Micología Tropical - Micromycetes

Tropical Mycology:
Volume 2, Micromycetes
Tropical Mycology:
Volume 2, Micromycetes
Edited by
Roy Watling,
Juliet C. Frankland,
A.M. Ainsworth,
Susan Isaac
and
Clare H. Robinson
CABI Publishing
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Library of Congress Cataloging-in-Publication Data

Tropical mycology / edited by Roy Watling ... [et al.].
p. cm.
Selected papers of the Millenium Symposium held April 2000 at the Liverpool John Moores
University and organized by the British Mycological Society.
Includes bibliographical references and index.
Contents: v. 1. Macromycetes.
ISBN 0-85199-542-X (v. 1 : alk. paper)
1. Mycology- -Tropics- -Congresses. 2. Fungi- -Tropics- -Congresses. I. Watling, Roy.
QK615.7.T76 2001
616.9¢69¢00913- -dc21
2001025877
ISBN 0 85199 543 8
Typeset in Photina by Wyvern 21 Ltd.

Printed and bound in the UK by Biddles Ltd, Guildford and King’s Lynn.
Contents
Dedication vii
Contributors ix
Preface xi
1 Why Study Tropical Fungi? 1

D.L. Hawksworth
2 Key to Tropical Species of Nectria-like Fungi 13

G.J. Samuels, A.Y. Rossman and H.-J. Schroers
3 A Reassessment of the Taxonomy of Some Tropical Sooty Moulds 33

J.L. Faull, I. Olejnik, M. Ingrouille and D. Reynolds
4 Lignicolous Freshwater Higher Fungi with Reference to their

Teleomorph and Anamorph Stages 41
S. Sivichai, E.B. Gareth Jones and N.L. Hywel-Jones
5 The Pandanaceae – Does it Have a Diverse and Unique Fungal

Biota? 51
E.H.C. McKenzie, S.R. Whitton and K.D. Hyde
6 Aspects of Graminicolous Downy Mildew Biology: Perspectives for

Tropical Plant Pathology and Peronosporomycetes Phylogeny 63
M.A. Spencer and M.W. Dick
v
vi Contents
7 Invasive Neotropical Pathogens of Tree Crops 83

H.C. Evans
8 Lichens of Tropical Forests 113

B.J. Coppins and P. Wolseley
9 The Importance of Invertebrate-pathogenic Fungi from the Tropics 133

N.L. Hywel-Jones
10 Tropical Mycoses: Hazards to Travellers 145

E.G.V. Evans and H.R. Ashbee
11 Recent and Future Discoveries of Pharmacologically Active

Metabolites from Tropical Fungi 165
G. Bills, A. Dombrowski, F. Peláez, J. Polishook and Zhiqiang An
Index 195
Dedication
It is with great pleasure that this volume is dedicated to Dr Reginald W.G.

Dennis, who has made many important contributions to our understanding
of tropical fungi, especially in unravelling the complexities of members of the
large genus Xylaria and allies. His mycological interests range from the
hyaloscyphaceous and helotiaceous ascomycetes to pyrenomycetous taxa,
agarics, rusts and gasteromycetes. Visits to Trinidad and Venezuela, in 1949
and 1958, resulted in several papers, which were to be only a foretaste of his
Fungus Flora of Venezuela and Adjacent Countries, published in 1970 and illus-
trated with his own watercolours. It has become an indispensable companion
to any mycologist working in South America and is a useful introductory guide
for all tropical fungus researchers. He began his career as a plant pathologist
working in Scotland at the West of Scotland College of Agriculture and Dept
of Agriculture of Scotland, East Craigs, as they were then, with a sandwich
period between in the School of Agriculture, Cambridge. He moved to taxon-
omy from his earlier work on plant pathogens, including virus diseases of
cereal and root crops, when he joined the staff of the Royal Botanic Gardens,
Kew in 1944, where he still works even though retired. He is an Honorary
Member of the British Mycological Society, The Mycological Society of
America and the Swiss Mycological Society, and is a Corresponding Member
of the Botanical Society of Argentina.
A special Festschrift published in Vol. 31 of the Kew Bulletin was dedi-
cated to Dr Dennis on his retirement from service and includes therein an
account of his works and interests. He was 90 in July 2000.
Roy Watling, June 2001
(See Spooner, B.J. (2000) Mycological Research 104, 1410.)
vii
Contributors
Zhiqiang An, Centro de Investigación Básica, Merck, Sharp & Dohme de España S.A.,
Josefa Valcárcel 38, 28027 Madrid, Spain
H. Ruth Ashbee, Mycology Reference Centre, Division of Microbiology, University of
Leeds, LS2 9JT and General Infirmary, Leeds LS1 3EX, UK
Gerald Bills, Centro de Investigación Básica, Merck, Sharp & Dohme de España S.A.,
Josefa Valcárcel 38, 28027 Madrid, Spain
Brian J. Coppins, Royal Botanic Garden Edinburgh, Edinburgh EH3 5LR, UK
M.W. Dick, Centre for Plant Diversity and Systematics, School of Plant Sciences, The
University of Reading, Whiteknights, Reading RG6 6AS, UK
Anne Dombrowski, Natural Products Drug Discovery, Merck Research Laboratories,
Rahway, NJ 07065, USA
E.G.V. Evans, Mycology Reference Centre, Division of Microbiology, University of
Leeds, Leeds LS2 9JT, UK and General Infirmary, Leeds LS1 3EX, UK; present
address: Department of Medical Microbiology, University of Wales College of
Medicine, Heath Park, Cardiff CF14 4XN, UK
Harry C. Evans, CABI Bioscience, Silwood Park, Ascot, Berkshire SL5 7TA, UK
J.L. Faull, School of Biological and Chemical Sciences, Birkbeck College, University of
London, Malet Street, London WC1E 7HX, UK
E.B. Gareth Jones, BIOTEC–Mycology, Yothi Laboritories, 73/1 Rama 6 Road,
Rajdhevee, Bangkok 10400, Thailand
David L. Hawksworth, Departamento de Biología Vegetal II, Facultad de Farmacia,
Universidad Complutense, Plaza de Ramon y Cajal, Ciudad Universitaria, E-28040
Madrid, Spain
Kevin D. Hyde, Centre for Research in Fungal Diversity, Department of Ecology and
Biodiversity, The University of Hong Kong, Pokfulam Road, Hong Kong, SAR,
China
ix
x Contributors
Nigel L. Hywel-Jones, BIOTEC–Mycology, Yothi Laboratories, 73/1 Rama 6 Road,

