotal structure, which was
later confirmed by using more strains, as well as by other authors (77). For exam-
ple, as presented schematically in Fig. 2, the chromosomes in most of the strains
seemed to fluctuate around certain modal sizes. The range of sizes and the three
major groups of sizes—short, medium, and long chromosomes—remained ap-
proximately the same. This paradox still requires an explanation.
Thrash-Bingham et al. (77) used 22 markers to analyze six laboratory
strains and reported that three strains contained translocations, which were sug-
gested to be a natural source of variation in C. albicans. Perhaps more transloca-
tions would be uncovered with a larger number of probes. The same authors
hypothesized that translocations might arise by recombination between any of
several families of the repeated dispersed sequences; for example Rel-1, Rel-2,
27A, or Ca3 (77, 78). Iwaguchi et al. (79) suggested that the RPS1 sequence,
highly homologous to Ca3/27A, may be involved in chromosomal re-
arrangements and may in part explain chromosomal polymorphism. Finally, Chu
et al. (69) proposed that translocations have a tendency to occur at or near SfiI
sites within RPS1, and that such a mechanism may be a general means of generat-
ing translocations in C. albicans. It has to be noted, however, that the later hypoth-
esis was based on the analysis of three translocations from a single strain WO-
1, which has many unique features not observed in the other strains (47, 48), as
for example, two different sizes of the rDNA unit (65). In another strain 1006,
no translocation associated with SfiI sites was observed. It would be of interest
to determine if translocations occur at SfiI sites in other strains. The most recent
hypothesis attributed the chromosomal variability to the recombination between
retrotransposon-like sequence kappa (80), which is found in both CARE-2 and
Rel-2 repeats. The suggestion that repeats like Ca3/27A, RPS1 or kappa may be
the source of chromosomal variability does not agree with the analysis of mutants
with various spontaneous chromosomal alterations. When DNA digests of these
mutants were hybridized with a Ca3 probe, their signal patterns were no different
from the parental strain (46, 81; see also section III.C).
One more rare condition of the electrokaryotype can be illustrated with the
example of strain SGY-243, which has a banding pattern that has more complex-
ity than in the other strains in that it contains six additional chromosomes in the
positions of chromosomes 4, 5, 6, and 7. The chromosome R in this strain is
represented by an inseparable cloud of weakly staining bands instead of two
homologues (Fig. 2). This type of diffused band has been described previously
for chromosome 6 in strain 300 (48), and indicates a cumulative karyotype of a
highly heterogeneous population of corresponding molecules. Apparently there
is some poorly understood high instability in the genome that is limited to a
single chromosome. Although the instability is detectable on a gel and this ap-
proach reveals various sizes, it somehow fails to determine possible changes in
the copy number of the corresponding chromosome, which can vary in different
cells. Another kind of single chromosome instability leading to what can also be
called a cumulative karyotype, occurs when the band on a gel is represented by
a different number of the homologues of the same size in a mixed population of
cells. In this situation, the corresponding band has an intermediate brightness
between, for example, single and double copies (48).
The multiple aneuploidy exemplified by the strain SGY-243 could be either
natural or induced by exposure to UV light (82) formed under laboratory cultiva-
tion, or acquired by improper storage. (See, e.g., Refs. 46, 48.) In this regard,
three chromosomal alterations—the loss of two nonhomologous chromosomes
and the change in the length of chromosome R, which occurred in strain WO-1
during regular maintenance in the laboratory—is significant (83). One possible
explanation could be a subcloning of the culture after removal from the ⫺70°C
freezer. This method, when applied to Candida, often leads to the selection of
a mutant with altered chromosome R (47; E. Rustchenko, unpublished results).
For example, we currently have two C. albicans CAI4 strains received from dif-
ferent laboratories. These strains differ by the appearance of their chromosomes
R. Another example is strain 3153A, in which alteration of several chromosomes
occurred. Strains 3153A and 300 were originally derived from the same strain, but
after maintenance and preservation in different laboratories exhibited phenotypic
differences. Identical patterns of restriction fragments revealed with the Ca3
probe confirmed their common identity, however (48). These two strains were
compared for their electrophoretic karyotypes and three differences were found
(48), a result that is consistent with a number of differences in assimilating pro-
files of these strains (26). Because of the unstable condition of one of the chromo-
somes (see above), 300 was assumed to be an unstable mutant of 3153A. The
mutagenic genetic manipulations, such as transformation, could also affect the
chromosomal pattern by producing chromosomal instability, as may be the situa-
tion for CAI4 and SGY-243. The implication of supposedly genetic manipula-
tions was independently noted by several groups that used different strains (84;
F. Navarro-Garcia, L. Monteoliva, unpublished data). Larriba and colleagues re-
ported alterations of size of chromosome R due to gene disruption procedure, as
well as due to the exposure to 5-fluoro-orotic acid (5-FOA) (85), which is a
required step when two copies of gene are disrupted using the URA3-blaster.
We recently found that short exposure of 24 hrs to 5-FOA induced nonspecific
chromosomal alterations. The prolonged exposure, however, resulted, on one
hand, in the death of the majority of the cells, and on the other hand, in the
formation of 5-FOA-resistant mutants having specific alterations of either chro-
mosome 5 or chromosome 4 (M. Wellington and E. Rustchenko, unpublished
data). Although transformation procedure itself is also considered mutagenic by
many researchers, a reliable experimental proof has not been presented. In the
work of Ramsey et al. (86) using Southern blot analysis, UV light was shown
to induce instability of chromosome R in strain 3153A. The poor separation did
not allow an evaluation of the other chromosomes, however.
So far only a few cases of aneuploidy of 2n ⫹ x type have been observed
in laboratory strains, but none in clinical isolates. Nevertheless, it is premature
to conclude that this alteration is less frequent in a natural environment, and is
preferably occurring, for example, in the laboratory condition. As we recently
showed, the cultivation of the monosomic cells in a rich liquid medium selects
for the genetically balanced diploid of the 2n type (44). One explanation for the
lack of aneuploidy in clinical isolates thus can be simply the selection of a bal-
anced diploid state after maintenance in the laboratory. It is still possible, how-
ever, that laboratory strains, which may have been converted to aneuploidy, such
as WO-1 or SGY-243, have acquired an effective gene balance. Another explana-
tion could be a lack of resolution in the PFGE in early separations, which pro-
vided most of the documentation on the electrokaryotypes of clinical isolates.
This conclusion needs further clarification.
Overall, the comparison of a large number of C. albicans strains (72, 75,
76) established that there is a high degree of variability of every chromosome.
Frequent differences include deviated homologues of the same chromosome,
which can be due to either a deletion or a translocation. The deletion can be
identified by Southern blot analysis as lacking one or more chromosomal markers
on the shorter homologue (40, 87), whereas the translocation would give two
signals with two nonhomologous chromosomes (44, 46, 47, 77). As highly ho-
mologous repetitive sequences Ca3/27A and RPS-HOK-RB2—also called major
repetitive sequence (MRS), representing probably the same large segment—were
shown to vary in size and copy number per chromosome within the same strain
and between different strains (H. Chibana, unpublished data), these repetitive
sequences have to be considered as one of the sources of chromosomal polymor-
phism as well. The combination of several different events also has to be consid-
ered, although the current methods reveal only the final result of rearrangements,
and not the sequence of events. Obviously, electrokaryotyping and Southern blot
analyses are crude methods that probably detect only a portion of structural
changes in chromosomes. For example, an inversion, a small deletion or insertion,
as well as sequence changes may remain undetected. It is important to note that
because the native chromosomes are prepared from a cell mass, the cumulative
chromosomal pattern is produced on the PFGE gel. Only the electrokaryotype
representing the major portion(s) of the cells is visualized by staining, however,
and underrepresented chromosomal patterns are not observed.
It can be considered at this time that reports on two identical electrokaryo-
types of independently derived strains have not been published. In light of this
knowledge, it is important to correct the notion that some strains have ‘‘typical’’
and others have ‘‘atypical’’ electrophoretic karyotypes. A correct view would
be that each strain is represented by an individual karyotype. Another popular
misconception is to regard different strains as phenotypically uniform. In fact,
strains differ by multiple phenotypes, including, for example, the appearance of
streak culture or colonial appearance and assimilation profile (26, 45, 47, 48, 88,
89), to mention a few, in accordance with their unique genomic condition.
The complex relationship between stress and chromosomal instability, pos-
sibly leading to the diversification of strains, is just beginning to be understood.
(See Sec. III, ‘‘The Role of Chromosomal Instability in Adaptation to the Chang-
ing Environment.’’)
In summary, there are many results indicating that chromosomal polymor-
phism arises from different genomic rearrangements. The recent accumulation
of data suggests an important role of environmental stresses in chromosomal
variability. The mechanisms producing this variability are still obscure, however,
as is the mechanism(s) of strain diversification.
2. Spontaneous Mutants
Instability in Historical Perspective. The study of the C. albicans instabil-
ity was initiated in 1935, when Negroni (90) reported a ‘‘rough’’ variant, as
opposed to a ‘‘smooth’’ or ‘‘normal’’ one, with micro- and macromorphological
differences, as compared to the parental population under the same condition. A
series of similar reports followed, describing the individual morphology of either
the colonies or streaks of the mutants derived from the same strain (reviewed in
Ref. 45), as well as sectors developed within giant colonies (91). Soon after it
became obvious that a large number of possible morphological forms can arise
in the same population (45, 92). Early workers, however, attempted to produce
simplified classifications, and either did not analyze large collections of mutants
or did not report the full extent of the variations, as they tended to consider only
some types but not all forms. This approach systematically led to the assumption
that a certain defined number of colonial morphologies were produced from a
given strain. The extreme situation of this kind was probably that reported by
Slutsky et al. (39), who studied colonial morphology of mutants in lightly UV-
irradiated population of cells of strain 3153A, and who assigned colonial forms
to just seven types. On the other hand, Rustchenko-Bulgac (47) showed that the
same strain 3153A spontaneously produced a very large number of the colonial
morphologies, and the frequency of recovery of the particular form probably
depended on the rate of growth of the corresponding mutant. The comparative
study of a number of Candida species, including a total of 100 strains, suggested
that colony instability is a common property among isolates of many Candida
species (93). Early workers also systematically described different combinations
of multiple changes in other features related to these morphological mutants,
including traits used for taxonomic assignment, such as germination or carbon
utilization, and traits believed to be correlated with putative pathogenic factors,
such as adhesion and colonization, as well as virulence for laboratory animals
(reviewed in Ref. 26). Despite a substantial amount of data on phenotypic vari-
ability in morphological mutants, no underlying mechanism was proposed (re-
viewed in Refs. 28, 45, 91). From today’s prospective, the main result of the
early studies was a comprehensive documentation of multiple altered phenotypes
associated with a change in macromorphology.
It was also previously observed that much higher frequencies of various
colonial forms could be recovered from the oral cavity of patients than from
healthy carriers (94). The author discussed whether the altered morphologies oc-
curred in the oral cavity or were induced by the transfer. Subsequently, using
the Ca3 probe, the question of whether different morphologies belong to the same
strain and occur as a result of the instability or represent different strains in the
same patient, was addressed (95–97). At that time, however, the high instability
of C. albicans related to environmental stresses was not taken into account, and
care was not taken for protecting cells from low temperature, aging during stor-
age, or working with the whole population of cells versus traditional subclon-
ing.
For the purpose of this review, it is important to note that early workers
often reported the increased frequencies of morphological mutants under stressful
conditions, such as excessively alkaline medium, immune serum, lithium chloride
(94), aging (45, 98), cold shock, heat shock, UV light, or benzazoles (99). More
recently two additional significant stressful factors were suggested. Jones et al.
(100) obtained data on the increased instability of colony morphology in strains
recovered from patients with the invasive infection versus strains isolated from
superficial infection, and Odds (101) pointed out the gravity of general changes
in the environment, which Candida experiences after transfer from the human
microniche to a laboratory plate. We would like to note in this connection that
among the multiple differences between these two milieu (i.e., in vivo versus in
vitro), the excessive amount of primary sources of carbon and nitrogen under
laboratory conditions is an obvious contrast that would be expected to produce
changes in the expression of numerous genes. (See, e.g., Refs. 36, 102.)
The scope of the colonial instability and its possible relation with virulence
was the subject of some speculations, whose main idea was that multiple change-
able phenotypes serve for adaptation, as summarized by Odds (101).
Currently the prominent phenotypic instability in C. albicans is explained
by the high frequency of chromosomal instability. The series of papers by Rust-
chenko et al. (26) and Rustchenko-Bulgac et al. (46, 48) established a link be-
tween colonial appearance, multiple associated phenotypes, and alterations in
electrophoretic karyotype. Unstable and highly unstable morphological mutants
were shown to have correspondent levels of chromosomal instability (47). Karyo-
typic alterations consisted of a large repertoire of single and multiple changes,
as documented with the following C. albicans strains: 46 mutants from 3153A,
307 and 310; one mutant and its several phenotypic ‘‘revertants’’ from NUM961;
and 15 mutants from ATCC 32077 (reviewed in Ref. 26; M. J. McEachern, un-
published data). It is important to note that we never encountered a situation in
which the electrokaryotype of a morphological mutant was not altered. Some
mutants altered solely in chromosome R are discussed in ‘‘Chromosome R Insta-
bility’’ below. The simplest assumption is thus that the morphological changes
and the multiplicity of the related phenotypes are caused by chromosomal alter-
ations due to changes in dose or level of expression of a large number of genes.
Presently, differently appearing colonies can serve as a convenient means to iden-
tify chromosomal instability.
Because important physiological functions, such as the utilization of differ-

ent secondary nutrients, was associated with chromosomal instability manifested
by various chromosomal alterations, it was concluded that C. albicans developed
an unusual but effective means for controlling gene expression (26). This is in
direct support of the earlier hypothesis that chromosomal alterations in Candida
act as a means to achieve genetic variability (46).
Although recent findings on the increased frequency of chromosomal non-
disjunction during adaptation directly support early ideas on the scope of morpho-
logical instability, the role of instability in infection requires a more careful analy-
sis. In this connection the uncertainty of the contribution of instability through
the handling of clinical samples needs to be clarified.
Chromosomal Instability. Both Suzuki et al. (68) and Rustchenko-Bulgac