M. Ingrouille, School of Biological and Chemical Sciences, Birkbeck College,
University of London, Malet Street, London WC1E 7HX, UK
Eric H.C. McKenzie, Landcare Research, Private Bag 92170, Auckland, New Zealand
I. Olejnik, School of Biological and Chemical Sciences, Birkbeck College, University
of London, Malet Street, London WC1E 7HX, UK
Fernando Peláez, Centro de Investigación Básica, Merck, Sharp & Dohme de España
S.A., Josefa Valcárcel 38, 28027 Madrid, Spain
Jon Polishook, Natural Products Drug Discovery, Merck Research Laboratories,
Rahway, NJ 07065, USA
D. Reynolds, Botany Section, Natural History Museum, 900 Exposition Boulevard,
Los Angeles, CA 90007, USA
Amy Y. Rossman, Systematic Botany and Mycology Laboratory, United States
Department of Agriculture, Agricultural Research Service, Beltsville, MD 20705,
USA
Gary J. Samuels, Systematic Botany and Mycology Laboratory, United States
Department of Agriculture, Agricultural Research Service, Beltsville, MD 20705,
USA
Hans-Josef Schroers, Centraalbureau voor Schimmelcultures, 3508 AD Utrecht,
The Netherlands
Somsak Sivichai, BIOTEC–Mycology, Yothi Laboratories, 73/1 Rama 6 Road,
M.A. Spencer, Centre for Plant Diversity and Systematics, School of Plant Sciences,
The University of Reading, Whiteknights, Reading RG6 6AS, UK
Stephen R. Whitton, Landcare Research, Private Bag 92170, Auckland, New
Zealand
Pat Wolseley, Department of Botany, The Natural History Museum, Cromwell Road,
London SW7 5BD, UK
Preface
Half of the membership of the British Mycological Society resides abroad and
many of these members are from countries which are generally considered to
be situated in ‘The Tropics’. Since the Sixth General Meeting of the Society
held in Birmingham in 1988, the Society has recognized the increasing impor-
tance of tropical mycology and Council felt that it would be appropriate to
bring both these factors of membership and research interests together to
celebrate the millennium. A symposium therefore was organized for April
2000 to cover as many aspects as possible of the tropical mycobiota. This
volume is one of two which result from this Millennium Symposium held at
the Liverpool John Moores University. In order to broaden the scope and the
depth of the subjects covered, other chapters not formally presented at the
symposium are included. This present volume deals with the microfungi;
Volume 1 deals with the macrofungi.
The British Mycological Society has now organized two meetings specifi-
cally on Tropical Mycology and both were held in Liverpool, coincidentally a
city with a long history of contacts with the areas of the world which lie
between 25° North and South of the equator and known to us all as ‘The
Tropics’. The present pair of books will therefore act as a companion to the
Proceedings of the meeting held in 1992, which were published 1 year later
under the title Aspects of Tropical Mycology. The chapters included here have
expanded the ideas first discussed therein and each is a summary of the state
of knowledge in the respective field. Some of the studies have indirect
connections to the two expeditions the Society has organized to tropical areas,
the first to Ecuador in 1993 and the second to Thailand in 1997.
In the selection of subjects, attempts have been made to bring together
xi
xii Preface
papers which offer a wide spectrum of information, linking results from

studies in the New World to data from the Old World tropics. Contributions
by 26 researchers provide 11 chapters covering the major area of the tropics.
Although our knowledge of the tropical mycotas is far from adequate, the
surge of interest in the fungi of these areas has expanded our knowledge
several-fold since 1992, as documented in the first chapter, the contents of
which could equally apply to both the macro- and the microfungi. However,
in this first chapter, the micro-forms have been emphasized by the author and
so it acts as a convenient opening statement to the book.
The additional chapters undoubtedly interlink, but they have been
arranged in such a way as to bring together like interests for easy reference.
It is refreshing to see that several of the chapters cover various aspects of
mycogeography placed in a geological timescale. Chapters 2–4 cover broad
issues of systematics whilst Chapters 5–7 explore the interrelationships
between fungi and plants, a theme which is continued in Chapters 8–10, but
in these the interacting organisms are either algae or from the animal king-
dom. The concluding chapter explains recent discoveries of pharmacologically
active metabolites isolated from tropical fungi and what the future holds in
this area of exploration.
It is essential in all fields of mycology to have a firm taxonomic base-line
from which to work and a good key by which researchers can identify the
organisms they are studying. This has been made possible for the first time
for the nectriaceous fungi by the authors of Chapter 2. Theirs has been a wide-
reaching study, which will place many unfamiliar and obscure names into
context and make it possible to address the relationships of some of our
widespread nectriaceous plant pathogens both in the tropics and in the tem-
perate world. For instance, the ‘Panama disease’ of bananas, which is
currently showing a resurgence, is caused by a race of the nectriaceous
Fusarium oxysporum and is a devastating pathogen on a crop on which many
tropical economies rely.
Chapter 3 deals with the finer details of the systematics of sooty moulds,
a group of fungi widespread in the tropics but also a familiar sight in
glasshouse conditions in the temperate world. The authors extend the patient
work commenced by a former vice-president of the British Mycological Society,
Stan Hughes. It was he who first unravelled the relationships for this group
of fungi, the various sexual and asexual stages, the latter often multiple,
and made sense of the biology. More importantly, many of the herbarium
collections languishing in national herbaria under the broad name of
Hormiscium etc. were finally determined. Reynolds expanded this work and
with his colleagues in Chapter 3 continues to tease apart the complex
typification of some of these obscure fungi.
Terence Ingold and John Webster, two former presidents of the British
Mycological Society, have explored the relationships of aeroaquatic
hyphomycetes. Their studies were based mainly, but not exclusively, on
Preface xiii
temperate taxa, and it was they who encouraged the search for the sexual
stages of these fungi which produce such beautiful branched water-borne
conidia. Several links are now known, but the authors of Chapter 4 extend
these observations to the anamorphic and teleomorphic stages of lignicolous
fungi in the tropics of South-East Asia.
In 1995, the author of the first chapter of this volume expressed the view
that there were of the order of 1,500,000 fungi worldwide. The authors of
Chapter 5 examine this estimate in connection with the fungi found on mem-
bers of the Pandanaceae, a tropical family of flowering plants resembling the
palms in many ways in form and structure. They found that there is a multi-
tude of fungi on members of this family, many of which are restricted in their
host range. This theme is continued in Chapter 6 where various aspects of downy
mildews of grasses are considered. With again so much specificity, it was only
logical for the authors to examine the coevolution of the host, the grasses, and
the downy mildews. This allowed them to extrapolate and place these fungi in
the same frame as the flowering plants when enquiring into the geographical
area of origin of these fungi and pattern of migration in geological time. Equally,
such evolutionary discussions provoke further questions as to the uniformity of
the mildew taxa, and the opportunity is taken to address this issue.
Chapter 7 deals with invasive neotropical pathogens of two major crops,
the export of products of which many developing countries depend on for
foreign exchange. The crops are cacao and rubber, the fungi ranging from
ascomycetes to basidiomycetes. The chapter also unravels some of the
taxonomic problems encountered in naming the causal organisms.
Lichens have only recently been incorporated into general discussions
about fungi, but they should be considered even though they are only
lichenized forms. ‘Only’ really does not reflect their unique role in plant assem-
blages or their long history of being recognized as organisms in their own
right. It is fitting therefore to include a chapter on lichens within the frame-
work of this publication. Chapter 8 tackles various aspects of tropical lichens,
based on extensive fieldwork, especially in South-East Asia. Their diversity, dis-
tribution within and outwith the rainforest canopy, and the possible environ-
mental and anthropogenic factors which affect their continual growth in
specific sites are all discussed.
Animals have long been associated with fungi but generally the interactions
which they establish, except for perhaps the medical fungal pathogens, are much
less publicized than those for plant diseases caused by fungi. However, these fungi
do play an important role in naturally occurring invertebrate and vertebrate
populations. In addition, as described in Chapter 9, the estimate of world fungi
by Hawksworth will certainly have to be reconsidered when a full study of
the fungi associated with invertebrates is taken into account, for there appears
to be great specificity expressed in these fungi. There are estimated to be
about three times, if not more, insects than fungi! The author of Chapter 9 has
taken into account, in the title of his contribution, that there are many species
xiv Preface
pathogenic on spiders and mites and that entomogenous or even entomopath-