et al. (46) initiated the analysis of spontaneous chromosomal instability. Suzuki
et al. (68) found a different looking colony, which was called a semirough variant,
among approximately 3 ⫻ 10 5 colonies of a parental strain. When cells from the
variant were subcloned, ‘‘revertants’’ to the original colonial appearance were
derived with a high frequency of 5 ⫻ 10⫺3. The parental strain, semirough variant,
and revertants were compared for the cell DNA content, the production of acid
proteinase, and for their electrophoretic karyotypes. Of approximately 10 re-
solved bands in their chromosomal patterns, the sizes of five did not change in
any of the strains, whereas the remainder varied. The suggested interpretation of
the mechanism of chromosomal instability was based on the observation of multi-
ple spindles in the nucleus of the semirough mutant and changes in the DNA
content in the mutant and some of the revertants. It was assumed that chromosome
reorganization in the derivatives of the original strain was coupled with the ploidy
shift. It was also suggested that chromosomal instability plays a role in natural
chromosomal polymorphism. Similarly, Rustchenko-Bulgac et al. (46), who stud-
ied spontaneous chromosomal instability with numerous morphological mutants
or variants, found one mutant with an approximate twofold increase of DNA
content. This finding could be explained, however, by the combined effect of the
presence of 2–5% dikaryotic cells in the population with multiple chromosomal
duplication. Although the authors also speculated that multiple chromosomal du-
plication could be the result of polyploidization followed by partial restoration
of the original ploidy, the hypothesis that ploidy shift was responsible for the
chromosomal rearrangements was neither rigorously proved nor dismissed.
The rich YPD medium used by Suzuki et al. (68) is for the most part unable
to distinguish morphological mutants. For example, some mutants give rise to
smooth colonies on YPD medium, but rough ones on LBC medium. Similarly,
the laboratory strains have identical-appearing colonies on YPD medium, but
have individual morphologies on LBC plates (47). It is likely that the semirough
variant was a highly unstable mutant dissociating into various morphological
forms, similar to the many mutants analyzed by Rustchenko-Bulgac (47). These

mutants are known to segregate large numbers of the new colonial forms, as
presented in Fig. 3, each of which possesses unique spontaneous changes in their
electrokaryotype.
Rustchenko et al. (46, 47) obtained a collection of 384 spontaneous colony
form mutants or so-called morphological mutants from five separate cultures,
each derived in turn from a separate clone of the original strain. This procedure
ensured that mutants arising from separate clones would be the result of indepen-
dent mutational events, and not arising from siblings. The mutants were detected
on a glucose-containing synthetic medium, LBC (103), due to their unusual look-
ing colonies, which included a wide variety of shapes and sizes (46–48), and
sometimes, after repeated subcloning, segregated colonies of opaque, yellow,
brown, or dark red colors (E. Rustchenko, unpublished results). It is of interest
to note that some mutants with small colony size analyzed earlier by Bianchi
(104) had slow rates of growth, but were not, for example, ‘‘petite’’ mutants
having mitochondrial defects. The coloration was due to unknown factors, and
was not due to auxotrophy (E. Rustchenko, unpublished results 1989). The spec-
trum of morphologies produced by each of the five original clones included some
common forms, as well as unique forms. Overall each clone seemed to display
an individual spectrum of morphologies, which might indicate that clones in C.
albicans populations have unique genomic conditions. In order to corroborate
that the mutants derived from the same strain and to exclude contamination, 14
of them, representing all five original clones and having the most rare morpholog-
ies, were analyzed with the molecular probe Ca3 (105). These results revealed the
authenticity of the original strain 3153A and fourteen mutants (46). The mutants
appeared at approximate frequencies of 3.1 ⫻ 10⫺4 to 1.4 ⫻ 10⫺2. Mutation rates
were not determined, however, and a quantitative analysis needs to be undertaken.
As it became gradually evident, multiple factors, especially temperature, aging,
or nutrient depletion, could influence the frequency of mutation (46, 48). For
example, the highest mutation frequency of 1.4 ⫻ 10⫺2 was obtained after pro-
longed incubation at 4°C. The data on the role of aging or low temperature are
consistent with the reports by early authors on the increased frequencies of mor-
phological mutants in aged cultures (45, 98) or after cold shock (104). More
examples of the environmental factors inducing Candida instability are described
in section III.A.2a. A subset of 223 mutants was analyzed for stability of their
colonial morphologies by repeated subcloning, and it was found that one-third
of the mutants were unstable or highly unstable, segregating different colonial
forms in an individual manner (46). Some of the instability patterns were reported
in detail (47).
A systematic study of a total of 46 colony morphology mutants revealed
that each electrokaryotype was individually altered, containing a broad range of
single or multiple chromosomal changes, which resembled the natural karyotypic
Figure 3 The appearance of colonies of two unstable morphological mutants, m504

(top) and m505 (bottom), derived from independent original subclones of C. albicans
strain 3153A. The LBC plates were incubated at 23°C for 3 weeks.
variability among laboratory strains and clinical isolates described above in Sec.
‘‘Chromosomal Polymorphism’’ (46–48). Spontaneous changes included aneu-
ploidy, deletions, translocations, and variation in the length of the rDNA cluster
and sometimes complex rearrangements of the whole chromosomal pattern. We
wish to note that aneuploidy was mostly of a type 2n ⫹ x, with only two instances
of 2n ⫺ x type reported in early studies (47). Subsequently it became clear that
growing spontaneous mutants with a reduced chromosome copy number in a rich
liquid medium leads to selection of the compensatory duplication of the re-
maining homologue (44). The corresponding cells have higher rates of growth,
thus overgrowing other cells in the culture. It is reasonable to assume that the
spontaneous loss and gain of a chromosome is based on nondisjunction, in which
both events occur with the same probability. The future use of controlled growth
conditions may further demonstrate the role of selection.
Multiple changes, which included either chromosomal duplications or a
combination of different kinds of alterations, occurred in one-half of the mutants
at frequencies higher than expected for independent single events. Multiple alter-
ations can result either from a special mechanism, which affects the stability of
several chromosomes at once, or accumulate during subcloning due to a high
frequency of single sequential alterations. Ploidy shift, followed by a partial resto-
ration of the diploid state by the reduction or duplication of some chromosomal
copies, can also be considered as an explanation of multiple aneuploidy.
The observation of unstable and highly unstable mutants adds to the com-
plexity of the phenomenon of spontaneous chromosomal alterations. Of two such
unstable mutants analyzed in more detail, both gave rise on subsequent replating
to mutants that had diverse colony morphologies and unique chromosomal pat-
terns (47). In addition, these two unstable mutants differed from each other by
producing different arrays of morphological mutants. Some—but not all—of the
mutants subsequently stabilized after subcloning, a property reminiscent of mo-
bile elements. It was demonstrated that in some cases a single spontaneous mutant
could continuously produce an overwhelming range of altered karyotypes, re-
sulting in a wide variety of different phenotypes.
Because at the time of initiation of the studies of the electrophoretic karyo-
types, Candida was not known to mate and to possibly produce genetic variabil-
ity in a conventional manner, high-frequency spontaneous chromosomal re-
arrangements were suggested to be a substitute for generating new phenotypes
in populations (46), and thus to be a source of natural variability (47). As mating
was recently demonstrated with Candida diploid strains, which were either genet-
ically manipulated or ‘‘forced’’ to mate, we evaluated the possible role of this
process in the Candida life cycle in the section ‘‘Mating.’’ We came to the con-
clusion that although the mating can occur at a low frequency, thus contributing
to genetic variability, the chromosomal rearrangements remain the prominent
means for introducing the diversity. Consistent with this view, a large variety of
phenotypes was found to be associated with spontaneous chromosomal alter-
ations. Some features, such as cellular or colonial morphology, were always
changed, although they did not appear to be advantageous for the organism. Some
other traits seemed to represent either destroyed or diminished functions; for
example, the inability to form germ tubes or chlamydospores, or the diminution of
adherence or virulence that appeared in some but not all mutants. The phenotypic
changes were usually multiple, and arose in different combinations, reflecting
large genomic changes due to chromosomal rearrangements.
A particular class of phenotypic changes was especially significant. A study
of utilization of 21 carbon and three nitrogen sources at three different tempera-
tures in more than 100 spontaneous mutants showed multiple changes in their
assimilation profiles compared with the parental strain (26). The differences in-
cluded both the gain and loss of multiple assimilation functions, as well as tem-
perature dependence, with an almost endless array of new combinations. Each
of the spontaneous mutants had a differently altered chromosomal pattern and a
different assimilation pattern, a finding that established for the first time a rela-
tionship between chromosomal alterations and vital functions.
The natural diversity among C. albicans strains in utilization of different
carbon sources is well documented. (See, e.g., Ref. 89.) The repertoire of chromo-
somal differences, and the related differences in assimilation profiles in naturally
occurring strains and spontaneous mutants is similar, consistent with the proposed
view that natural chromosomal variability results from chromosomal instability.
Nevertheless, diversification under laboratory conditions is considerably less pro-
nounced than that observed among different naturally occurring strains. For ex-
ample, probing with the repetitive sequence Ca3, as well as other repetitive se-
quence probes, clearly distinguishes among independent strains, but fails to reveal
differences between the parental strain and spontaneous or selected mutants, in
spite of karyotypic changes. (See section C on regular repeated sequences.)
Scherer and Stevens (106), however, reported that probing with 27A, which is
identical with Ca3, showed one instance of polymorphism during laboratory sub-
culture. Also, Franz et al. (107) observed slight differences in series of geneti-
cally related clinical fluconazole-resistant isolates tested with epidemiological
probe CARE-2.
In summary, genetic instability is a means to achieve a wide range of pheno-
types within a population. The available data provided an insight on ‘‘regular’’
diversity within populations. Perhaps a certain permanent level of instability
within a population can be viewed as a resource of preadapted variants, which
will be ready to proliferate in different environments. Although extensive results
on spontaneous instability have accumulated, and some means of instability were
elucidated, its mechanisms are still unknown.
Chromosome R Instability. Sadhu et al. (105) were the first to report that
the chromosomes carrying rDNA fluctuate in size at a very high rate from one
colony to another in the same strain. Iwaguchi et al. (108) subsequently reported
that the alteration of chromosome R occurred in approximately 10% of the sub-
clones in the same population and is due to different lengths of rDNA clusters.
Rustchenko et al. (65) reported a physiological condition that controls this type
of instability. In a slowly growing population, chromosome R of all the subclones
were identical. In a rapidly growing population the distribution among subclones
was shifted to an increase in the number of rDNA units. The authors suggested
that there normally is a distribution in the number of rDNA units per cell in
population due to unequal crossing over (109) or gene conversion within the
rDNA cluster during mitotic growth (H. Zou, S. Gangloff, and R. Rothstein,
unpublished results), as seen with S. cerevisiae. Perhaps a certain optimum num-
ber of rDNA units exists for every growth condition. Enrichment of a small num-
ber of cells with the appropriate number of rDNA units is expected after a shift
to a different growth condition. In other words, the representative karyotypes of
a cell population can be controlled by growth conditions.
The comparison of spontaneously altered karyotypes in morphological mu-
tants showed that chromosome R, which carries rDNA, was at least twice as
unstable as any other chromosome (47). More detailed analysis demonstrated that
in approximately 92% of cases the change in the length of chromosome R resulted
from a change in the length of its rDNA cluster (60). The other rearrangements
can be exemplified by a mutant with three chromosomes that each carries an
rDNA cluster. One of the chromosomes was about 700 kb shorter in the region
outside the cluster and failed to hybridize with four genes available as probes for
chromosome R. This rearrangement can be interpreted either as a large deletion of
the additional third copy of chromosome R or a transposition or translocation of
the entire rDNA cluster to another unknown duplicated chromosome.
There is an apparent difference between rDNA instability in regular sub-
clones of normal strains, which preserve the parental colonial morphology and
the instability of rDNA in morphological mutants. For example, approximately
one-third of the morphological mutants analyzed by Rustchenko-Bulgac (47) had
alterations only in chromosome R, even though there were distinct changes in
colonial morphology (46, 47). Also, the rDNA cluster lengths in morphological
mutants had a wide distribution under growth conditions, which produced no
instability among the regular subclones (Fig. 4). Although it is unknown if the
differences in rDNA cluster length are directly responsible for the phenotypic
changes in morphological mutants, it is reasonable to assume that rearrangements
occurring in their rDNA cluster differ in that they lead to the altered expression
of rDNA or other genes. In fact, morphological mutants can arise as a result of
the insertional-deletional events affecting all or a certain portion of units similar
to many cases of rDNA rearrangements in other organisms described below. (See
Figure 4 The comparative distribution of the deduced number of genomic rDNA units
in different morphological mutants and subclones from population of C. albicans 3153A
that were grown under the same conditions: (A) Morphological mutants m3, m7, m16,
m17, m20, m500, and m500-3; (B) Random subclones C.a.1 to C.a.10. The number of
subclones with the designated number of rDNA units in shorter homologue of R (open
bars) and longer homologue of R (filled bars) are indicated. The number of rDNA units
was deduced from the size of a single unit and the lengths of HindIII fragments that
encompassed tandem rDNA units. The strains were grown on LBC plates at 22°C for 4
weeks. Source: Adapted from Ref. 65.
Sec. III.B.2, ‘‘rDNA Unit Size and Number of Units in Genome.’’) One of these
examples is the regulation of drug resistance in Candida (41, 42). In contrast,
rearrangements in rDNA cluster occurring in the subclones of a population may
be caused by looping out of a portion of the cluster or unequal crossing-over,
which changes the number of units in the cluster but not their structure. Such
a type of innocuous change is expected to affect overall protein synthesis, but
presumably not expression of specific genes.
B. Regular Repetitive Sequences