ogenic does not cover the real world. This chapter also focuses heavily, although
broadly, on results of work in South-East Asia, introducing the reader to these
fungi as potential biocontrol organisms and as sources of useful secondary
metabolites, a fact developed further in the last chapter.
Humans, although parasitized by fungi, as demonstrated in the penulti-
mate chapter, are organisms scarcely mentioned in fungal texts, and especially
tropical taxa. Thus, Chapter 10 is doubly important as it places another type
of fungal/animal interrelationship into context with the rest of general mycol-
ogy and demonstrates the increase in these diseases as a result of freer world
travel between tropical and temperate destinations. This chapter summarizes
the state of knowledge in this potentially threatening scenario where many
practising mycologists and doctors, although well aware of the symptoms of
ringworm and athlete’s foot, are unaccustomed to the conditions caused by
some of these tropical agents.
For a long time it has been argued that we must conserve the biodiversity
of the world because it may hold hidden secondary metabolites which will cure
disease. Although this is a commendable reason, it is folly and unsubstantiated
to base the conservation of the world’s fauna and flora purely on this single sell-
ing point, although it is true that some of the most effective pharmacological
compounds have been isolated from nature. Indeed, the first three top-selling
pharmaceutical compounds are derived from fungi. The last chapter looks at
some aspects of novel compounds isolated from fungi and the possible future of
such activities in industrial mycology. The development of new technologies
based on what we already know of active fungal compunds is discussed.
Scientists, including many botanists, are blissfully unaware of the role of
fungi in the world’s ecosystems, and it is hoped that this collection of chap-
ters along with the sister volume will emphasize their importance, bringing
mycologists up to date in areas with which they are less familiar. It is also
hoped that these two books will stimulate students of mycology and be help-
ful to a whole range of biologists interested in tropical biodiversity. It would
be outstanding if they could act as a pump primer to support research pro-
grammes and even necessitate a demand to launch a third British Mycological
Society tropical expedition.
It is my great pleasure to thank an industrious team of editors: Martyn
Ainsworth, Susan Isaac, Clare Robinson, who have all given their all
unselfishly since April 2000 in reviewing and editing the chapters for this
publication and its sister volume, and especially Juliet Frankland, who has
helped me finalize the drafts ready for technical adjustment and presentation,
so they could be brought together as soon after the meeting as possible in
order to act as a base-line for future studies.
Roy Watling
Caledonian Mycological Enterprises, Edinburgh and
Royal Botanic Garden, Edinburgh, Scotland, UK
Why Study Tropical Fungi? 1
D.L. HAWKSWORTH
Departamento de Biología Vegetal II, Facultad de Farmacia,

Universidad Complutense, Plaza de Ramón y Cajal, Ciudad
Universitaria, E-28040 Madrid, Spain
Introduction
The question ‘Why study tropical fungi?’ can be addressed in different ways,
and at a variety of levels, depending on both the perspective of the person
being asked and also its relevance to the interests and concerns of the
questioner. The reasons why tropical fungi should be studied are numerous.
Here in the author’s view are the ten most important. Tropical fungi are a
major component of biodiversity, essential to the survival of other organisms,
crucial in global ecological processes, a source of novel bioactive compounds,
a source of biocontrol agents, a source of plant pathogens, a threat to human
health, able to contribute to sustainable development, a part of human
culture, ‘and are there...’. Each reason will be justified and a prognosis of trop-
ical mycology presented.
The subject has attracted fresh interest, and is now more active than at
any time in the last 50 years. However, support for basic mycological work in
developed countries has fallen at a time when the demand from a wide range
of ecologists, conservationists, and those interested in sustainable develop-
ment is greater than it has ever been. Encouragingly, the focus of work on
tropical fungi is moving to centres in the tropics where active research schools,
workshops, symposia, and mycological organizations and publications are
being established. Mycologists are now encouraged to commit to actively pro-
moting the study of tropical fungi, in order to achieve a steady improvement
in both knowledge and utilization.
The rekindling of interest in tropical mycology, particularly in fungal diver-
sity and utilization, has mainly occurred in the last 10 years. The subject has
© CAB International 2002. Tropical Mycology, Vol. 2, Micromycetes 1

(eds R. Watling, J.C. Frankland, A.M. Ainsworth, S. Isaac and C.H. Robinson)
2 D.L. Hawksworth
risen to a level not paralleled since the colonial period came to an end in the
1940s and 1950s. This revival is exemplified by several books and symposia
focusing on tropical mycology, particularly in the wake of the British
Mycological Society’s pioneering meeting on Aspects of Tropical Mycology held
in Liverpool in April 1992 (Isaac et al., 1993), and including Tropical Mycology
(Janardhanan et al., 1997) and Biodiversity of Tropical Microfungi (Hyde, 1997).
A new journal devoted entirely to the topic, Fungal Diversity, started pub-
lication in 1998. Other books have focused on particular areas in the tropics
(e.g. Hennebert, 1994; Rossman et al., 1998) or on exploitation (Palm and
Chapela, 1997; Pointing and Hyde, 2001). In addition, fungi have received a
heightened profile in general assessments of diversity in tropical and other
areas (Heywood, 1995).
At the 1992 meeting, I surveyed the extent of the tropical fungal biota,
examined the resource base available for its study, touched on its pertinence,
suggested what might be done, and cautioned that, if mycologists did not
become involved in the biodiversity arena, mycology would be increasingly
marginalized (Hawksworth, 1993). It is gratifying to have witnessed the
progress made over the last 8 years.
Fungi, while still ‘orphans’ and trailing in the biodiversity stakes
(Hawksworth, 1997), are now being noticed by a wider range of international
and national bodies, government departments, conservation agencies,
ecologists, biodiversity scientists, and naturalists than ever before. See, for
example, Kaul (2002). This is not an inconsiderable achievement, but needs
to be followed through. There is no room for complacency if we are to
capitalize on our investments to date. The next task is to enthuse the array of
potential sponsors, and newly found supporters, with impelling reasons why
work on tropical fungi should be incorporated into their portfolios and
programmes.
I should stress that the following points are necessarily selective and what
other cogent reasons can be found elsewhere in this and the companion vol-
ume (Watling et al., 2002) as to why tropical fungi should be studied.
Because they are . . .
(1) . . . a major component of biodiversity
Fungi are now generally accepted as the largest group of organisms on Earth
after the insects; as working figures, the Global Biodiversity Assessment
(Heywood, 1995) accepted estimates of 1.5 million fungi and 8 million insects,
respectively. Dissensions from this view are primarily from non-mycologists
who do not appreciate the long-term studies necessary to determine how
many fungi are present in a site or the extent of host specificity and geo-
graphical differences (e.g. May, 2000). An analysis of estimates of species
numbers made since the 1.5 million figure was first proposed (Hawksworth,
1991) suggests that, if anything, the figure is an underestimate (Hawksworth,
2001).
A remarkable 179 governments are committed to the conservation and
sustainable use of biodiversity, through their ratification of the Convention
on Biological Diversity (UNEP, 1992). This was drawn up in 1992, after
the Society’s Aspects of Tropical Mycology meeting, and entered into force on
29 December, 1993. Article 7 requires signatory countries (and the European
Union) to identify the components of biodiversity important to the
Convention’s objectives. This therefore embraces fungi, as will be stressed later,
and many developed countries in particular are now taking fungal conserva-
tion more seriously than ever before. Governments of some tropical countries
are also starting to take a broader view, including fungal components in
projects supported through agencies such as the World Bank’s Global
Environment Facility.
(2) . . . essential to the survival of other organisms
Fungi can be essential to the survival of other organisms at the individual,

species and ecosystem levels. At the individual level, the role of fungi in
mutualisms is well known, especially in the case of mycorrhizas formed with
perhaps 90% of the world’s plants, and the extent of involvement with insects
is becoming clearer. In the tropics, a surprising proportion of beetles, for
example, are fungus-feeders, over 40% of 1.2 million collected in Sulawasi,
Indonesia (Hammond, 1990). This is not just an issue for plants and smaller
organisms. Herbivorous mammals need anaerobic fungi in their stomachs to
aid the breakdown of cellulosic materials on which they feed.
Termites culturing Termitomyces mushrooms, and the gardens of leaf-
cutter ants (Fisher and Stradling, 2002) are increasingly featured in museum
displays and even introductory textbooks. Images of moths and spiders
camouflaged against lichens are also becoming increasingly familiar (e.g.
Gilbert, 2000).
However, it is the role of fungi in ecosystem functioning overall that is the
most significant for the survival of other organisms in tropical forests (Lodge
et al., 1996). They are basal in complex food webs, not least in soil where they
are the key food of many nematodes and collembolans, for instance, which in
turn are devoured by larger invertebrates. In their absence, crucial soil
processes, including decomposition and nutrient cycling, would fail and above-
ground ecosystems be endangered. Further, there are indications that elevated
carbon dioxide levels lead to some changes in populations of soil fungi, which
in turn affect the numbers of fungus-feeding invertebrates (Jones et al., 1998).
Our understanding of interactions between fungi and other organisms is
in its infancy. The significance of endophytic fungi is still only starting to be
4 D.L. Hawksworth
appreciated (Bacon and White, 2000), connections and nutrient transfers