Similar to other lower eucaryotes (110, 111), about 13% of the C. albicans ge-
nome is identified as repetitive sequences. Regular repetitive sequences, which
are present in all cells as rDNA, mitochondrial DNA, or telomeres, are a source
of natural variability in many microbes, including C. albicans. In many lower
eukaryotes, this variability is associated with some important regulatory functions

in addition to the normal cellular functions of these sequences or organelles.
Examples include age-associated accumulation of rDNA circles, which are re-
leased from the cluster of chromosomal rRNA genes in S. cerevisiae (112), and
senescence in fungi of the genus Neurospora, which is determined by the integra-
tion of plasmids in mitochondria (reviewed in Ref. 113).
1. Telomeres
C. albicans telomeres were analyzed by McEachern and Hicks (114), who cloned
them as a middle repetitive sequence designated Ca7. The telomeres consisted
of unusually large and not so common repeats of 23 bp, which superficially re-
sembled the mitochondrial telomeres of members of the genus Tetrahymena more
than other known chromosomal termini. This result places C. albicans in a group
of budding yeasts such as C. tropicalis and Kluyveromyces lactis that also have
large telomeric repeats (114, 115). The telomeric repeats had a remarkable unifor-
mity among different strains, as well as clones from the same strain. In marked
contrast, the adjacent subtelomeric DNA sequences were the subject of interstrain
variability in the form of frequent deletions and changes. Although overall the
telomeric repeats were stable, a physiological condition controlling their instabil-
ity in a population was identified. Their number increased when cells were grown
at a higher temperature. The functional implication of the temperature-controlled
number of telomeric repeats is not known.
2. rDNA Unit Size and Number of Units in Genome

rDNA units exist as a cluster of multiple repeated copies in the genomes of the
majority of eukaryotes. The variation in rDNA cluster length and composition
were reported in earlier studies of S. cerevisiae and many other organisms. The
mechanisms involved were reported to be unequal crossing-over (109), gene con-
version during mitotic growth (H. Zou, S. Gangloff, and R. Rothstein, unpub-
lished data), short deletions, and insertions at regular intervals within the nontran-
scribed spacers of rDNA units (116), as well as the insertion of a single large
segment of DNA (117) or Ty element (118). It is noteworthy that rDNA can be
a recipient of mobile elements in different species. Xiong and Eickbush (119)
reported the precise insertion of functional retrotransposons that inactivated a
fraction of 28S RNA genes in Drosophila melanogaster and Bombyx mori. The
insertions of mobil group I intron Pp LSU3 in all rDNA units followed the cross
between intron-bearing and intron-lacking strains of the slime mold Physarum
polycephalum (120). Taken together these results suggest that the rDNA cluster
of many different organisms is a site of dynamic rearrangements that can control
the expression of the genes.
Genomic rDNA from C. albicans has been studied less than that from S.
cerevisiae. The results obtained so far, however, are indicative of the similarly
dynamic nature of rDNA cluster in C. albicans and of its high potential for pro-
ducing new phenotypes. A total of 120 clinical isolates of C. albicans were stud-
ied for the presence of the group I intron in the large subunit 25S rDNA precursor
molecule, which was subsequently denoted CaLSU (41). About 40% of the
strains harbored CaLSU in each copy of their 25S rDNA coding sequences. In-
tron-bearing C. albicans strains all exhibited a high degree of susceptibility to
antifungal drugs, 5-fluorocytosine, or 5-fluorouracil. The other groups demon-
strated the heterogenous condition of rRNA genes, with and without CaLSU in-
tron, in some of C. albicans strains (42, 121, 122), as well as sensitivity of the
intron-bearing strains to other antibiotics, pentamidine (42), and flucytosine
(122).
The variation in the number of C. albicans chromosomal rDNA units was
first analyzed with HindIII digest of genomic DNA in unrelated studies on the
natural chromosomal variability (123). Currently the number and organization
of rDNA units can be conveniently and accurately assessed by digesting either
agarose blocks or agarose beads with the intact chromosomes with restriction
endonucleases that do not cut within the rDNA unit, as for example HindIII and
XhoI, followed by examining the length of the fragments with PFGE (48, 108).
On the other hand, a restriction enzyme such as NotI, which cleaves at a single
site within an rDNA unit, allows the determination of the size of the single unit
(108). Subsequently the data from two groups demonstrated the large variability
in sizes of the clusters from different laboratory strains (65, 108). The estimated
number of rDNA units can vary between 9 and 176 per cluster (65, 108), thus
determining the cluster size and ultimately the size of chromosome R.
The size of a single unit is also variable. Rustchenko et al. (65) determined
two different units, 12.2 kbp and 11.6 kbp, in three different strains. It is interest-
ing that not only laboratory strains differ among themselves by the length of their
rDNA units, but also that a single strain can contain two different sizes of rDNA
units, as exemplified by WO-1, which contains 11.5 and 12.5 kbp units in approx-
imately equal proportion. Iwaguchi et al. (108) also reported two different sizes
among three subclones of the same C. albicans TCM297 laboratory strain. The
difference in the ranges of size reported by two groups, however, 14.3 to 15.2
kbp (108) and 11.5 to 12.5 kbp (65), was relatively large and could not be easily
attributed solely to the differences between strains, but may have been due to
differences in the methods of measurement. There are preliminary results on the
sources of differences in unit sizes, which probably do not include all possible
sources of the variability and instability. Mercure et al. (41) found a 379-bp group
I intron, denoted CaLSU, in a highly conserved region, 25S nuclear rRNA-encod-
ing gene, in which most large ribosomal subunit introns of other organisms have
also been mapped. The presence or absence of the intron is reflected in a well-
characterized EcoRI digest banding pattern, as a 4.2 kbp band or 3.7 kbp band
dimorphism, respectively. It is interesting to note that the CaLSU intron does

not closely resemble other introns in these generally conserved regions, which
makes it unusual. The question of whether or not CaLSU might be a mobile
sequence has not been addressed. On the other hand, the study of the 25S rRNA
in two C. albicans strains revealed the difference of 3361 bp versus 3363 bp in
length, as well as seven nucleotides substitutions (124). These differences cannot
account for the differences in rDNA units mentioned above.
3. Extrachromosomal rRNA Genes

The presence of rRNA genes on extrachromosomal molecules has been reported
in organisms throughout the phylogenetic tree, including circular or linear plas-
mids in lower eukaryotes (reviewed in Ref. 125). Recently extrachromosomal
copies of rRNA genes were found in two types of plasmids of C. albicans, which
had circular and linear forms, and which coexisted in laboratory strains (125).
The number of extrachromosomal rRNA genes varied with the growth cycle. The
copies of the linear plasmid per cell accumulated in abundance in actively grow-
ing cells, and strongly declined during the stationary phase of growth. It was
suggested that the total copy number of rDNA units in C. albicans cells is con-
trolled in part by variation in the copy number of the linear extrachromosomal
plasmid. It was also suggested that rapid change in the total number of rDNA
copies is important for adaptation, and ultimately, better survival in the mamma-
lian host.
The preliminary data indicate that the copy number of extrachromosomal
rRNA genes may be regulated and not determined simply by selection. The varia-
tion of rRNA genes in C. albicans is reminiscent of the well-known cases of
physiologically related amplification of the cellular copy number of rDNA in
other organisms (reviewed in Ref. 125).
4. Mitochondrial DNA
The mitochondrial genome of C. albicans is circular with a contour length of
approximately 41 kbp, thus representing a middle-size range for fungi (126–
128; see also 70). The restriction site polymorphism of mitochondrial DNA was
analyzed in 300 strains of C. albicans, and the variability found was less than
expected by comparison with similar analysis of S. cerevisiae (53). It was sug-
gested that the inverted repeat of 5 kbp in C. albicans mtDNA was acting as a
stabilizing element in a manner similar to the stabilizing element in chloroplasts
(126, 129, 130).
C. Dispersed Repetitive Sequences

The work of Hicks and co-workers (95–97), and Scherer and coworkers (106),
who cloned C. albicans middle-repetitive identical sequences, Ca3 of 12 kbp and
27A of 15 kbp, initiated the use of molecular epidemiological probes to distin-

guish between species of the genus Candida, as well as strains of the species C.
albicans. Both probes were also successfully used to address an important clinical
problem of the identity of the strains from multiple sites of infection in the body.
In the decade that followed, several more repetitive sequences were reported,
and some of them were also used as molecular probes.
Three sequences, so-called RPSs whose sizes varied between 2.1 and 2.9
kbp (79, 131, 132), HOK of 5.3 kbp, and RB2 of undetermined size (133), were
homologous to a portion of Ca3/27A (79, 133, 134). In addition, the following
sequences have been reported: the so-called MspI fragment of 2.9 kbp (135);
CARE-1 and CARE-2 of 0.47 kbp and 1.6 kbp, respectively, with no homology
between them (136, 137); a Ca24 family of a size comparable to that of Ca3 and
sharing some sequences in common but made up mostly of non-Ca3 sequences
(105); EOB1 and EOB2 of 1.0 kbp and 0.38 kbp, respectively (138); and Rel-1
and Rel-2 of 0.22 kbp and 2.8 kbp, respectively, which are different but share
two short subrepeat sequences (78). Recently it was found that CARE-2 and
Rel-2 share a retrotransposonlike sequence-designated kappa, which is believed
to be partially responsible for their repetitious nature (80). The MspI fragment
is thought to be present as tandem copies rather than dispersed. Additional data
on interstrain variability related to MspI are needed to clarify this suggestion,
however. It appears that Ca24, CARE-1, Rel-1, MspI fragment, and Ca3/27A/
RPS/HOK/RB2 represent independent families of the repeats. Some of the
above-mentioned sequences hybridized only to C. albicans DNA, whereas others
produced various degrees of weak signals with different species of the genus
Candida (105, 132, 135, 138). This result simply confirms relatedness of species
within the genus. The copy number of repetitive sequences ranged from few to
approximately 100 per cell, and the number of the repeats differed from strain
to strain. For example, there are 80 copies of RPS1 in the C. albicans FC18
strain, but much less in NUM812 (79). The repetitive DNA of Rel-2 is present
on most and perhaps all chromosomes in every strain studied, suggesting that
this sequence is important for chromosome functioning. The unique portion of
Rel-2, however, maps to only a few nonhomologous chromosomes, which are
variable between strains (77). The repeats are usually dispersed over the majority
of the chromosomes, but they also could be tandemly amplified on the same
chromosome (78, 105, 132, 136; H. Chibana and P. T. Magee, unpublished re-
sults). When repetitive sequences are labeled and hybridized to the blots of re-
striction digests of the genomic DNA from independent strains, they produce
specific patterns of multiple signals for each strain, as illustrated in Fig. 5a for
sequence CARE-2. It was found that morphological mutants with various types
of spontaneously altered electrokaryotypes preserved the same pattern of signals
as their parental strain (46, 48, 81), thus allowing distinction from independent
strains with different unique electrokaryotypes. Similarly, identical hybridization
patterns were reported for morphological mutants and a parental strain by Lasker
Figure 5 The comparison of signal patterns by molecular epidemiological probes. (A)

The two unrelated C. albicans laboratory strains (1) 3153A and (2) SGY-243, as analyzed
with the probe CARE-2. (B) The fluconazole resistant mutants fzE5 (3) and fzD5 (4)
derived from strain SGY-243, as analyzed with the probe 27A. See section IV.A.2 for
the chromosomal alterations in the mutants. See Ref. 40 for the origin of the strains. Total
DNA was digested with restriction enzymes EcoRI, HindIII or ClaI, as indicated at the
top of the figure, and was separated by conventional electrophoreses. The Southern blot
analysis was performed by hybridization with probes CARE-2 and 27A labeled with
Digoxigenin (Genius Kit, Boehringer-Mannheim, Indianapolis, IN).
et al. (137), although electrokaryotypes were not presented. Also, as presented

in Fig. 5b, a parental strain and its fluconazole resistant mutants with altered
chromosomes (see IV.A.2) showed identical patterns with molecular probe. In
one instance, DNA polymorphism was observed among mutants selected on 5-
fluorocytosine solid minimal medium with subsequent overnight incubation in a
rich medium, as probed with 27A (106). A total of two signals out of 12 was
changed. Another group published several series of the sequential isolates from
patients undergoing fluconazole treatment, in which some members of the series
exhibited one or two changes in CARE-2 pattern (107, 139). Despite a basic
similarity of patterns of the signals between the original strain and the mutant
or the original isolate and its sequential derivatives, such cases may require the
use of several epidemiological probes; for example, 27A and CARE-2, combined
with several enzymatic digests of the total DNA to rigorously exclude possible
contamination. Recently, PCR techniques have been used for species and strain
identification (140). These techniques allow rapid screening of a larger number

of isolates. Because repetitive-sequence probes have been more carefully studied
and controlled, however, they remain a practical tool for strain delineation.
The function, if any, of the above-mentioned repetitive sequences in the
Candida genome remains obscure. (See Sec. III.A.1b, and, e.g., Ref. 132.)
During the past 10 years several repetitive sequences with homology to
the known transposable elements in S. cerevisiae were identified in the Candida
genome. Tca1 consists of two direct repeats of the alpha element separated by
approximately 5.5 kb of DNA (141) containing three open reading frames that
are no longer functional (142). Tca1, however, is transcriptionally active, produc-
ing an abundant mRNA at room temperature but not at 37°C, which suggests
that this element might be involved in the mechanism of sensing environmental
changes. It was argued that the above-mentioned property of Tca1 is significant
for virulence, and also that such elements as Tca1 may have a role in the evolution
of genomic diversity in C. albicans (141). In relation with the latter suggestion
it is interesting to note that the frequency of chromosomal rearrangements in
morphological mutants substantially increases at low temperatures. (See Sec. III.
A.2b.) Tca1 was also used for the identification of C. albicans (143). Another
retrotransposonlike element, pCal (or Tca2), is considered to be unusual, as it
produces a high level of its preintegrative double-stranded DNA, which can be
identified as a band on the conventional electrophoresis gel (142). It is important
to note that this unusual property was found in a strain subjected to the action
of two mutagens, which might have created a rare genomic condition. The same
laboratory characterized one more retrotransposonlike sequence, kappa (see
above) (80). A recent screen of a C. albicans genomic sequence provided by the
Stanford University database helped to identify more than 350 insertions homolo-
gous to retrotransposons (144). At present the function of Candida retrotranspo-
sonlike elements remains unknown. It is also not clear if all of them are truly
transposable elements that change positions in the genome. The specific regula-
tion of the tRNA genes, by the analogy with other organisms, was suggested by
the identification of a 399-bp beta element, 9-bp upstream of a seryl-tRNACAG
gene, in several fresh clinical isolates, as well as immediately adjacent to
tRNASER, tRNAASP, tRNAALA, and tRNAILE genes in one laboratory strain, but not
in others (145).
IV. THE ROLE OF CHROMOSOMAL INSTABILITY IN

ADAPTATION TO THE CHANGING ENVIRONMENT
Under regular circumstances, Candida infectivity is undoubtedly defined by the

condition of the host, and is restricted to the mucous surfaces of the gastrointesti-
nal tract and genitalia by a healthy immune system. Nevertheless, within this
comparatively restricted habitat, a wide variety of conditions occur, requiring

microbial phenotypic plasticity in the form of tolerance to different pH, nutrients,
and coexisting natural microflora and mucous conditions. Although the patho-
genicity of the fungi is poorly understood, the ability of the opportunistic species
to survive on mucosal surfaces in a healthy host can be compared with the sur-
vival of the pathogens, whose pathogenetic factors are well defined. Furthermore,
in the course of migration from mucosal surfaces to deeper tissues of the immuno-
compromised host, Candida encounters unfamiliar environments, which require
rapid adaptation. In the course of the past few years, as the methodology and the
separation of chromosomes continued to improve, a role of one process, nondis-
junction, emerged as an important means in adaptation to the new environments.
It was found that the copy number of different specific chromosomes is altered
in different environments, perhaps as a response to the different specific stresses.
Because the chromosome copy number was involved in such important functions
as utilization of nutrients or resistance to antibiotics (44, 40, 87), this novel means
for regulating gene expression in microbes may be general in C. albicans. On
the other hand, the role of other types of chromosomal rearrangements remains
to be elucidated, including, for example, deletions and translocations, largely
documented in altered electrokaryotypes of spontaneous morphological mutants
(46, 47). Finally, the detailed investigation of the fate of Candida cells on a
selective plate changed our understanding of this microbe’s reaction to the new
environment (27). As will be described below, the appearance of the advanta-
geous mutants having a specific chromosomal nondisjunction highly increased
after the contact with either an alternative carbon source or a drug. These types
of experiments may serve as a model of Candida behavior in the course of pro-
gressive dissemination in the body or under the conditions of the antibiotic treat-
ment.
A. Chromosome Copy Number: A New Principle of Gene

Regulation
The understanding of the causal relationship between chromosome alteration and
gene expression came from the study of a specific chromosomal nondisjunction,
which occurred in mutants selected on L-sorbose medium (42). In the cases ana-
lyzed so far, including utilization of the alternative carbon sources L-sorbose and
D-arabinose, as well as resistance to fluconazole, many mutants, each derived as
an independent mutational event, had the gain or loss of chromosomes specific
to the given change in the environment (40, 44, 78). We speculate that the well-
known phenomenon of nondisjunction (146), which is frequent and well tolerated
in lower fungi, is responsible for the decrease or increase of the copy number
of a specific chromosome.
The aneuploidy condition created by the change in the chromosome copy
number obviously leads to gene imbalance and diminution of many functions.

We suggest that additional compensatory mutations partially or fully correct the
functional damages under the continuing selective pressure. In case the pressure
is removed, the opposite second nondisjunction may occur as a relatively frequent
event, thus recovering euploidy, and the mutant regains a competitive rate of
growth. The evolutionary possibility of the monosomy was discussed by Janbon
et al. (27). It was concluded that new strains could evolve by selection in the
changing environment.
Because the diminution of the copy number of a particular chromosome
was observed, the concept of the negative regulation was introduced (44). This
hypothesis was recently substantiated by cloning a number of the negative-regula-
tors genes of the utilization of l-sorbose that are distributed over different chro-
mosomes (147; Y.-K. Wang et al., unpublished results). In addition, an indepen-
dent group reported two negative regulators of fluconazole resistant (148, 149).
It was suggested that C. albicans contains a resource of potentially beneficial
genes, as well as their positive and negative regulators, which are distributed over
chromosomes in such a way that their expression can be activated by changes in
the chromosome number (40). This mechanism is based on the ratio between the
structural and regulatory gene number, and is controlled by a simple but effective
process; that is, the change in the chromosome copy number that is tolerable in
this and other lower fungi. It remains to be seen if this novel means of regulation
by changes in the chromosome number operates in organisms other than C. albi-
cans.
1. Utilization of the Alternative Carbon Sources L-sorbose

and D-arabinose
Some but not all strains of C. albicans cannot assimilate L-sorbose as a carbon
source. Such normal strains give rise to L-sorbose utilizers at high frequencies,
however. The mutants, which acquired the ability to utilize L-sorbose, were se-
lected in such a way that each mutant was derived as an independent mutational
event (44, 87). The SOU1 gene (named for sorbose utilization) responsible for
the utilization of L-sorbose was subsequently cloned from the C. albicans geno-
mic library (44). It was shown that the copy number of chromosome 5 controls
the expression of SOU1, so that strains disomic and monosomic for this chromo-
some were, respectively, nonutilizers and utilizers of L-sorbose (Fig. 6). This
conclusion was based on the examination of 69 electrophoretic karyotypes of
sorbose-positive and -negative derivatives of two laboratory strains, 3153A and
CAF4-2, including four complete series; that is
Sou⫺ → Sou⫹ → Sou⫺ → Sou⫹

Disomic Monosomic Disomic Monosomic
Figure 6 Chromosomal patterns from two C. albicans laboratory strains, 3153A and
CAF4-2, showing sequential sorbose-utilizing and sorbose-non-utilizing derivatives. (a)
and (b): OFAGE separation of chromosomes of typical-representative sequential-series of
derivatives from strains 3153A and CAF4-2, respectively. (1) 3153A (Sou⫺); (2) Sor55
(Sou⫹); (3) Sor55-1 (Sou⫺); (4) Sor55-1-1 (Sou⫹); (5) CAF4-2 (Sou⫺); (6) Sor19 (Sou⫹);
(7) Sor19-1 (Sou⫺); (8) Sor19-1-1 (Sou⫹). For the origin of mutants, see Ref. 44. Arrows
indicate alternating chromosome 5. For explanations, see Fig. 1. (c) and (d): Schematic
representation of chromosomes 5 from the Sou⫺ and Sou⫹ strains shown in (a) and (b),
respectively. The gene CSU51, a hypothetical negative regulator carried on chromosome
5, is designated by the symbol 䊉. Source: Adapted from Ref. 44.
as well as nine incomplete series of the type Sou⫺ (parental) → Sou⫹ → Sou⫺. The
analysis of these series also proved that this relationship could be continuously
perpetuated with each of the mutants derived from the opposite type. In our origi-
nal report on chromosome 5 alteration leading to the Sou⫹ phenotype, we did
not appreciate the monosomy of this chromosome, because the cultures were
routinely grown in liquid medium. This condition allowed for the selection of
reconstituted chromosome 5 disomics. Of the examined independent Sou⫹ mu-
tants, 47 retained a single copy of either one of two homologues of chromosome
5, as summarized in Fig. 7. One additional mutant, however, retained both copies,
Figure 7 Schematics of representative electrophoretic karyotypes of independently de-

rived sorbose- and arabinose-utilizing, as well as fluconazole-resistant mutants and two
C. albicans parental strains. (A) Chromosomal patterns of strain 3153A and its sorbose
(Sou⫹)- and arabinose (Aru⫹)-utilizing mutants. The chromosomal assignment of SOU1
gene, which preserved the same positions in the Sou⫹ mutants (44), is indicated only in
strain 3153A. (B) Chromosomal patterns of strain SGY-243 and its fluconazole-resistant
mutants (Flu R ). See Fig. 2 for explanations. The chromosomal numbers in Sor1, Sor2,
and Ara4 indicate a remaining homologue; in Ara7 an additional homologue. The numbers
in parentheses above chromosomes of the fluconazole-resistant mutants indicate the copy
numbers. The numbers at the bottom of the figure denote the number of independently
derived mutants that were analyzed. These numbers do not include mutants from the series
of sequential derivatives presented in Fig. 6. †One mutant, fzE5, obtained after a short
exposure, contained only one shorter homologue of chromosome 4 instead of two.
but instead had an approximately 300-kbp deletion on one of the chromosome

5 homologues. Another mutant of strain CAF4-2 acquired alteration of chromo-
somes R and presumably 3 but had no visible change in any other chromosome
(E. Rustchenko, unpublished data). While there may be other genetic changes
that produce a sorbose-positive phenotype, the monosomy of chromosome 5 is
a major means. Because SOU1 does not reside on alternating chromosome 5, it
was suggested that chromosome 5 contains a hypothetical negative regulatory
factor, CSU51 (control of sorbose utilization), which inhibits expression of SOU1
in two but not one copy. Obviously, the deletion, which would encompass
CSU51, would also lead to the utilization of L-sorbose. This work initiated the
study of the negative regulation in C. albicans. By using a total genomic li-
brary a number of the genes encoding negative regulators of the utilization of
sorbose were cloned, characterized, and shown to be situated on different
chromosomes (147; Y.-K. Wang, G. Janbon, and E. Rustchenko, unpublished
data). We found that these genes are additional negative regulators, whose ac-
tion is weaker than the action of major negative regulator CSU51, which is con-
trolled by chromosome 5. This result revealed the complexity of the control of the
utilization of food supplies. Apparently these different genes represent different
cellular levels of repression of structural genes for the utilization of secondary
carbon sources. The identification of CSU51 is currently being pursued in our
laboratory.
Another case of specific chromosomal alterations, either monosomy of
chromosome 6 or trisomy of chromosome 2 with one homologue out of three
being greatly truncated, resulted in growth on D-arabinose medium in each of
two groups of D-arabinose-positive mutants (87; D. H. Huber, J. Smith and E.
Rustchenko, unpublished observation) (Fig. 7). Although both types of nondis-
junction—either leading to the monosomy or to the trisomy—were observed,
one of the specific changes, the addition of one copy of a chromosome with the
large deletion of approximately 1 Mbp, cannot be viewed simply as a trivial
increase in the copy number of all the genes carried on this chromosome. Al-
though less studied, the case of D-arabinose utilization by means of monosomy
of a specific chromosome is a direct parallel of the causal relationship uncovered
in mutagenesis on L-sorbose. The understanding of this mechanism, which is
alternative to the monosomy and, respectively, negative regulation, will be the
subject of a future study.
2. Resistance to Fluconazole
Fluconazole is the main antifungal agent used to control candidosis. The high
frequency of occurrence of fluconazole-resistant mutants has hampered its use,
however. The possibility of electrokaryotypic changes associated with flucona-
zole resistance in clinical C. albicans isolates was investigated in several labora-
tories (reviewed in Ref. 40), and no differences in chromosomal patterns were
reported. The lack of revealing altered electrophoretic karyotypes apparently was
due to the limitations in the resolution of the chromosomal separation. As subse-
quently found by Perepnikhatka et al. (40), exposure of a C. albicans laboratory
strain SGY-243 to fluconazole on a petri plate resulted in the nondisjunction
of two specific chromosomes in 17 drug-resistant mutants, each obtained as an
independent mutational event. The changes were related to the duration of the
drug exposure. The loss of one homologue of chromosome 4 occurred after incu-
Figure 8 Precise separation of the chromosomes of the representative fluconazole-resis-

tant mutants fzE5 and fzD5, obtained after 7 and 35 days of exposure to fluconazole on
the plates, respectively. Electrophoretic karyotypes obtained by two running conditions
are presented in (A) and (B). (See Fig. 1 for explanations.) Arrows indicate chromosome
4, in which two out of three copies are lost, and chromosome 3, in which one copy was
acquired. No other detectable chromosomal changes are seen in these separations. Source:
Adapted from Ref. 40.
bation on fluconazole medium for 7 days (Figs. 7, 8a). A second change, the
gain of one copy of chromosome 3, which carried the genes CDR1 and CDR2
affiliated with fluconazole resistance, was observed after exposure for 35 or 40
days (Figs. 7, 8b). The fluconazole-resistance phenotype, electrokaryotypes, and
transcript levels (see below) of mutants were stable after growth for 112 genera-
tions in the absence of fluconazole. For the first time the resistance to fluconazole
was reported to be commonly caused by chromosomal changes. Throughout the
work of many groups, fluconazole resistance in clinical isolates of C. albicans
has been associated with a combination of several distinct mechanisms (139, 150,
151). These include mutations at the active site of the drug target 14α-sterol
demethylase, encoded by the ERG11 gene; overexpression of ERG11 (152–154);
alterations in the ergosterol biosynthetic pathway (152, 155); and overexpression
of the genes involved in energy-dependent drug efflux (139, 151, 156, 157). The
overexpression of two relevant genes, CDR1 and CDR2, which encode proteins
homologous to ATP-binding cassette (ABC) drug pumps, as well as MDR1
(BEN R ), which encodes a protein similar to pumps of the major facilitator super-
family (MFS), has been associated with fluconazole resistance in clinical isolates
(156, 158, 159). When the transcription levels of the candidate fluconazole-resis-
tance genes, ERG11, CDR1, CDR2, and MDR1, were measured in the mutants
with the specific chromosomal nondisjunctions, it was found that they either re-
mained the same or were diminished, except that expression of CDR1, carried
on the duplicated chromosome, increased in mutants obtained after the longer

exposure. The copy number of chromosomes 4 or 3 carrying transcriptional regu-
lators, as well as the structural gene CDR1, can explain these changes. The lack
of substantial overexpression of putative drug pumps or the drug target indicated
that some other mechanism(s) might be operating. Significantly, none of eight
mutants of strain 3153A possessing various single or multiple chromosomal re-
arrangements were altered in their sensitivity to fluconazole. Because the mutants
analyzed in our study were obtained as independent mutational events, we suggest
that a change in chromosomal copy number is a common means for producing
resistance. This mechanism might be based on a similar response to stress, which
lead to the formation of the mutants utilizing alternative nutrients.
Despite the importance, a systematic analysis of chromosomal patterns in
clinical isolates has not been undertaken. Such a study requires reliable series of
genetically related strains, which have developed resistance, and which have been
adequately collected, handled, and preserved in order to avoid the induction of
irrelevant chromosomal instability. In addition, the chromosomal separations
have to be of a high quality.
It is important to note that there is a remarkable similarity between the
results obtained under laboratory conditions by Perepnikhatka et al. (40) and the
results obtained by several other groups from patients undergoing treatment with
fluconazole. A number of independent studies revealed that a series of C. albicans
strains isolated sequentially in time from patients showed a progressive increase
of resistance, and that overexpression of efflux pumps or point mutations of the
target gene, ERG11, was not initially manifested, or in fact did not appear at all
during the study (107, 157, 160, 161). In one of these studies of five different
patients (160), sequentially obtained fluconazole-resistant mutants from one pa-
tient did not exhibit overexpression of MDR1, CDR1, CDR2, nor did the mutants
have point mutations in the ERG11 gene, even though strains were isolated and
tested for a period extending to nearly 10 months. Strains from the other patients
showed an alternation in overexpression of the MDR1 and CDR genes. In addi-
tion, a recent study showed that cross-resistant mutants, including resistance to
fluconazole, were obtained in strains with deleted genes for drug-pumps after
exposure to the itraconazole (162). Another recent study with clinical isolates
demonstrated that high constitutive expression of the MDR1 gene in fluconazole-
resistant strains was due to a transregulatory factor, but not, for example, a muta-
tion in the promoter region of MDRI (163). Perhaps the latter result might be
interpreted in the context of chromosomal alterations. We similarly found a sub-
stantial overexpression of MDR1 gene in highly fluconazole-resistant mutant FR2
having five chromosomal alterations, including a chromosome carrying MDR1
(40). Another explanation might be that the change of MDR1 expression was due
to a change of copy number of a regulatory gene on an altered chromosome. A
combined effect of several chromosomal alterations, as well as gene mutations,
also cannot be excluded. Thus, the laboratory study conducted by us and the
clinical studies undertaken by the other groups both revealed that at early stages
of infection, fluconazole resistance arose by mechanisms other than overexpres-
sion of known efflux pumps or mutations of a target gene. Because chromosomal
nondisjunction produced high frequencies of relatively low-level fluconazole-re-
sistant mutants, it is reasonable to suggest that a similar alteration is causing
primary resistance under clinical treatments. The higher resistance levels of the
latter clinical isolates can be viewed as a secondary event, which occurred as a
result of the accumulation of gene mutations under prolonged selective pressure.
The identification of the resistance genes controlled by chromosomal nondisjunc-
tion will be the subject of future research. The importance of the discovery of
chromosome copy number as a general response controlling drug resistance lies
in revealing new genes for the resistance, which in combination with mutations
in the target gene, genes for the efflux pumps, or alone, can allow a single strain
to adapt to antifungal treatment and establish a persistent infection. Together with
the fact that the same type of regulation was observed for the utilization of differ-
ent nutrients, these data further establish the hypothesis that change in the copy
number of a chromosome is a general means of regulating physiologically impor-
tant genes in Candida.
3. Adaptive Mutagenesis as a Means to Cope with