between different plant species via mycorrhizas have only recently been rec-
ognized (Linderman, 1997), and the role of pathogenic fungi in both limiting
species and creating fungal habitats and microenvironments in natural
ecosystems is scarcely studied. The complexities to be found can only be
guessed at. Who would have thought that soil Pythium species could influence
tree patterns in mixed forests (Packer and Clay, 2000)? The significance of
such phenomena in tropical forests awaits exploration.
(3) . . . crucial in global ecological processes
Fungi have a key role in the global carbon cycle in particular. They are the
only organisms able to break down materials composed of lignin, releasing
methylated gases during the decay process. Quantative data on litter and wood
decay in tropical forests remain scant (Lodge et al., 1996), but the rates
achieved must be enormous as humic layers remain shallow in relation to the
volume of material to be processed.
In addition fungi are often ignored as a carbon sink, tying up what must
be gigatonnes of carbon in their mycelia, and further by converting rock and
soil minerals to oxalates. It is also not always remembered that, in lichen
associations, fungi are involved in fixing substantial amounts of carbon
dioxide photosynthetically through the included algae or cyanobacteria.
Cyanobacteria in lichens have another role too, fixing atmospheric nitrogen,
a nutrient often in short supply in rainforests.
(4) . . . a source of novel bioactive compounds
Ever since penicillin hit the headlines in the press in 1942, fungi have been
recognized as a source of novel compounds important for human health. The
probabilities of finding a drug that will become a world leader are small but
the rewards are immense. Leading pharmaceutical companies have been well
aware of the potential benefits from screening tropical fungi, and encouraged
by successes achieved. Many companies probably hold isolates of undescribed
species in their collections as description and identification are not necessary
prior to screening. As so many of the fungi yet to be discovered are tropical,
it follows that exploitable compounds are likely to be present in tropical species
as well as in those from other regions. See Bills et al. in this volume.
In addition to providing pharmacologically active compounds, tropical
fungi may also be sources of new antifungal agents and enzymes for indus-
trial use, including bioconversions. The full array of uses to which fungi can
be put is now immense, and overviews of many aspects are provided in
Pointing and Hyde (2001).
Most tropical fungi have yet to be collected and grown in culture, some
may never be, and others grow only very slowly. However, this is now less of
a constraint to exploitation of useful products since the pertinent genes can
be cloned and expressed through bacterial and fungal (especially yeast) species
that are easily grown.
The Convention on Biological Diversity is concerned with the equitable
sharing of benefits from biodiversity and requires ‘prior informed consent’ to
bioprospecting and screening. Partnerships between tropical countries and
pharmaceutical companies are the way forward, and individual countries can
do much to facilitate the search and discovery process.
(5) . . . a source of biocontrol agents
The biocontrol of insects and weeds is high on the agendas of developed

and developing countries. The prospects of environmentally friendly specific
controls that do not require repeated applications are attractive. Tropical
insects have long been considered a source of fungal strains for testing against
agricultural pests, but more recently attention has focused on weed and even
crop pathogens (see Evans, this volume).
Plants originating in particular places where they are a benign normal
component of the vegetation can become aggressive weeds if they become
established in areas where their usual pest and disease organisms are absent,
or rather have not been introduced with them. The endemic sites of weeds are
those that have the greatest diversity of associated organisms, including fungi,
and where species that have potential as biocontrol agents need to be sought.
In the case of weeds originating in particular tropical countries, they may
have the key to their control elsewhere. For example, fungi new to science
meriting evaluation for the control of Lantana camara L. have been discovered
in Brazil (Barreto et al., 1995).
(6) . . . a source of plant pathogens
Reasons for studying tropical fungi are not because species are always
beneficial. Fungi that can cause diseases in agricultural crops, garden plants,
and native species in one country may be present in others. This can be at the
species or strain levels.
A recent example pertinent in the UK is the rust of Bellis perennis L., which
originated in Australia, became established in continental Europe, and has
now spread first to cultivated and then to native daisies (Preece et al., 2000).
More complex threats can arise when previously isolated fungal species are
brought together by human interference and hybridize, posing threats to hosts
previously immune from their effects (Newcombe et al., 2000).
6 D.L. Hawksworth
Potentially serious for tropical countries are strains of species that could
threaten economically important crops, for example the special form of
Fusarium oxysporum Schlecht that could devastate the oil-palm industry in
Malaysia, f.sp. elaeidis, should it get into South-East Asia from West Africa
(Holliday, 1980).
The risks of pathogens arriving in a new country are accentuated with
increased travel and movement of plants and seeds; see Evans, this volume.
Tropical and other countries need to take steps to increase their vigilance over
material entering the country that could affect wild or cultivated plants.
Regrettably, insufficient awareness of the risks invariably results in token
controls at national boundaries and other points of entry.
(7) . . . a threat to human health
There is always a need to know the poisonous fungi in an area, to document

these, and to ensure public awareness. However, they are only a very small
proportion of the species present. Of more widespread concern is the recog-
nition and limitation of mycotoxins in stored foodstuffs in the tropics. The
expertise to recognize the species involved needs to be developed; this a pre-
requisite for action. The extent of concern over the threat of mycotoxins to
human health is reflected in the large number of pertinent papers appearing
in journals such as the African Journal of Mycology and Biotechnology.
The spectrum of human pathogens is greater in tropical than in temper-
ate regions, whether of dermatophytes or species causing serious deep-seated
mycoses; see Evans and Ashbee, this volume. There is a need for heightened
awareness of species that are endemic in certain tropical regions, such as
Neotestudina rosatii Segretain & Destombe, which can be detected at early
stages when their effects may not be so severe. With a warming climate and
increased travel, some fungal problems now mainly associated with tropical
countries may become of increasing concern in temperate regions. Medical
pathology laboratories need to be aware of what species may be present in
particular countries in order to facilitate rapid diagnoses. However, the train-
ing of medical mycologists and identification guides for medical laboratories
tend to focus on the well-known disease-causing fungi.
(8) . . . able to contribute to sustainable development
Mushrooms for human consumption are perhaps one of the greatest hopes
for feeding a spiralling world population (see Härkönen, 2002). Their
nutritional values rival those of most vegetables apart from legumes, and, in
addition to being low in calories and saturated fatty acids, they are rich
in amino acids and vitamins, including some otherwise obtainable only from
animal products. It has been eloquently argued that the devloping world now
needs a mushroom-based Non-Green Revolution (Chang, 1999).
Mushroom production can be a household or a factory enterprise, and
use lignocellulosic wastes, such as paddy straw and wood chips, which oth-
erwise themselves cause pollution problems.
The use of fungi in biocontrol (see above) also means reduced inputs of
pesticides, which can be costly, uncertain in availability, and need repeated
applications. A successful biocontrol programme can operate in perpetuity fol-
lowing a successful introduction.
Using fungi for food, biocontrol, and the degradation of wastes are
‘planks’ of what is currently the drive for ‘White Agriculture’ in China. Indeed,
it is because of these multifarious contributions fungi can make to sustain-
ability that a book on the role of fungi in sustainable development concludes
with a cartoon captioned ‘No fungi. No future’ (Palm and Chapela, 1997).
(9) . . . a part of human culture
Certain mushrooms have long histories as a part of human cultures: in