New Environment
The ability of the microbial cells to adapt to the changing environment has been
the subject of numerous studies. A number of characteristic features of microbial
adaptability to selective conditions have been investigated as a result of renewed
interest during the last decade, primarily due to a prominent paper by Cairns et
al. (164). It was originally suggested that mutations arise in nondividing micro-
organisms subjected to selective pressures, and this phenomenon was denoted
as ‘‘directed mutation’’ or ‘‘adaptive mutation.’’ Currently, however, adaptive
mutations denote mutations that are formed at higher frequencies in response to
the selective conditions, even though they may not necessarily be ‘‘directed’’
(165). Under the current usage, mutants resistant to and induced by UV light
would be considered to arise by adaptive mutations, as would nutritionally se-
lected mutants induced by starvation or stress. The first attempt to document the
adaptive mutagenesis in Candida involved experiments on the resistance to heavy
metals (166). A detailed analysis, as well as crucial evidence for adaptive muta-
genesis, came from studies of the comparison of the daily rates of formation of
Sou⫹ mutants (27). As presented in Fig. 9, there was a striking increase of four
orders of magnitude in the rates of mutant formation immediately after contact
with the selective medium to days 4 and 8, clearly suggesting that the vast major-
ity of the mutational events appeared gradually after contact with the selective
Figure 9 The adjusted rates of sorbose-utilizing mutants of each of the 27 clones of

strain 3153A per viable cell per day. The adjusted values were calculated from the daily
number of colonies, divided by the number of viable cells 4 days prior to the appearance
of colonies. Both the deduced time of formation of the mutation (top row) and the time
of the appearance of the corresponding colonies (bottom row, in parentheses) are pre-
sented. Source: Adapted from Ref. 27.
condition. Taken together with the time-dependent manner of the appearance of

the mutants, the increased rates of formation of at least the late mutants were
indicative of adaptive mutagenesis. The comparison of the electrokaryotypes of
the mutants, which appeared on the experimental plate on different days within
a 2-week period, showed that, as expected, all of them had a common alteration,
a monosomy of chromosome 5. Some random additional changes in a few of the
mutants were not considered relevant. Most important, the control experiments
designed to test in several different ways if there was any turnover, residual
growth, or formation of microcolonies on sorbose-containing plates, all gave neg-

ative results.
Even if one assumes some reasonable turnover during the death of the Sou⫺
cells, in which the rate of death exceeds the rate of growth and that this turnover
is required for producing Sou⫹ mutants, the high rate of formation of approxi-
mately 10⫺2 Sou⫹ mutants per viable cell per day clearly established that these
mutants arose by adaptive mutagenesis. With a normal mutation rate of 10⫺5 per
cell per generation, one would have to assume that the dying cells are dividing
every minute in order to accumulate the number of Sou⫹ mutants observed during
the sharp increase of the rates. At the present time, the study of the C. albicans
cells’ behavior on L-sorbose plates is the most striking example of the adaptive
mutagenesis. Other, mostly bacterial models, showed approximately a tenfold
difference between the frequencies of classic and adaptive mutagenesis. Earlier,
Borst and Greaves (158) pointed out that the reaction of a microbe to major
changes in the nutrient supply is expected to be of two kinds, a preadaptation of
a fraction of the population through DNA rearrangements, or adaptation after an
environmental change. Because starvation is not immediately lethal, it is more
efficient for the organism to use alternative genes when needed. The study of the
adaptive mutagenesis to the condition of a new source of carbon can be consid-
ered as a model of Candida reaction to the outside stress and change. Such con-
trolled laboratory studies may represent situations in the host during a progressive
disseminate infection or treatment with the antibiotic.
As was previously realized, ‘‘C. albicans should be considered as a fungus
possessing a multiplicity of properties, each with a low propensity for enhancing
fungal infectivity but none necessarily dominant, and all, even in combination,
unlikely to overcome fully intact host defenses’’ (28). The genes, which promote
survival in the host, and particularly genetic diversity and plasticity, are as impor-
tant as virulence factors of other pathogens under these circumstances. Any of
the potentially unstable features of the genome listed in this chapter could change
the expression of the relevant genes.
V. PHENOTYPIC FEATURES AS A SUBJECT

OF VARIABILITY AND INSTABILITY
A. ‘‘Switching’’ Colonial Morphologies
The term switch was introduced in 1985 by Soll and co-workers (37) to describe
reversible changes of colonial morphologies in populations of the same strain.
Subsequently Soll (159) defined all reversible high-frequency mutations oc-
curring in C. albicans and related species as ‘‘switching,’’ independent of any
mechanism. As originally used, switching refers to two or a few alternative ge-
netic states that are reversible. The most extensively studied examples include
mating-type switching in S. cerevisiae (160), inversion control of gene expression

in Salmonella (161), and P⫹ and P⫺ antigenic variation in Neisseria gonorrhoeae
(162). Clearly, denoting any high rates of mutation switching can be inappropri-
ate. For example, Das et al. (163) uncovered cyc1 mutations in S. cerevisiae that
reverted with the frequency of 10⫺4 and had a reversion rate of over 10⫺5 per
generation.
Soll and co-workers (159) have investigated and applied the term switching
primarily to two types of instability, colony morphology mutants studied mainly
with strain 3135A, as described below, and to the white–opaque transition studied
with strain WO-1 and its derivatives, as discussed in the next section.
Spontaneously arising colonial forms, as well as those induced by mild UV
irradiation, were postulated to switch with certain defined frequencies between
seven morphologies (37). The normal parental colony type is ‘‘smooth,’’ whereas
variants arising from the parental strain 3153A were denoted as ‘‘star,’’ ‘‘ring,’’
‘‘irregular wrinkle,’’ ‘‘stipple,’’ ‘‘hat,’’ and ‘‘fuzzy.’’ This first report already
contained data inconsistent with the proposed nomenclature, as two independent
photographs of the ‘‘ring’’ morphology were not exactly the same. In later papers
some visibly different morphologies were given the same name introduced by
Slutsky et al. (37); compare, for example, the photograph of ‘‘fuzzy’’ reported
by Soll (159). ‘‘Revertant smooth’’ denoted the ‘‘smooth’’ revertants from each
of the variants. The normal strain was reported to give rise to variants at a fre-
quency of approximately 10⫺4 spontaneously or approximately 10⫺2 after UV
irradiation. ‘‘Revertant smooth,’’ however, spontaneously gave rise to variants
at a frequency of approximately 10⫺2, indicating the lack of true reversion. The
frequencies of a variant producing another type of variant varied from approxi-
mately 10⫺2 to approximately 10⫺4, although one type could not be interconverted
(37). In addition, ‘‘revertant smooth’’ types segregated morphologies, which the
authors could not fit into the above-mentioned seven types.
The spontaneous switching of colony morphology studied with 3153A by
Soll and co-workers is obviously the same phenomenon systematically studied
by Rustchenko and co-workers, who, however, demonstrated an association with
chromosomal alterations. Whelan (164) also suggested in his review the basic
similarity between the results of the Soll group and the early author Brown-
Thomsen. Our contention is that Soll and his co-workers have not used rigorous
criteria to define specific phenotypes, and their results do not fulfill the concept
of ‘‘reversibility.’’ Furthermore, our studies with the same strain 3153A estab-
lished that morphological mutants, including highly unstable representatives, do
not return to the truly original form, nor do they alternate between a limited
number of forms. The number of distinguishable types of colony morphologies
certainly exceeds seven. In addition, the unstable morphological mutants studied
by Rustchenko and co-workers gave rise to a large number of different forms
specific to each mutant. Our conclusion, based not only on our own extensive
work and numerous reports published since 1935 (see Ref. 28), but also on the
work of Pomés et al. (165) and on published work by Soll and his co-workers,
is that colonial forms do not readily revert, and therefore do not switch. Further-
more, the variation of colony forms can be simply explained by the gene imbal-
ance due to chromosomal alterations.
Pérez-Martı́n et al. (166) recently reported that double disrupted sir2-∆/
sir2-∆ strains of C. albicans produced mutants with altered chromosomal patterns
and colony morphologies at frequencies as high as one in 10. In the yeast S.
cerevisiae, the SIR2 gene maintains the inactive chromatin domains required for
transcriptional repression at the silent mating-type loci and telomeres. Such re-
pression is associated with specialized chromatin structures whose integrity de-
pends upon a complex combination of cis-acting sites, several shared transact-
ing factors, such as Sir1p, Sir2p, Sir3p, and Sir4p, as well as histones. Because
sir2-∆/sir2-∆ could affect the expression of any of a number of genes, the in-
creased frequencies of mutants with altered karyotypes can be explained by nu-
merous models (166). More important, J. Pérez-Martı́n (unpublished result) has
been unable to consistently reproduce the effect of SIR2 disruptions on colony
morphologies in C. albicans. This variation of instability resembles the findings
of Rustchenko-Bulgac (47), who observed the periodic stabilization of initially
unstable chromosomal alterations in morphological mutants. Chromosomal insta-
bility adequately explains the results reported by Pérez-Martı́n et al. (175) and
J. Pérez-Martı́n (unpublished result).
More recently, Lachke et al. (167) reported that the related species, Candida
glabrata, undergoes a reversible, high-frequency switching between three colony
types that were distinguished on CuSO 4 indicator plates. The three colony types,
white (Wh), light brown (LB), and dark brown (DB), were associated with differ-
ent levels of MT-II (metallothionen) and HLP (hemolysinlike protein) mRNAs.
The statistical nature of the transitions, the electrophoretic karyotypes, and the
mechanisms responsible for producing the three types of colony forms were not
presented. Because of an earlier report on the ability of a natural haploid species
C. glabrata to undergo chromosomal rearrangements (168), it would be of partic-
ular interest to examine the electrophoretic karyotypes of the above-mentioned
mutants.
B. White-Opaque Transition
As introduced above, the extensively studied white-opaque ‘‘switching’’ in the
strain WO-1 and its derivatives consists of transitional changes between the white
phase, in which colonies appear as smooth and white on solid media, and the
opaque phase, in which colonies appear flattened and gray, (178–180). Opaque
cells differ from white cells in a number of properties, including the following:
cell shape (in which the opaque cells appear elongated and rod shaped, superfi-
cially resembling pseudomycelium); budding pattern; response to different tem-

peratures; growth rate; frequency of transition; sensitivity to UV irradiation, sul-
fometuron methyl, and 5-fluoroorotic acid; accumulation of bismuth sulfide on
bismuth medium; interaction with host cells; and virulence (179, 181). Intercon-
version of the white and opaque phases occurs spontaneously and can be induced
by temperature shifts. For example, temperature shift from 24°C to 34°C results
in mass conversion of the opaque form to the white form (179) within two cell
divisions (J. Hicks, personal communication). Conversely, at 24°C a population
of white cells produces white colonies with opaque sectors at the periphery (179).
These opaque sectors become visible after 5 days of incubation at 24°C on YPD
medium and subsequently develop into large clusters of opaque cells (Huber and
Rustchenko, unpublished results). The frequency of mutual interconversion at
24°C was estimated by Anderson et al. (178) to be between 10⫺2 and 10⫺3, and
the rates were estimated by J. Hicks (personal communication) to be less than 5
⫻ 10⫺3 per cell division for opaque-to-white conversion and 5 ⫻ 10⫺2 per cell
division for white-to-opaque conversion. On the other hand, Rikkerink et al. (179)
reported that opaque cells converted to white cells at 24°C with a frequency of
10⫺4 to 10⫺5 and that from 0.1% to 5% of white colonies arise from population of
opaque cells formed at room temperature. Superimposed on the interconversion
between opaque and white morphologies is the formation of various colony mor-
phology mutants (180), presumably due to chromosomal alteration, as described
in the previous section. There are no systematic reports that the interconversion
between white and opaque phenotypes involves all of the other multiple pheno-
types, including estimated frequencies of the white-opaque transition. Further-
more, genes that are specifically expressed in the white phase, WH11, or in the
opaque phase, OP4, SAP1, and CDR3, have been reported, and portions of the
regulatory regions within the promoter regions have been characterized (182–
187). Srikantha et al. (187) presented evidence that common regulatory processes
determine white-opaque switching and budding-hyphal dimorphism of C. albi-
cans. Wh11p, an abundant cytoplasmic protein, is induced both by the opaque-
white transition, and the hypha-budding morphogenesis in WO-1, and both of
these repressional events require identical promoter segments (187). Furthermore,
opaque-phase cells of WO-1 and hyphal cells both contain certain specific anti-
gens not found in white-phase cells (169).
In addition, Sonneborn et al. (188) suggested that the Efg1p transcription
factor, which is required for hyphal induction (17, 23), is also an essential element
for white-opaque switching in WO-1. EFG1 is transcribed as a 3.2-kb mRNA
in white-phase cells and as a less abundant 2.2-kb mRNA in opaque-phase cells,
due to different transcription start sites (189). After the commitment event, the
efg1 null mutant forms daughter cells that have the smooth (pimpleless) surface of
white-phase cells, but maintain the elongate morphology of opaque-phase cells,
indicating that Efg1p is not essential for the switch event per se, but is essential
for a subset of phenotypic characteristics necessary for the full expression of the
phenotype of white-phase cells. Srikantha et al. (189) concluded that EFG1 is
not the site of the switch event, but that it is controlled downstream of the switch
event.
Klar et al. (190) and Srikantha et al. (191) have suggested that white-to-
opaque transitions in strain WO-1 is mediated through histone deacetylases,
which regulates chromatin structure by selective histone deacetylation, leading
to changes in chromatin folding and interactions between DNA and DNA-binding
proteins. Klar et al. (190) demonstrated that the frequency of white-to-opaque
transitions in WO-1 was greatly enhanced by exposing the strain for 48 hr on
agar containing 10 µg/l of the histone deacetylase inhibitor trichostatin-A (TSA).
In addition, Srikantha et al. (191) examined the expression of five histone deace-
tylase genes in white and opaque phases of the white-opaque transition, and they
examined the frequency of switching and the expression of white-phase and
opaque-phase specific genes in mutants deleted in one or an other of the histone
deacetylase genes. The results of their study suggested that the two deacetylase
genes, HDA1 and RPD3, play distinct roles in the suppression of switching, and
that the two distinct and selective roles in the regulation of phase-specific genes.
Furthermore, they suggested that the white-opaque transition is due to epigenetic
changes in chromatin structure, presumably involving the inhibition of gene ex-
pression by chromatin modification through the deacetylation of histones at a
key gene, which has not yet been identified.
Although the molecular mechanisms that determine white-opaque switch-
ing is still unclear, because opaque-phase cells are elongated and resemble pseu-
dohyphae, and because common components are required for both processes, it
appears that white-opaque transition and hyphal-budding morphogenesis are re-
lated phenomena that are conceptually distinct from the other types of ‘‘general
instability’’ discussed in this review.
ACKNOWLEDGMENTS
We thank Dr. M. Wellington for obtaining signal patterns with molecular probes
27A and CARE-2. The studies from our laboratories cited in this review were
supported by grants AI29433 and GM12702 from the National Institutes of
Health.
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Index
Absidia, 4, 67, 70, 77, 87–88 A. spinosum, 317, 384–385

key to species of, 88–89 A. strictum, 318, 385–387
Absidia corymbifera, 89–90 A. terricola, 376
A. hyalospora, 90 Acrophialophora, 567
A. ramosa, 89 Acrophialophora fusispora, 567–568
A. repens, 88 Agaricales, 9–10
Achaetomium, 651 Agonomycetes, 11
Achaetomium cristalliferum, 654 Ajellomyces, 200–201
A. strumarium, 651, 654 Ajellomyces capsulatus, 7, 197, 201,
Achlya, 19 204
Achorion, 143 A. crescens, 7, 197, 214–215
Acremonium, 368 A. dermatitidis, 7, 197, 211
identification of, 369–370 Allescheria boydii, 639
laboratory characters of, 317–318 Alternaria, 567–568
unidentified species associated with Alternaria alternata, 568–569, 572
disease, 368–369 A. chartarum, 567
A. alabamense, 317, 370–372 A. chlamydospora, 569–570
A. atrogriseum, 372 A. infectoria, 567
A. blockii, 317, 372–373 A. longipes, 570–572
A. curvulum, 373–374 A. tenuis, 567
A. falciforme, 317, 374–375 A. tenuissima, 568, 570–572
A. hyalinulum, 375–376 Ancylistaceae, 4–5, 129
A. implicatum, 376 Antibiotic resistance among fungi,
A. kiliense, 317, 376–380 693
A. portronii, 317, 380–382 Aphanoascus, 225
A. recifei, 317, 382–384 Aphanoascus fulvescens, 219–221
A. roseogriseum, 384 A. keratinophilus, 198
777
778 Index
Aphanomyces astaci, 45–46 Ascosubramania, 650

Aphanomyces frigidophilus, 44–45 Aspergillosis, case reports of, 253
A. invadans, 44 Aspergillus, 7, 240–241
A. pisci, 44 locator list, 244–245
A. piscicida, 44 natural (teleomorph-based) groups,
Aphenomycopsis sexualis, 49 242–243
Aphyllophorales, 9–10, 668 Aspergillus albiaceus, 298
Apophysomyces, 4, 67, 70, 90–91 A. alliaceus, 298
Apophysomyces elegans, 91 A. amstelodami, 252
Arachnomyces nodosetosus, 199 A. avenaceus, 247, 303–304
Arthroderma, 6, 141, 149, 151–152, A. caesiellus, 264–266
Arthroderma benhamiae, 152, 154–155, A. candidus, 248, 290–293
168, 188 A. carneus, 249, 288–290
A. borellii, 178 A. chevalieri, 246, 256–257
A. cajetani, 155–156 A. clavatonanica, 315
A. ciferrii, 179 A. clavatus, 247, 313–314
A. cookiellum, 180 A. conicus, 266
A. corniculatum, 180 A. deflectus, 250, 283–285
A. curreyi, 151 A. flavipes, 248, 285–287
A. flavescens, 181 A. flavus, 247, 298–301
A. fulvum, 156–157, 181 A. fumigatus, 246, 310–313
A. gertleri, 182 A. glaucus, 246, 257–262
A. gloriae, 183 A. granulosus, 250, 277–279
A. grubyi, 157–158 A. hollandicus, 252
A. gypseum, 158, 170 A. janus, 278–279
A. incurvatum, 160–161, 170 A. japonicus, 247–248, 306–307
A. insingulare, 183–184 A. nidulans, 249, 267–270
A. lenticulare, 184, 188 A. nidulans var. dentatus, 270
A. obtusum, 161 A. nidulans var. echinulatus, 270–271
A. otae, 161–164, 170 A. niger, 248, 304–307
A. persicolor, 164–165 A. nivea, 286–288
A. quadrifidum, 184, 188 A. niveus, 248, 286–288
A. racemosum, 165–166 A. ochraceus, 247, 294–296
A. simii, 166–167, 175 A. oryzae, 301–303
A. tuberculatum, 205 A. parasiticus, 247
A. uncinatum, 153, 185–186 A. penicillioides, 266–267
A. vanbreuseghemii, 167–168 A. quadrilineatus, 271
Arthrodermataceae, 6, 8, 141–189 A. reptans, 262
anamorph-teleomorph states of the A. restrictus, 246, 263–265
saprophytic forms of, 147 A. rubrobrunneus, 263
comparisons to Onygenaceae, 202– A. rugulosus, 272
203 A. rugulovalus, 272–273
Arthrographis kalrae, 655–657 A. sclerotiorum, 248, 295–297
Arxiozyma telluris, 515, 520 A. sydrowii, 250, 279–281
Ascomycetes, 6–9 A. tamarii, 247, 303–305
Ascomycota, 2, 6–9 A. terreus, 249, 290–292
Index 779
A. tetrazonus, 271–272 Botryomyces caespitosus, 575–576

A. thermomutatus, 308–310 Botryosphaeria rhodina, 664
A. unguis, 250, 274–275 Botryosphaeriaceae, 8
A. ustus, 250, 281–284 Botryostrichum, 210
A. versicolor, 250, 275–278 Byssochlamys, 240
A. varians, 280–281
A. vitis, 246, 252–256 Candida, 515
Atkinsiella dubia, 53 Candida albicans, 13–14, 516, 520
Aureobasidium, 571 genetic instability of, 723–765
Aureobasidium pullulans, 571–573 mating of, 725–728
Auxarthron, 225 Candida albicans, chromosomal instabil-
ity and adaptations of, 751–760
Basidiobolaceae, 4–5, 129 Candida albicans, genome of, 728–751
Basidiobolus, 129–133 chromosomes, ploidy and cell DNA,
taxonomy of, 131,133 content of, 728–745
Basidiobolus haptosporus (see B. dispersed repetitive sequences of,
ranarum) 748–750
B. microsporus, 128 regular repetitive sequences of, 745–
B. ranarum, 5, 127–128 748
Basidiomycetes, 668–669 Candida albicans, phenotypic variability
Basidiomycetous yeasts, 535–564 of, 761–765
phylogeny of, 536 switching colony morphology, 760–
physiological patterns of, 538 762
Basidiomycota, 2, 9–10 white-opaque transition, 763–765
Beauveria alba, 658 C. ciferrii, 519–520
B. bassiana, 657 C. dubliniensis, 516, 520
Bipolaris, 9, 572–573 C. famata, 517, 520
Bipolaris australiensis, 572–574 C. glabrata, 516, 520
B. hawaiiensis, 574–575 C. globosa, 516
B. spicifera, 574–575 C. guilliermondii, 518, 520
Blastocladiales, 3, 10 C. haemulonii, 520
Blastomyces dermatitidis, 7, 196, 201, C. inconspicua, 516
205, 207, 211–214 C. kefyr, 518, 520
Blastulidium paedophthorum, 53–54 C. krusei, 516–517, 520
Blastomycetes, 10–11, 499–533 C. lipolytica, 519–520
methods for identification of, 501– C. lusitaniae, 516, 520
506 C. norvegensis, 518, 520
molecular epidemiology of, 511–512 C. parapsilosis, 520
phylogeny of, 506–508, 513–514 C. pelliculosa, 517
species identification of, 506–511, C. pintolopesii, 515
514–515 C. pseudotropicalis, 518
taxonomy of, 499–502 C. pulcherrima, 518
Blastoschizomyces capitatus, 515, 520 C. tropicalis, 516, 520
Blastulidiopsis chattonii, 53 C. utilis, 520
Botryodiplodia theobromae, 664 C. viswanathii, 520
Botryomyces, 575 C. zylanoides, 520
780 Index
Capronia semiimmersa, 608 C. werneckii, 606

Catenaria, 3 Clavispora capitatus, 516, 520–521
Catenaria ramosa, 50 C. lusitaniae, 516
C. spinosa, 50 Coccidioides immitis, 7, 196, 198, 209,
C. uncinata, 49 217–219
Cephalosporium acremonium, 385 Cochliobolus, 9
C. roseogriseum, 384 Cochliobolus australiensis, 573
C. serrae, 449 C. hawaiiensis, 573
Chaetomiaceae, 637, 651–655 C. spicifera, 574
Chaetomium, 9, 651 Coelomomyces, 3, 50
Chaetomium atrobrunneum, 652–653, 655 species in the genus, 51
C. globosum, 652, 654 Coelomomycetaceae, 15
C. histoplasmoides, 205 Coelomycetes, 11, 662–668
C. Strumarium, 654 Cokeromyces, 67, 70, 121
Chlamydomyzium, 54 Cokeromyces recurvatus, 5, 76, 121
Chrysosporium, 7, 152, 198, 219 Colletotrichum, 663–664
Chrysosporium articulatum, 198, 224 Colletotrichum coccodes, 663
C. carmichaelii, 199, 224 C. crassipes, 663
C. gourii, 221 C. dematium, 663
C. keratinophilum, 198, 223 C. gloecosporioides, 663–664
C. lobatum, 198, 224–225 C. graminicola, 663
C. merdarium, 219 Columella, types of, 75
C. thermophile, 660 Completoriaceae, 4–5, 129
C. zonatum, 221–222 Conidiobolus, 5, 133–137
Chytridiales, 3 taxonomy of, 136
Chytridiomycetes, 3–4 Conidiobolus coronatus, 5, 127–128
associated with Aschelminthes, 21–25 C. incongruus, 5, 127–128
associated with protozoan Protoctista, Coniochaeta, 658
31–32 Coprinus cinereus, 9, 669
Chytridiomycota, 2–4 Corynascus heterothallicus, 660–661
Circinella umbellata, 86 Couchia circumplexa, 48–49
Citeromyces matritensis, 516, 521 Crypticola clavulifera, 49
Cladophialophora, 7, 576–577 C. entomophaga, 49
Cladophialophora arxii, 579 Cryptococcus, 9, 521, 535–537
C. bantiana, 576–578 characteristics of, 537
C. carrionii, 576–579 Cryptococcus albidus, 541
C. devriesii, 578–580 C. ater, 541–542
C. emmonsii, 579, 581 C. bacillisporus, 540
C. modesta, 576 C. curvatus, 541
Cladosporium, 576, 579–581 C. humicola, 546
Cladosporium. bantiana, 576 C. laurentii, 539, 541
C. carrionii, 576 C. luteolus, 541
C. cladosporiodes, 579–582 C. neoformans, 537, 540
C. herbarum, 581–583 C. neoformans var. gattii, 539–541
C. sphaerosperumum, 579–580, 582–583 C. neoformans var. grubii, 516–517,
C. trichoides, 576 539–541
Index 781
C. neoformans var. neoformans, 516– [Dermatophytes]

517, 539–541 spore types in the three major genera
C. uniguttulatus, 542 of, 149
Cunninghamella, 5, 67, 70 taxonomy of, 147–148
Cunninghamella bertholletiae, 5, 78, Deuteromycota, 2, 10–11
117–118 Didymosphaeriaceae, 7–8
C. elegans, 117–118 Diplorhintrichum gallopavum, 586
Cunninghamellaceae, 4–5, 117–118 Dipodascaceae, 6, 8
key to the species of, 117 Dipodascus, 6
Curvularia, 9, 582, 584 Discosphaerina fulvida, 572
Curvularia brachnyspora, 584 Dissitimurus, 588
C. clavata, 584 Dissitimurus exedrus, 588
C. geniculata, 584–585 Doratomyces, 649
C. lunata, 584–586 Dothideaceae, 8
C. pallescens, 585, 587 Dothideales, 7–8, 459–461
C. senegalensis, 585, 586 Drechslera, 588–589
C. verruculosa, 585–586, 588 Drechslera biseptata, 589, 591
Cylindrocarpon, 387
identification of, 388 Echinobotryum, 638
laboratory characters of, 318 Emericella, 240, 266–267
Cylindrocarpon cyanescens, 318, 388– Emericella dentata, 270
389 E. echinulata, 270–271
C. destructans, 318, 389–391 E. nidulans, 249, 267–270
C. lichenicola, 318, 391–393 E. quadrilineata, 249, 271–272
C. tonkinense, 391 E. rugulosa, 249, 272–273
C. vaginae, 394 E. unguis, 274–275
Cystofilobasidiales, 9–10, 535 Emmonsia, 196
Cystofilobasidium, 542 Emmonsia parva, 7, 197, 208, 215–216
E. crescens, 7, 197, 201, 207, 214
Dactylaria, 586 E. pasteuriensis, 197, 208, 216
Dactylaria constricta, 586 Emmonsiella, 200, 204
D. gallopaury, 586 Emmonsiella capsulata, 201
Debaryomyces hansenii, 517, 520 Endochytrium, 3
Delacroxia coronata (see Conidiobolus Endomycetes, 6, 499–533
coronatus, 127) methods for identification of, 501–
Dematiaceous hyphomycetes 11, 565– 506
626 molecular epidemiology, 511–513
clinical overview of, 565–566 phylogeny of, 506–508, 513–514
description and natural habitat of, species identification of, 506–511,
565 514–515
identification of, 566 taxonomy of, 499–502
Dermatophytes, 141, 144 Endosphaerium funiculatum, 55
anamorph-teleomorph states of, 145 Engyodontium album, 658
epidemiology of, 146–147 Entomophthora, 128–129
geographic distribution of, 148 Entomophthoraceae, 4–5, 129
identification of, 144, 146 Entomophthorales, 4–5, 127–139
782 Index
[Entomophthorales] F. neoformans var. neoformans, 539–