traditional medicine, religion, and art. In China, for example, an amazing
array of fungi is used in traditional medicine, some of which are clearly
effective even if the pharmacological basis of the effects has not always been
clearly elucidated, as is the case for Ganoderma-based preparations (Buchanan
et al., 1995; Härkönen, 2002).
The involvement of mushrooms with hallucinogenic effects in the
development of religions is increasingly being recognized and is deeply rooted,
even in western countries, although rarely acknowledged or its extent fully
appreciated (Wasson et al., 1986). The religious interest spills over into art
and artefacts, in which it is the hallucinogenic species that invariably feature
the most strongly. The array of artefacts utilizing the mushroom form is stag-
gering (Taylor-Hawksworth, 2000), although the deep-rooted historical rea-
sons for this empathy are not always recognized.
(10) . . . and there
Scientific curiosity and explorative instincts are a part of human nature. The
need to search for new stars, send uncrewed spacecraft to planets and to pass
close to occasional comets, characterize ever smaller parts of atoms, map
areas where no people live and the floors of oceans, and climb hitherto uncon-
quered peaks is something ingrained in the human pysche and attracts con-
siderable media interest and major funding. So few of the world’s fungi are
known, even in temperate regions, let alone tropical forests, that there is
always the excitement of discovery. When mycologists make a microscope slide
8 D.L. Hawksworth
of that small black speck on a decaying stem, or look in detail at the features
of a freshly collected mushroom, they can have the joy of seeing an organ-
ism no human has ever observed or documented before. It can have novel and
even aesthetically beautiful features, or unsuspected combinations of known
features, that can affect our current views and understanding of the range
of, and relationships between, the organisms with which we share planet
Earth.
Now that the larger animals and plants are relatively well-known, the
excitement of finding an unknown organism is most likely to be found in the
fungi and other microbial groups. The fascination of the new and the
unknown is such that the leading biodiversity scientist, sociobiologist and
entomologist Edward Wilson, in his autobiography, indicated that, if he had
his time again, he would spend it as a microbiologist (Wilson, 1995).
A Personal Prognosis
In 1992 I forecast that, for tropical mycology to progress, the prerequisite

organization and planning needed to be in place by 1995 (Hawksworth,
1993). While some progress was made, a sufficiently concerted campaign for
particular activities did not materialize, some doors are now closed, and, sadly,
opportunities have been missed.
However, the subject has not become as marginalized as feared at that
time. The heightened publicity that has been achieved and the general
increase in awareness of fungi already referred to have been gratifying. The
estimate that there might be as many as 1.5 million fungi on Earth reappears
even in the most semi-popular articles and also in radio and television items
worldwide. Fungi scarcely even entered the arena of the ecologist and con-
servation scientist in the late 1980s. In addition, the role of fungi recognized
by work undertaken as part of the International Biological Programme (e.g.
Kjøller and Struwe, 1982) in the previous decade seemed to be passed over.
Further, information being promulgated in the 1980s was often erroneous
and not contributed by mycologists. The major change that has occurred since
that time is exemplified by the book from the symposium that launched the
word ‘biodiversity’ in 1986 (Wilson, 1988). That publication had a mere six
index entries on fungi (Wilson, 1988). In contrast, the proceedings of its suc-
cessor symposial meeting 10 years had not only a whole chapter devoted
entirely to fungi, but 28 other references to them, not including mentions of
particular taxa (Raven and Williams, 2000). This is a major change mycolo-
gists have effected.
Nevertheless, I remain pessimistic over the situation of mycology in
developed countries. The number of positions and the standing of organismal
biology generally, including mycology, continue to be eroded against a tide of
recognition of increased importance. There are currently fewer professional
mycologists now active in the UK than at any time this century; indeed, there
is now only one involved in identification and systematic work in the entire
UK university system and he will retire this decade.
This situation arises because of historical patterns of funding and differ-
ing responsibilities and perspectives of the responsible agencies, departments
and other official institutions. Further, there is a preoccupation with reduc-
tionist, and thus simplistic, science generally in developed countries, and a
lack of appreciation of the value of work on complex systems that cannot be
reduced to short-term experiments with few variables. Fortunately, the situa-
tion in developed countries is not mirrored in all parts of the world. Several
individuals have had major roles in developing schools of mycology in areas
where there was previously no or little activity in the field. Amongst these are
Drs K.D. Hyde in Hong Kong and N. Hywel-Jones in Thailand, and Profs R.K.
Mibey in Nairobi and G. Guzmán in Mexico.
I have been impressed by the number of new mycologists in tropical coun-
tries, and by both the quantity and quality of fungal data now being gener-
ated from, and projects that are running in, such countries. Encouraged by
the International Mycological Association, African, Asian and Latin American
associations have all been formed in the last decade, and they now organize
their own series of congresses, workshops, directories, and even journals.
These groups now need to share their experiences, knowledge and ideas; forg-
ing south–south as well as north–south partnerships.
As mycologists we need to face the challenges of asserting our identity,
having a clear mission, and taking individual as well as corporate action; this
is something we all need to contribute to on a regular basis – 2 h a week spent
by each active mycologist equates to numerous full-time campaigners
(Hawksworth, 1995). This is a matter for each of us and not only for corpo-
rate society-organized action; we should not expect outside help but appreci-
ate any that comes as a bonus, and work both separately and together to
realize the potential mycology holds for so many areas of human concern.
If we do have this shared commitment, my prognosis is of one of steady
improvement in the tropical situation, but more slowly than ideal and at a
lower rate than might have been possible – the rate sadly being hindered by
a decreased ability to help from declining numbers of mycologists and myco-
logical institutions in developed countries.
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Key to Tropical Species of 2
Nectria-like Fungi
G.J. SAMUELS,1 A.Y. ROSSMAN1 AND H.-J. SCHROERS2
1
Systematic Botany and Mycology Laboratory, United States
Department of Agriculture, Agricultural Research Service, Beltsville,
MD 20705, USA; 2Centraalbureau voor Schimmelcultures, 3508 AD
Utrecht, The Netherlands
Introduction
Fungi historically identified as Nectria are a conspicuous element of the

tropical biota. The sexual fruit bodies of these hypocrealean ascomycetes are
primarily light- to bright-coloured, uniloculate perithecia that can occur
singly on decaying leaves or in masses of bright red or orange aggregations
bursting through the bark of recently dead trees. The fungi may colonize the
plant substrata in various ways, e.g. via the soil or plant roots, possibly long
before perithecia are formed. As invaders, endophytes, plant pathogens, or
saprotrophs, they play important ecological roles. In addition to decaying
woody and herbaceous debris, Nectria-like fungi occur on insects, pyreno-
mycetous stromata and other ascomycetes, lichens, basidiomycetous fungi and
living leaves. These fungi are often encountered as isolations from soil and
diseased plant tissue and are manifested in vitro in their asexual forms. This
chapter consists of a key to the Nectria-like fungi frequently encountered in
tropical regions.
Until recently, the genus Nectria included over 1000 species described over
a period of 100 or more years, most of which have not been characterized in
the modern sense. These taxa are known to be linked to a diverse array of
anamorphs. To understand this large genus, groups of related species were
recognized in an informal sense initially by Weese (1916, 1919), followed by
Booth (1959), Samuels (1976), and Rossman (1983). These groups were cor-
related with characteristics of the ascomatal wall morphology and anamorph
as well as biological features, particularly of the host. Rossman (1989) cir-
cumscribed the genus Nectria in a narrow sense to include only one group of

14 G.J. Samuels et al.
related species based on the type species, Nectria cinnabarina (Tode : Fr.) Fr. and
its anamorph, Tubercularia vulgaris Tode : Fr. This left many hundreds of orphan
species that did not belong in Nectria but had not been placed elsewhere.
In a comprehensive account of three major families of the Hypocreales,
many of these species described in Nectria were placed in segregate genera in
the two families Bionectriaceae and Nectriaceae (Rossman et al., 1999). Many
of the tropical Nectria-like species were placed in segregate genera and are
included in this key. Additional Nectria-like species are included here that do
not belong to the narrowly circumscribed genus Nectria and have not been
placed in segregate genera. For these species, the generic name is placed in
quotes and the affinity with a segregate genus is indicated if known. No new
combinations are proposed in this key.
This key to tropical Nectria-like fungi is limited to those species belonging
to the Bionectriaceae and Nectriaceae that are relatively common in tropical
regions. In general, species that are known only from the type specimen are
not included in the key, although there are exceptions especially for distinc-
tive or well-characterized species. The authors have collected primarily in the
Neotropics, with brief forays in the Asian and African tropics. Although dis-
tribution data are limited at present, it is suspected that many of these species
are pantropical. Reference is given to a full description of the species, although
often under another generic name as indicated. Users are urged to delve into
the literature for similar taxa if a specimen of a Nectria-like fungus from the
tropics cannot be identified using this key.
Key to Common Tropical Species of Nectria, Genera