families of, 130–131 541
propagative and vegetative structures Filobasidiales, 9–10, 535
of, 128 Fissuricella filamenta, 545, 547
Epidermophyton, 6, 143, 146, 148, 168 Flagellate fungi, 17–66
Epidermophyton floccosum, 146, 168– basic biology of, 17–20
169 biochemistry of, 41–42
macroconidia of, 169 classification of, 18
E. simii, 166 host-parasite relationships of, 20, 30
E. stockdaleae, 186 methods for detection of, 30, 33
Eremomyces langeronii, 655–657 sexual reproduction of, 38–41
Eremomycetaceae, 8, 240 thallus morphology of, 33–35
Eupenicillium, 325 zoosporangia of, 35–36
Eurotiales, 7–8, 195, 240–359 zoospores of, 36–38
Eurotium, 240–241, 245, 251–252 Fonsecaea, 7, 596–597
Eurotium amstelodami, 246, 252–256 Fonsecaea pedrosoi, 597–598, 601
E. chevalieri, 246, 256–258 F. compacta, 597–598, 600
E. herbariorum, 246, 257–262 Fungi imperfecti, 9–12
E. repens, 246, 262 Fusarium, 9, 394–395
E. rubrum, 248, 263 general pattern of involvement by,
Exophiala, 7, 590–591 395–396
Exophiala castellanii, 592 identification of, 397–401
E. jeanselmei, 592–594 laboratory characters of, 318–320
E. jeanselmei var. heteromorpha, 590 unidentified species associated with
E. jeanselmei var. jeanselmei, 592–594 disease, 396–397
E. jeanselmei var. lecanii-corni, 592– Fusarium anthrophilum, 401
594 F. aquaeductum, 402
E. mansonii, 592 F. chlamydosporum, 318, 402–403
E. moniliae, 590, 594 F. coeruleum, 319, 403–405
E. pisciphila, 590, 594–595 F. dimerum, 319, 405–406
E. salmonis, 591 F. episphaeria, 405
E. spinifera, 590, 594–596 F, equiseti, 406–407
E. werneckii, 590, 606 F. fujikoroi, 407
Exserohilium, 573 F. incarnatum, 407–409
Exserohilium longirostratum, 596–597 F. lacertarum, 409–410
E. mcginnisii, 596–598 F. moniliforme, 410, 429
E. rostratum, 596–597, 599 F. moniliforme var. subglutinans, 427
F. napiforme, 319, 411–412
F. nivale, 431
Fennellia, 240, 284–285 F. nygamai, 319, 412–414
Fennellia flavipes, 285–286 F. oxysporum, 320, 414–418
F. nivea, 286–288 F. pallidoroseum, 407
Filobasidiella bacillispora, 540 F. proliferatum, 320, 418–421
Filobasidiella neoformans, 537, 540 F. sacchari, 320, 421–422
F. neoformans var. bacillispora, 539– F. sacchari var. subglutinans, 427
541 F. semitectum, 407
Index 783
F. solani, 320, 422–427 H. capsulatum var. capsulatum, 204–

F. solani var. coeruleum, 403 206, 210
F. subglutinans, 427–429 H. capsulatum var. dubosii, 197, 204,
F. vasinfectum, 434 206, 210–211
F. verticillioides, 320, 429–431 H. capsulatum var. farciminosum, 197,
Fusidium terricola, 376 207
Hormographiella aspergillata, 669
Galactomyces geotrichum, 521 H. verticillata, 669
Genetic diversity among fungi, 693 Hormonema, 571, 598–599
Genetic variation among fungal popula- Hormonema dematioides, 598–599, 601
tions, 715–719 Hortaea werneckii, 590, 606
examples, 719 Hymenomycetes, 9–10, 535
issues and analytical methods, 715– Hyphomycetes, 10–11
719 Hypocrea koningii, 444
Geomyces pannorum, 199, 230 H. rufa, 447
Geotrichum, 6 H. schweinitzii, 440
Geotrichum candidum, 517 Hypocreaceae, 8–9
Gibberella, 9 Hypocreales, 8–9, 359–450
Gibberella fujikoroi, 407
G. fujikoroi var. subglutinans, 427 Icthyasporea, 11
G. intricans, 406 Issatchenkia orientalis, 517, 522
G. moniliformis, 429
G. nygami, 412–414 Keratinomyces ajelloi, 185
Glomerella, 663 Kernia, 638
Gonimochaeta, 54 Kingdom Fungi, 3–12
Graphium, 638–639 Kingdom Protoctista, 2, 12–13
Graphium eumorphum, 638–641 Kingdom Straminipila, 2, 12–13
Gymnascella dankaliensis, 199 Kluyveromyces maxianus, 518, 522
G. hyalinospora, 199, 229
Gymnoascaceae, 7–8, 149, 195, 227 Labyrinthista, 12–13
Gymnoascus arxii, 221 Labyrinthula, 12
G. gypseus, 143,158 Labyrinthula thaidis, 53
L. thaisi, 53
Haliphthoraceae, key to species of, 26– L. jeremarina, 53
29 Labyrinthuloides, 12, 18
Haliphthoros, 53 Labyrinthuloides haliotidis, 53
Halodaphnea, 53 Lacazia (Loboa) laboi, 11
Hansenula anomala, 517, 521–522 Lagenidium giganteum, 48, 50, 52
Haptoglossa, 13, 54 Lasiodiplodia theobromae, 664
Haptoglossales, 13 Lecythophora, 658
Helminthosporium, 573 Lecythophora hoffmannii, 608, 658–659
Hemicarpenteles, 240, 313 L. mutabilis, 608, 656–659
Herpotrichiellaceae, 7–8 Leptolegnia chapmanii, 48
Heterokonta, 12–13 Leptomitales, 12–13
Histoplasma capsulatum, 196–197, 210, Leptosphaeraceae, 8–9
204 Leptosphaeria, 461
784 Index
Leptosphaeria senegalensis, 461–464 M. manginii, 644–647

L. tompkinsii, 9, 463–466 Microdochium, 431
Lomentospora prolificans, 640, 642 M. niger, 644
Lophotrichus, 638 M. nivalis, 431–433
M. nivalis var. major, 433
Madurella, 599–602 M. oryzae, 433–434
Madurella grisea, 599–602 Microsphaeropsis olivaceus, 667
M. mycetomatis, 599–602 Microsporum, 6, 143, 146, 148, 152, 169
Majocchi’s granuloma, 141 Microsporum amazonicus, 178
Malassezia, 535, 547–548 M. audouinii, 143, 146, 163, 169–170
Malassezia furfur, 518, 522, 548–550 M. boullardii, 181
M. globosa, 522, 549, 551–552 M. canis, 161, 163, 170–171
M. obtusa, 522, 550 M. canis var. canis, 163
M. pachydermatis, 518, 548, 550–551 M. canis var. distortum, 162
M. restricta, 522, 552 M. cookei, 155
M. slooffiae, 522, 530, 549–550 M. de Clercq, 180
M. sympodialis, 522, 551, 578 M. distortum, 161
Malasseziales, 9, 535 M. equinum, 170
Malbranchea, 7, 198, 225 M. ferrugineum, 171, 176
Malbranchea gypsea, 226–227 M. fulvum, 156, 181
M. pulchella, 225 M. gallinae, 171–172
Massospora, 128 M. gypseum, 146, 158–160, 170
Meristhacraceae, 4, 6, 129 M. magellanicum, 186–187
Mating, zygomycetes, 75, 77–81 M. mentagrophytes, 142
Medically important fungi, typing of, M. nanum, 161–162
678–691 M. persicolor, 164
DNA-DNA hybridization of, 680 M. praecox, 172
DNA sequencing of, 689–691 M. racemosum, 165
electrophoretic karyotype of, 680 M. ripariae, 187
isozyme electrophoresis of, 618– M. rivalieri, 169
679 M. vanbreuseghemii, 157–158
PCR-based methods for, 682–687, Mitosporic Fungi, 2, 5, 9–12
689 Monoblepharidales, 3–4
restriction fragment length polymor- Monopodial branching, 74
phism, 687–689 Molecular epidemiology, 692–693
Merosporangia, 75 Moniliaceous hyphomycetes, 11, 655–
Metarhizium, 659 662
Metarhizium anisopliae, 659–660 Monographella albescens, 433
Metschnikowia pulcherrima, 518, 522 M. nivalis, 431
Microascaceae, 8, 638–650 Mortierella, 5, 67, 70
Microascales, 8, 638–651 key to the sections and relevant spe-
Microascus, 637–638 cies of, 114–116
Microascus brevicaulis, 643 pathogenic species of, 116–117
M. cinereus, 647–649 Mortierella alpina, 117
M. cirrosus, 649–651 M. polycephala, 117
M. desmosporus, 649 M. wolfii, 5, 7, 116–117
Index 785
M. zychae, 117 N. cajetani (‘‘cajetana’’), 155

Mortierellaceae, 4–5, 111, 116–117 N. cookiella, 180
key to the genera of, 111 N. corniculata, 180
Mrakia, 542 N. fulva, 156
Mucor, 4, 67, 70, 87, 91–99 N. grubyi (‘‘grubyia’’), 157
differential characters of the patho- N. gypsea, 158
genic species of, 101 N. gypsea var. fulva, 156
key to species of, 92–97 N. gypsea var. gypsea, 158
Mucor amphibiorum, 70 N. incurvata, 150, 160
M. bainieri, 79 N. obtusa, 161
M. caninus, 92 N. otae, 161
M. circinelloides, 86, 98, 101 N. persicolor, 164
M. indicus, 70, 98–99, 101 N. quinckeani, 164
M. genevensis, 79 N. racemosa, 165
M. hiemalis, 78–79, 99–101 Nannizziopsis vriesii, 198, 222–223
M. racemosus, 99–101 Nattrassia mangiferae, 662, 664–667
M. ramosissimus, 77, 98, 101 Nectria, 9
M. stolonifer, 106 Nectria cinnabarina, 448
Mucoraceae, 4 N. episphaeria, 402
differential characters of the genera N. haematocca, 422
of, 87 N. radicicola, 389
key to the genera of, 84–87 Nematophthora gynophila, 54
Mucorales, 4–5, 67–121 Neocallimasticales, 3
cultivation of, 69–71 Neocallimasticaceae, 18
direct examination for, 68–96 Neocallimastix, 18
history of, 68 Neocosmospora, 434
key to the families of, 83–84 Neocosmospora vasinfecta, 434–436
Mucormycoses, 67, 72–73 Neopetromyces, 240, 293–294
cutaneous, 70, 72 Neosartorya, 240, 307–308
disseminated, 73 Neosartorya fischeri, 247, 308–310
gastrointestinal, 73 N. pseudofischeri, 247, 308–310
pulmonary, 72 N. spinosa, 247
rhinocerebral, 72 Neotestudina, 459
Myceliophthora thermophila, 660–661 Neotestudina rosatii, 7, 459–460
Myconcentrospora acerina, 602–603 Neozygitaceae, 4, 6, 129
Mycoleptodiscus, 603 Neurospora, 9
Mycoleptodiscus indicus, 603–604
Mycosphaerellaceae, 8 Ochroconis gallopava, 586
Myriodontium keratinophilum, 668 Olpidium, 3
Myxotrichaceae, 7–8, 149, 195, 229 Olpidium/Rhizophydium, 47–48
Myxotrichum deflexum, 199, 230–231 Omnidemptus, 603
Myzocytiopsidiales, 12–13 Onychocola canadensis, 199, 227–
Myzocytiopsis, 54 228
Onygenaceae, 6–8, 149, 195, 200
Nannizzia, 149–150 comparison to Arthrodermataceae,
Nannizzia borellii, 178 202–203
786 Index
Onygenales, 6–8, 141, 149, 195–236 P. janthinellum, 331

Ophiostoma, 7, 457 P. lilacinum, 363
Ophiostoma stenoceras, 457–459 P. marneffei, 240, 316, 344–346
Ophiostomataceae, 7–8, 638 P. melinii, 331
Ophiostomatales, 7–8, 451–459 P. notatum, 333
identification of, 452 P. oxalicum, 331–332
Ovadendron sulphureo-ocheaceum, 199, P. piceum, 346–348
231–232 P. puberulum, 337
P. purpurogenum, 347–349
Paecilomyces P. rugulosum, 348–349
identification of, 354–355 P. spinulosum, 328–329
unidentified species associated with P. verruculosum, 349–350
disease, 353–354 Peronosporomycetes, 12–13, 17–66
Paecilomyces (Eurotialean Part), 352– association with Aschelminthes, 21–
353 25
Paecilomyces (Hypocrealean Part), 361 associated with protozoan Protoctista,
Paecilomyces fumoseroseus, 361–362 31–32
P. javanicus, 362–363 classification of, 19
P. lilacinus, 240, 317, 363–367 Peronosporomycetidae, 12–13
P. marquandii, 366–368 Petriella, 637–638, 642
P. terricola, 376 Petriellidium boydii, 639
P. variotii, 240, 317, 355–358 Petromyces, 240, 296–297
P. viridis, 357–359 Petromyces albiaceus, 298–299
Paracoccidioides brasiliensis, 7, 13, Phaeococcomyces, 571
198, 209, 216–217 Phaeoacremonium, 604, 658
Pathogenic fungi, molecular methods Phaeoacremonium inflatipes, 604
for identification of, 677–694 P. parasiticum, 604–605, 658
Penicillium, 315–324 P. rubrigenum, 604–605
identification techniques for, 321–323 Phaeoanellomyces, 605
laboratory characters of, 317 Phaeoanellomyces elegans, 605–606
natural (teleomorph-based) group of, P. werneckii, 590, 592, 605–608
316 Phaeotrichonis, 606
unidentified species associated with Phaeotrichonis crotariae, 606–607, 609
disease, 323–324 Phialemonium, 661–662
Penicillium brevicompactum, 332–333 Phialemonium curvatum, 662
P. chysogenum, 333–337 P. dimorphosporum, 662
P. citreonigrum, 325–326 P. obovatum, 662
P. citrinum, 329–330 Phialophora, 7, 607
P. commune, 337 Phialophora americana, 607–608,
P. cyclopium, 337 610
P. dangeardii, 342–343 P. bubakii, 607
P. decumbens, 326–328 P. hoffmannii, 658
P. dupontii, 343–344 P. repens, 607–611
P. emersonii, 340–341 P. richardsiae, 609, 611–612
P. expansum, 338 P. verrucosa, 607, 612
P. griseofulvum, 338–340 Phlyctochytrium, 3
Index 787
Phoma, 667 [Populations of fungi]

Phoma cruris-hominis, 667 issues and rationales concerning,
P. eupyrena, 667 705–707
P. glomerata, 667 variation analysis of, 705–715
P. hibernica, 667 Protista, 2, 12–13
P. leveillei, 668 Prototheca, 545
P. minutella, 667 Pseudallescheria, 637–639
P. minutispora, 667 Pseudallescheria boydii, 8, 639–643
P. ocula-hominis, 667 P. angusta, 640
P. sorghina, 667 P. ellipsoidea, 640
Phomopsis, 668 P. fusoidea, 640
Phycomycoses, 67 Pseudoaracniotus orissi, 221
Pichia, 518, 523 Pseudodelitschia coriandri, 459
Pichia fermentans, 523 Pseudoeurotiaceae, 8, 466–467
P. guilliermondii, 518, 523 Pseudoeurotium, 466
P. jadinii, 523 Pseudoeurotium ovale, 466–467
P. membranaefaciens, 523 Pseudomicrodochium, 612
P. norvegensis, 518, 523 Pseudomicrodochium suttonii, 612–613
Piedraia, 461 Pseudomycetoma, 141
Piedraia hortae, 7, 461–462 Pseudophaeotrichum sudanense, 459
Piedraiaceae, 7–8 Pseudosphaerita, 55
Pinoyella simii, 166 Pseudozyma, 553
Pithoascina langeronii, 655 Pyrenochaeta, 668
Pithoascus, 638 Pyrenochaeta mackinnonii, 668
Pithoascus langeronii, 655 P. romeroi, 668
Pityrosporum, 518, 548 Pythiales, 12–13
Pityrosporum obiculare, 551 Pythium insidiosum, 12, 46–47
P. ovale, 552
Plasmodiophoromycetes, 12–13 Ramichloridium, 613
Plasmodiaphoromycetes, association Ramichloridium obovoideum, 613–614
with Aschelminthes, 21–25 Renispora flavissima, 205, 223
Pleosporaceae, 8–9 Rhinocladiella, 614–615
Pleosporales, 8–9, 461–466 Rhinocladiella aquaspersa, 615–616
Pleurophoma, 667–668 R. atrovirens, 616
Pleurophomopsis lignicola, 667 Rhinosporidium seeberi, 11
Pneumocystis carinii, 11 Rhipidiomycetidae, 12–13
Polypaecilum, 351 Rhizoctonia, 11
Polypaecilum insolitum, 351–352 Rhizoids, 73
Populations of fungi, 703–719 Rhizomucor, 4, 67, 70, 87, 99–103, 106
clonality and recombination of differential charcteristics of the spe-
diploid species, 713–714 cies of, 106
clonality and recombination of key to the species of, 102
haploid species, 707–713 Rhizomucor miehei, 103–104
definition of, 703–704 R. parasiticus, 102
epidemic clones of, 714–715 R. pusillus, 79, 102–103
genetic markers of, 705 R. tauricus, 103, 105
788 Index
Rhizophydium, 3 Scolecobasidium constrictum, 618–619

Rhizopus, 4, 67, 70, 87, 105–111 S. gallopava, 617–618
diagnostic characters of, 110 Scopulariopsis, 8, 637–639, 647, 650
key to species of, 108–109 Scopulariopsis acremonium, 649
Rhizopus arrhizus, 109 S. asperula, 644
R. homothallicus, 79 S. brevicaulis, 643–645
R. microsporus, 70, 74, 106, 109–110, S. brumptii, 644
112–113 S. candida, 644–647
R. oryzae, 70, 74, 106, 109 S. divaricata, 351
R. sexualis, 79 S. koningii, 643
R. schipperae, 110–111 Scytalidium, 662
R. stolonifer, 70, 74, 106 Scytalidium dimidiatum, 662, 664–667
Rhodotorula, 9, 535, 552 S. hyalinum, 662, 666
Rhodotorula glutinis, 518, 523, 553 S. lignicola, 666–667
R. minuta, 552–553 Sepedonium, 205
R. mucilaginosa, 518, 552–553 Slopalinida, 17
R. rubra, 518, 523, 552 Sordariales, 8–9, 651–655
Sphaerita, 55
Sabouraudites gypseum, 143 Spizellomycetales, 3–4
S. langeronii, 164 Sporangia, 73, 75
Saccharomycetaceae, 7–8 Sporangioles, 75
Saccharomycetales, 6, 8 Sporangiophores, 73
Saccharomyces cerevisiae, 519, 523 Sporidiales, 9–10, 535
S. exiguus, 523 Sporobolomyces, 9, 535, 553
Saksenaea, 67, 70 Sporobolomyces holsaticus, 519, 523,
Saksenaea vasiforme, 5, 117–119 553
Saksenaeaceae, 4–5, 118–119 S. johnsonii, 553
Salilagenidiaceae, key to the species of, S. roseus, 519, 523, 553
26–29 S. salmonicolor, 519, 523, 553
Salilagenidiales, 12–13 Sporothrix, 454, 619
Salilagenidium, 53 Sporothrix cyanescens, 619
Saprolegniales, 12–13 S. schenckii, 7, 454–456, 619–621
Saprolegnia parasitica, 42–44 S. schenckii var. luriei, 619–621
Saprolegniomycetidae, 12–13 Sporotrichum schenkii, 454
Sarcinomyces, 545, 617 Sporotrichum thermophile, 660
Sarcinomyces phaeomuriformis, 617– Stolons, 73
618 Stephanoascus ciferrii, 519, 523–524
Sarcinosporon, 545 Sydowia polyspora, 599
Scedosporium, 637, 639 Sympodial branching, 74
Scedosporium apiospermum, 8, 639– Syncephalastraceae, 4–5, 119
643 Syncephalastrum, 67, 70, 119
S. inflatum, 640 Syncephalastrum racemosum, 5, 76, 119
S. prolificans, 640–643
Schizophylaceae, 668 Taeniolella, 621
Schizophyllum commune, 9, 668–669 Taeniolella aristata, 622–623
Scolecobasidium, 586, 617 T. exilis, 621–622
Index 789
T. stilaspora, 621 T. georgia, 179–180

Talaromyces, 340 T. gloriae, 183
Talaromyces emersonii, 339–341 T. gourvilii, 173
T. flavus, 342–343 T. interdigitale, 152, 155
T. thermophilus, 343–344 T. kanei, 145
Tetraploa, 622 T. krajdenii, 145
Tetraploa aristata, 622–623 T. longifusum, 188
Thamnidiaceae, 4–5, 8, 120–121 T. mariatii, 188
key to the genera of, 120–121 T. megninii, 173–174
Thermoascaceae, 8, 126, 240 T. mentagrophytes, 152, 154–155, 163,
Thermoascus, 240 167–168, 172–173, 175
Thermomucor, 67, 70, 87, 105 T. mentagrophytes var. nodulare, 145
Thermomucor indicae-seudaticae, 104– T. persicolor, 165
105, 107 T. phaseoliforme, 188–189
Thermomyces, 622 T. quinckeanum, 152, 155
Thermomyces lanuginosus, 622–623 T. raubitschekii, 145
Tilletiopsis, 553 T. rubrum, 167, 174–175, 188
Tilletiopsis albescens, 553 T. schoenleinii, 146, 175
T. minor, 553, 659 T. simii, 166
Torulaspora delbrueckii, 517 T. soudanense, 173, 175–176
Torulopsis candida, 515 T. terrestre, 183–184
T. pintolopesii, 515 T. thuringiense, 184
Tremellales, 9–10, 535 T. tonsurans, 143, 176
Trichocomaceae, 7, 240 T. tonsurans var. tonsurans, 176
Trichoderma, 437 T. tonsurans var. sulfureum, 176
identification of, 437–440 T. vanbreuseghemii, 182
Trichoderma citrinoviride, 440–441 T. verrucosum, 146, 176–178
T. harzianum, 441–444 T. violaceum, 173, 178
T. koningii, 444–445 T. yaoundei, 178
T. longibrachiatum, 445–447 Trichosporon, 9, 535, 542–544
T. pseudokoningii, 447 Trichosporon asahii, 519, 524, 543–
T. viride, 447–448 545
Tricomomaceae, 7–8 T. asteroides, 524, 543, 547
Trichomycetes, 4 T. beigelii, 519, 542–543
Trichophyton, 6, 143–144, 146, 148, T. capetatum, 515
152, 172 T. coremiiforme, 543
Trichophyton ajelloi, 153, 185 T. cutaneum, 519, 524, 543, 547
T. ajelloi var. nanum, 186 T. domesticum, 543, 547
T. concentricum, 172 T. faecale, 543
T. equinum, 172–173 T. inkin, 524, 543, 545–546
T. equinum var. autotrophicum, 173 T. montevideense, 543
T. erinacei, 152, 155 T. mucoides, 519, 524, 542–543, 546
T. ferrugineum, 171 T. ovoides, 524, 542–543, 546–547
T. fischeri, 188 T. pullulans, 542
T. flavescens, 181 Trichosporonales, 9–10, 535
T. fluviomuniense, 174 Trichurus, 638
790 Index
Triterachium album, 658 Wardomyces, 638

Tubercularia, 448 Wardomycopsis, 638
Tubercularia vulgaris, 448–449
Xylohypha, 576
Xylohypha bantiana, 576
Ulocladium, 624 X. emmonsii, 576
Ulocladium chartarum, 624
Uncinocarpus, 225 Yarrowia lipolytica, 519–520, 524
Uncinocarpus orissi, 221
U. reesii, 199, 225–226 Zoopathogenic fungi, introduction to the
Urediniomycetes, 9–10, 535 taxonomy of, 1–13
Ustilaginomycetes, 9–10, 535 Zopfia rosatii, 459
Zygomycetes, 4, 67, 82
key to the orders of, 82
Verticillium, 449
mating by, 75, 77–81
Verticillium nigrescens, 449–450
zygospore production in pathogenic
Volutella, 451
species, 81
Volutella cinerescens, 451–452
Zygomycoses, 67
Zygomycota, 2, 4–6
Wangiella, 624 Zygorhynchus moelleri, 79
Wangiella dermatitidis, 590, 624–626 Z. japonicus, 79
W. heteromorpha, 590, 626 Zygospores, 75, 81

(Mycology Series 16) D.H. Howard-Pathogenic Fungi in Humans and Animals-Marcel Dekker (2003)

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Pathogenic Fungi

Marcel Dekker, Inc. New York • Basel

Copyright © 2002 by Marcel Dekker, Inc. All Rights Reserved.

Library of Congress Cataloging-in-Publication Data

This book is printed on acid-free paper.

Eastern Hemisphere Distribution

World Wide Web

Copyright  2003 by Marcel Dekker, Inc. All Rights Reserved.

Current printing (last digit):

PRINTED IN THE UNITED STATES OF AMERICA

1. Viruses and Plasmids in Fungi, edited by Paul A. Lemke

Additional Volumes in Preparation

In 1947, Walter J. Nickerson edited a volume on the Biology of Pathogenic Fungi,

impossible for any single author to give an in-depth presentation. Accordingly,

phagocytic cells and, in an Addendum, a reconsideration of the genus Exophiala,

Preface to the Second Edition iii

Part One Classiﬁcation and Nomenclature

1. An Introduction to the Taxonomy of Zoopathogenic Fungi 1

2. The Peronosporomycetes and Other Flagellate Fungi 17

3. Zygomycetes: The Order Mucorales 67

4. Zygomycetes: The Order Entomophthorales 127

5. Onygenales: Arthrodermataceae 141

6. Ascomycetes: The Onygenaceae and Other Fungi from the

7. Aspergillus, Fusarium, Sporothrix, Piedraia, and Their Relatives 237

8. Yeasts: Blastomycetes and Endomycetes 499

9. Basidiomycetous Yeasts 535

10. Dematiaceous Hyphomycetes 565

11. Miscellaneous Opportunistic Fungi: Microascaceae and Other

Part Two Fungal Populations

12. Molecular Methods to Identify Pathogenic Fungi 677

13. Population Genetic Analyses of Medically Important Fungi 703

14. Genetic Instability of Candida albicans 723

Teun Boekhout, Ph.D. Centraalbureau voor Schimmelcultures, Institute of the

Michael W. Dick, D.Sc. Department of Botany at Earley Gate, School of Plant

Frank M. Dugan, Ph.D. USDA-ARS Western Regional Plant Introduction

Eveline Guého, Ph.D. Mauves sur Huisne, France

Kevin C. Hazen, Ph.D. Department of Pathology, University of Virginia

Dexter H. Howard, Ph.D. Departments of Microbiology, Immunology, and

Susan A. Howell, B.Sc., Ph.D. Department of Medical Mycology, St. John’s

Shung-Chang Jong, Ph.D. Department of Microbiology, American Type Cul-

Thomas G. Mitchell, Ph.D. Department of Genetics and Microbiology, Duke

Arvind A. Padhye, Ph.D. Mycotic Disease Branch, Division of Bacterial and

Elena Rustchenko, Ph.D. Department of Biochemistry and Biophysics, Uni-

Wiley A. Schell, M.S. Department of Medicine, Duke University Medical Cen-

M. A. A. Schipper, Ph.D. Centraalbureau voor Schimmelcultures, Institute of

Fred Sherman, Ph.D. Department of Biochemistry and Biophysics, University

Lynne Sigler, M.S. Microfungus Collection and Herbarium, Devonian Botanic

J. A. Stalpers, Ph.D. Centraalbureau voor Schimmelcultures, Institute of the

Richard Summerbell, Ph.D. Centraalbureau voor Schimmelcultures, Institute

Irene Weitzman, Ph.D. Department of Pathology, Columbia University Col-

Jianping Xu, Ph.D. Department of Biology, McMaster University, Hamilton,

I. THE CLASSIFICATION OF FUNGI

Table 1 The Kingdoms and Phyla

II. NOMENCLATURE OF HIERARCHAL GROUPS

The pathogenic organisms traditionally considered as fungi* are now distributed

* Fundamental descriptions of fungi may be found in the textbooks on medical mycology by K. J.

III. THE KINGDOM FUNGI

1. Neocallimasticales. The members of the order differ from other mem-

Table 2 The Phylum

Note: For a more complete treatment of this

4. Spizellomycetales. The members of this order are diverse ecologically,

Table 3 The Phylum Zygomycota

b. Syncephalastraceae. One species, Syncephalastrum racemosum,

e. Meristacraceae. The family Meristacraceae contains obligate para-