Segregated from Nectria, and Nectria-like Fungi
Species with yellow-orange, orange, red to purple perithecia, becoming
darker in 3% KOH, primarily belonging to the Nectriaceae
Go to p. 22
Species with white, yellow, orange or brown perithecia, not turning

dark in 3% KOH, primarily belonging to the Bionectriaceae
1. Ascospores averaging 15 µm or less in length ..........................................2

1. Ascospores averaging more than 15 µm in length.................................37
2. Ascospores averaging 3.5 µm or less in width .......................3
2. Ascospores averaging more than 3.5 µm in width ..............22
3. On myxomycetes.........................................................................................4
3. On diverse substrata but not myxomycetes ...............................................5
4. Perithecial hairs tapering from base to tip; ascospores
4.5–5.5 ¥ 2–3 µm..................Nectriopsis exigua (Pat.) W.Gams
(Samuels, 1988a)
Key to Tropical Species of Nectria-like Fungi 15
4. Perithecial hairs cylindrical, hyphal; ascospores 5.5–7 ¥

2.5–3.5 µm ................Nectriopsis oropensoides (Rehm) Samuels
(Samuels, 1988a)
5. On living leaves, sometimes on Meliolaceae ...............................................6
5. Lignicolous or fungicolous but not on leaves..........................................11
6. Perithecia not on fungi; stiff, thick-walled setae arising from
perithecial surface; ascospores 13.5–16.5 ¥ 3–3.5 µm,
smooth.......................... Trichonectria erythroxylifoliae Samuels
(Samuels, 1988a)
6. Perithecia on Meliolaceae .........................................................7
7. Ascospores striate, 12.5–16.5 ¥ 3–4 µm .......Dimerosporiella leucorrhodina
(Mont.) Rossman & Samuels (Samuels, 1988a as Nectriopsis leucorrhodina)
7. Ascospores smooth or spinulose ................................................................8
8. Ascospores spinulose, 8–10 ¥ 2.5–3 µm ...........Dimerosporiella
guarapiensis (Speg.) Rossman & Samuels (Samuels, 1988a as
N. guarapiensis)
8. Ascospores smooth ..................................................................9
9. Perithecia glabrous; ascospores 14–16 ¥ 2–3.5 µm....Dimerosporiella sensi-
tiva (Rehm) Rossman & Samuels (Samuels, 1988a as N. sensitiva)
9. Perithecia with saccate or hyphal hairs arising from the ostiolar
region ....................................................................................................10
10. Perithecia with saccate hairs arising from around the ostio-
lar region; ascospores 9–11 ¥ 3–4 µm............Dimerosporiella
pipericola (Henn.) Rossman & Samuels (Samuels, 1988a as
N. pipericola)
10. Perithecia with thick-walled hyphae arising from around
the opening; ascospores 11.5–15.5 ¥ 2.5–4 µm....................
Dimerosporiella cephalosporii (Hansf.) Rossman & Samuels
(Samuels, 1988a as N. cephalosporii)
11 (5). On bark or decaying herbaceous debris .........................................12
11. On Lachnum or on other fungi including superficial or immersed
pyrenomycetes, Stereum, polypores or lichens ......................................16
12. Perithecia smooth or slightly roughened ...........................13
12. Perithecia with solitary hyphae or triangular fascicles of
hairs arising from perithecial surface ................................14
13. Perithecia solitary or in small groups; ascospores 10–12.5 ¥
2.5–3.5 µm, verruculose, rarely smooth; anamorph Sesquicillium sp.
......................Bionectria sesquicillii (Samuels) Schroers & Samuels
(Schroers, 2001)
13. Perithecia on a well-developed, erumpent stroma; ascospores 9.5–11 ¥
3–3.5 µm, spinulose; conidiophores dimorphic, either verticillium-like
or penicillate, Clonostachys rosea .............Bionectria ochroleuca (Schwein.)
Schroers & Samuels (Schroers et al., 1999; Schroers, 2001)
14. Perithecia yellow to orange-yellow, covered with white to
pale golden hyphae; ascospores 7.5–10 ¥ 1.5–2.5 µm,

smooth.........................Nectriopsis squamulosa (Ellis) Samuels
(Samuels, 1988a)
14. Perithecia orange to orange-brown with solitary to
fasciculate hairs ..................................................................15
15. Perithecia orange with orange, solitary to fasciculate hairs; ascospores
11–15 ¥ 3–4 µm, smooth .......Lasionectria sylvana (Mouton) Rossman &
Samuels (Rossman et al., 1999)
15. Perithecia orange to brown-orange with white to brown, fasciculate
hairs; ascospores 12.5–17 ¥ 3–4 µm, spinulose .......................................
Hydropisphaera rufofusca (Penz. & Sacc.) Rossman (Samuels et al., 1990
as Nectria brasiliensis)
16 (11). Ascospores averaging more than 7 µm in length .....17
16. Ascospores averaging less than 7 µm in length ................20
17. On Lachnum; ascospores 11–20 ¥ 2–3 µm ...............Nectriopsis discophila
(Rogerson & Samuels) Samuels (Samuels, 1988a)
17. On other fungi .......................................................................................18
18. Perithecia pale orange; ascospores 13–15 ¥ 2–3 µm.............
Nectriopsis ostiolorum (Berk. & Cooke) Samuels (Samuels,
1988a)
18. Perithecia yellow; ascospores averaging less than 14 µm in
length ..................................................................................19
19. Ascospores 10.5–13 ¥ 2.5–3 µm ................Nectriopsis epimyces Samuels
(Samuels, 1988a)
19. Perithecia yellow, with chains of thin-walled, globose cells arising from
the perithecial surface; ascospores 6.5–11 ¥ 2–3 µm, smooth to spinu-
lose; on ascomata, lichens or non-fungal substrata.................Nectriopsis
mindoensis (Petr.) Samuels (Samuels, 1988a)
20 (16). Perithecia yellow, smooth; ascospores 5.5–6.5 ¥ 2–2.5
µm, smooth; on perithecia and stromata of Nectria, Bertia
and Xylariaceae...........................Nectriopsis epimycota Samuels
(Samuels, 1988a)
20. Perithecia white, with hyphal hairs arising from perithecial
apex or surface; ascospores smooth to finely spinulose.....21
21. Perithecia white, with white, unbranched, septate hairs forming a
fringe around the apex; ascospores 5.5–7.5 ¥ 2–2.5 µm, smooth to
finely spinulose; on perithecia of Hypoxylon, Nectria and Xylaria ............
...............Nectriopsis perpusilla (Mont.) Samuels (Samuels, 1988a)
21. Perithecia white, with spinulose hyphae arising from the perithecial
surface; ascospores 5.5–7 ¥ 2.5–3.5 µm, spinulose; on diverse sub-
strata including Stereum, polypores and myxomycetes .............................
Nectriopsis oropensoides (Rehm) Samuels (Samuels, 1988a)
22 (2). Ascospores averaging more than 3.5 and less than 5
µm in width...........................................................................23
22. Ascospores averaging 5–6 µm in width.............................33

23. On stromata of Balansia or immersed ascomycetous structures on grass;
ascospores 9.5–11.5 ¥ 3.5–4 µm, smooth to slightly spinulose...............
Bionectria epichloë (Speg.) Schroers (Schroers, 2001)
23. On herbaceous or lignified, monocotyledonous or dicotyledonous mate-
rial including bamboo, lichens and fungi .............................................24
24. Ascospores smooth or spinulose ........................................25
24. Ascospores striate ...............................................................28
25. On perithecia of Neonectria; perithecia pale yellow; ascospores 9.5–13
¥ 3.5–4 µm, smooth; anamorph gliocladium-like....................Nectriopsis
byssotecta (Rehm) Samuels (Samuels, 1988a)
25. On non-fungal substrata .......................................................................26
26. Perithecia orange, with large white to off-white warts;
warts made of cells that are unevenly thickened on one
side; ascospores 11–13 ¥ 3.5–4.5 µm, smooth or
spinulose...................... Bionectria byssicola (Berk. & Broome)
Schroers and Samuels (Schroers et al., 1999)
26. Perithecia smooth, granulose or slightly scaly ..................27
27. Perithecia smooth to granulose; ascospores 9.5–17 ¥ 3.5–6 µm, spinu-
lose; anamorph synnematous; synnemata grey to black, forming
orange drops of conidia ......................Stilbocrea gracilipes (Tul. & C. Tul.)
Samuels & Seifert (Rossman et al., 1999)
27. Perithecia smooth to scaly; ascospores 12–16 ¥ 4–5 µm, spinulose;
conidiophores sporodochial, dendrodochium-like .....................Bionectria
Samuelsii Schroers (Schroers, 2001) (previously referred to as
Nectria aureofulva Cooke & Ellis, see Samuels et al., 1990)
28 (24). Perithecia caespitose, sometimes appearing as if
immersed in a superficial, hyphal stroma (Hypocrea-like);
ascospores 13–17 ¥ 4–5 µm, with few striations that extend
the full length of the spore .............Protocreopsis pertusa (Pat.)
Samuels & Rossman (Rossman et al., 1999)
28. Perithecia solitary or caespitose but not immersed within a
stroma ..................................................................................29
29. Ascospores 8.5–10.5 ¥ 3.5–4 µm, striate; perithecia yellow, globose,
thin-walled, collapsing by lateral pinching when dry, with spinulose
hairs or modified cells around apex of immature fruit bodies..................
Nectriopsis albofulta (Petch) Samuels (Samuels, 1988a)
29. Ascospores averaging more than 10 µm in length; perithecia not col-
lapsing by lateral pinching....................................................................30
30. Perithecia pale yellow with conspicuous warts; ascospores
11–13.5 ¥ 4.5–5 µm, striate; anamorph Clonostachys sp.
(Bionectria grammicospora (Ferd. & Winge) Schroers
& Samuels (Samuels et al., 1990)
30. Perithecia yellow to orange or brown, smooth to slightly

roughened or with fasciculate hairs ..................................31
31. Perithecia with triangular, fasciculate hairs arising from surface;
ascospores 12–17 ¥ 4–5 µm, finely striate ...........Hydropisphaera suffulta
(Berk. & M.A. Curtis) Rossman & Samuels (Samuels et al., 1990 as N.
suffulta)
31. Perithecia glabrous to slightly roughened ............................................32
32. Perithecia solitary, yellow, on decaying wood or herbaceous
debris; ascospores 14.5–17.5 ¥ 4–5 µm, initially smooth
but becoming finely to coarsely striate; anamorph syn-
nematous, forming dark greenish conidial masses,
myrothecium-like.......................‘Nectria’ chlorogloea Samuels
(Samuels, 1988b)
32. Perithecia solitary or in caespitose clusters, orange to
brown, on decaying monocotyledonous leaves; ascospores
14–16 ¥ 3.5–4 µm, smooth or striate; anamorph acremo-
nium-like ................Hydropisphaera arenula (Berk. & Broome)
Rossman & Samuels (Samuels, 1978 as N. arenula)
33 (22). Perithecia completely immersed within an effuse white stroma;
ostiolum orange; ascospores 10–14 ¥ 4–6 µm, verrucose; anamorph
synnematous, black or hyaline...........Stilbocrea macrostoma (Berk. & M.A.
Curtis) Höhn. (Rossman et al., 1999)
33. Perithecia superficial .............................................................................34
34. Ascospores pale green, 13–17 ¥ 5–7 µm; perithecia tan;
anamorph Penicillifer macrosporus...........................Viridispora
penicilliferi Samuels & Rossman, Nectriaceae
(Samuels, 1989b; Rossman et al., 1999)
34. Ascospores hyaline; perithecia yellow or orange;
anamorphs not Penicillifer ..................................................35
35. On non-fungal substrata; perithecia glabrous, globose, collapsing cupu-
late; ascospores 11–14 ¥ 5–7 µm, striate; anamorph acremonium-
like............................................Hydropisphaera peziza (Tode : Fr.) Dumort.
(Rossman et al., 1999)
35. On ascomycetous fruiting structures or stromata ................................36
36. On Xylariaceae; ascospores coarsely striate, 11.5–14 ¥
4.5–6 µm .......................Nectriopsis puiggarii (Speg.) Samuels
(Samuels, 1988a)
36. On perithecia of various pyrenomycetes but not Xylariaceae;
ascospores smooth, 9.5–13.5 ¥ 4–6 µm................Nectriopsis
lasioderma (Ellis) Samuels (Samuels, 1988a)
37 (1). Ascospores averaging more than 25 µm in length ........................38
37. Ascospores averaging 15–25 µm in length ..........................................59
38. Ascospores dictyosporous, 48–97 ¥ 10–16 µm; perithecia
white to orange, with long, triangular, fasciculate hairs
around the apex; on herbaceous and woody debris...............

Ijuhya dictyospora (Rossman) Rossman & Samuels
(Rossman, 1983 as N. dictyospora)
38. Ascospores 1- or more septate, not muriform ...................39
39. Ascospores 1-septate .............................................................................40
39. Ascospores 2- or more septate ..............................................................47
40. On lichens; asci each with four large ascospores
(34–50 ¥ 12–18 µm) and four small ascospores
(8–17 ¥ 3.5–7 µm) .....................................‘Nectria’ parmeliae
(Berk. & M.A. Curtis) D. Hawksw. (Hawksworth and Booth,
1976 as N. heterospora)
40. Ascospores of homogeneous size .......................................41
41. Ascospores 50–76 ¥ 6.5–9 µm, striate; perithecia immersed in a com-
pact white, hyphal stroma.....Protocreopsis fusigera (Berk. & Broome) Doi
41. Ascospores averaging 25–35 µm in length ..........................................42
42. On lichens; perithecia brown-orange, yellow in 3% KOH;
ascospores 22–25 ¥ 7–8 µm, smooth.....................................
‘Nectria’ lichenophila Speg.
42. On Xylaria or on bark of recently dead trees.....................43
43. On stromata of Xylaria spp.; perithecia immersed in a minute, yellow,
pulvinate stroma; ascospores 28–31 ¥ 4.5–6 µm, smooth.......................
‘Hypocreopsis’ xylariicola Samuels (Samuels, 1988a)
43. On bark of recently dead trees..............................................................44
44. Perithecia brown.................................................................45
44. Perithecia yellow to orange ................................................46
45. Perithecia brown, red in 3% KOH; ascospores 26–35 ¥ 10–13 µm,
striate .......................................‘Nectria’ (Neonectria) balansae, Nectriaceae
(Samuels and Brayford, 1994)
45. Perithecia brown, associated with a chrome-yellow Cylindrocarpon
anamorph; ascospores 25.5–31.5 ¥ 8–10 µm, smooth ...................
‘Nectria’ (Neonectria) cinnamomea Brayford & Samuels,
Nectriaceae (Brayford and Samuels, 1993)
46. Ascospores striate, 26–32.5 ¥ 8.5–10.5 µm; perithecia
orange, with orange to white warts; anamorph clono-
stachys-like ............... Bionectria lucifer (Samuels) Schroers &
Samuels (Samuels 1988b; Schroers, 2001)
46. Ascospores smooth, 24–29 ¥ 8–10 µm; anamorph
myrothecium-like ................Bionectria pityrodes (Mont.)
Schroers (Samuels et al, 1990; Schroers, 2001) Mont.
(Samuels et al., 1990)
47 (39). Ascospores three septate...............................................................48
47. Ascospores more than three septate .....................................................54
48. Ascospores conspicuously striate .......................................49
48. Ascospores smooth or only faintly striate..........................50

49. Ascospores 38–55 ¥ 11–13 µm; perithecia yellow to dark yellow,
smooth; anamorph Didymostilbe echinofibrosa........................Peethambara
spirostriata (Rossman) Rossman (Rossman et al., 1999)
49. Ascospores 24–35 ¥ 7–10 µm; perithecia white, grossly warted, cells of
warts unevenly thickened; anamorph Fusarium decemcellulare; on
diverse substrata including cacao pods .................Albonectria rigidiuscula
(Berk. & Broome) Rossman & Samuels, Nectriaceae
50. Ascospores 48–55 ¥ 6–7 µm; perithecia dark orange with
orange hairs........Hydropisphaera gigantea (Speg.) Rossman &
Samuels (Samuels, 1976 as N. gigantea)
50. Ascospores averaging less than 50 µm in length ..............51
51. Perithecia white .....................................................................................52
51. Perithecia orange to brown...................................................................53
52. Ascospores 40–48 ¥ 10–12.5 µm; perithecia white, grossly
warted, cells of warts unevenly thickened ............Albonectria
albosuccinea (Pat.) Rossman & Samuels, Nectriaceae Rossman
et al., 1999)
52. Ascospores 38–41 ¥ 8–11 µm; perithecia smooth.................
‘Nectria’ albida Rossman (Rossman, 1983)
53. Ascospores 18–29 ¥ 4–6 µm; perithecia orange to brown, smooth to
slightly roughened ............Hydropisphaera erubescens (Desm.) Rossman &
Samuels (Rossman, 1983 as N. erubescens)
53. Ascospores 25–38 ¥ 5–7 µm; perithecia brown with brown hairs..........
Hydropisphaera dolichospora (Penz. & Sacc.) Rossman & Samuels
(Samuels et al., 1990 as N. dolichospora)
54 (47). Perithecia pale yellow to yellow with a bright red
papilla; ascospores 56–95 ¥ 6–9 µm, 7–11-septate; on dead
culms of bamboo, often at the nodes...........................‘Nectria’
rubrostoma Rossman (Rossman, 1983)
54. Perithecia concolorous .......................................................55
55. Ascospores 11–19-septate, 50–70 ¥ 6–7 µm; perithecia smooth to
slightly roughened, orange to umber............Hydropisphaera multiloculata
(Samuels) Rossman & Samuels (Rossman, 1983 as N. multiloculata)
55. Ascospores 5–9-septate, averaging less than 60 µm in length ............56
56. Perithecia white to orange, with long, triangular, fascicu-
late hairs around the apex; ascospores 5–7-septate, 30–60
¥ 4–7 µm; on decaying herbaceous debris ...................Ijuhya
peristomialis (Berk. & Broome) Rossman & Samuels
56. Perithecia smooth or with conspicuous warts...................57
57. Ascospores 24–38 ¥ 4–5.5 µm, 7–9-septate; perithecia smooth, with
abundant, orange oily droplets in middle wall region ...........Ochronectria
calami (Henn. & E. Nyman) Rossman & Samuels (Rossman et al., 1999)
57. Ascospores longer than 40 µm; perithecia smooth or warty but with-
out oily droplets .....................................................................................58
58. Ascospores 40–60 ¥ 5–7 µm, 5–7-septate; perithecia
smooth........................‘Nectria’ pseudopeziza Rossman (1983)
58. Ascospores 42–62 ¥ 7–9 µm, 5–9-septate; perithecia
grossly warted, cells of warts unevenly thickened .................
Albonectria verrucosa (Pat.) Rossman & Samuels, Nectriaceae
59 (37). Ascospores averaging less than 5 µm in width............................60
59. Ascospores averaging 6–8.5 µm in width ............................................65
60. Perithecia densely caespitose, often immersed in a hyphal
stroma (Hypocrea-like); ascospores striate; typically on
monocotyledonous leaves ...................................................61
60. Perithecia solitary or in groups of a few but neither densely
caespitose nor forming Hypocrea-like stromata..................62
61. Perithecia clothed in tan to brown hyphae; ascospores 21–27 ¥
4–5 µm ....................Protocreopsis foliicola (Berk. & M.A. Curtis) Samuels
61. Perithecia clothed in white to tan hyphae; ascospores 15–18 ¥ 4–5 µm ...
Protocreopsis javanica (Höhn.) Rossman & Samuels (Rossman et al., 1999)
62. On meliolaceous fungi on living leaves; ascospores 17–22 ¥
3–4 µm, striate.......................Dimerosporiella oidioides (Speg.)
Rossman & Samuels (Samuels, 1988a as Nectriopsis
oidioides)
62. On decaying wood or herbaceous debris ...........................63
63. Perithecia orange, globose, wall pseudoparenchymatous; ascospores
14.5–17.5 ¥ 3.3–5 µm, striate; anamorph Septomyrothecium..................
‘Nectria’ septomyrotheciae Samuels (Samuels, 1988b)
63. Perithecia white to pale yellow, globose or flattened above, wall hyphal
in structure ............................................................................................64
64. Ascospores 14.5–20 ¥ 3–5 µm, spinulose..........Ijuhya parilis
(Berk. & Broome) Rossman & Samuels (Samuels, 1988a as
Peristomialis parilis)
64. Ascospores 21.5–24.5 ¥ 4–5 µm, coarsely striate .................
Ijuhya paraparilis (Samuels) Rossman & Samuels (Samuels,
1988a as Peristomialis paraparilis)
65 (59). Ascospores striate, 16.5–19 ¥ 5.5–7 µm .........‘Nectria’ (Bionectria)
subquaternata Berk. & Broome (Samuels et al., 1990)
65. Ascospores smooth to spinulose or warted...........................................66
66. On foliicolous ascomycetes; ascospores 19.5–24.5 ¥
5.5–6.5 µm, smooth to spinulose...............Bionectria tonduzii
Speg. (Rossman et al., 1999)
66. On wood or bark.................................................................67
67. Perithecia completely immersed in stroma, stroma appearing

Hypocrea-like; ascospores 15–22 ¥ 7–10 µm, spinulose.............Stilbocrea
impressa (Mont.) Samuels (Rossman et al., 1999)
67. Perithecia superficial; ascospores 16–33 ¥ 4.5–9.5 µm, verrucose..........
Bionectria apocyni (Peck) Schroers & Samuels (Samuels, 1976 as Nectria
apocyni)
Species with yellow-orange, orange, red to purple perithecia, becoming

darker in 3% KOH, primarily belonging to the Nectriaceae
1. Perithecia with spinulose, golden, rarely whitish, hairs arising from the
surface; ascospores striate..........................................................................2
1. Perithecia glabrous or hairs not spinulose; ascospores smooth, striate,
spinulose or tuberculate.............................................................................4
2. Ascospores 24–30 ¥ 7–9 µm; anamorph synnematous...........
Lanatonectria mammiformis (Chardon) Samuels & Rossman
2. Ascospores less than 20 µm in length; anamorph synnema-
tous or sporodochial ................................................................3
3. Ascospores 13.5–18 ¥ 4–6 µm; anamorph sporodochial.......Lanatonectria
flavolanata (Berk. & Broome) Samuels & Rossman (Rossman et al., 1999)
3. Ascospores 10–13 ¥ 3–4.5 µm; anamorph synnematous......Lanatonectria
flocculenta (Henn. & E. Nyman) Samuels & Rossman (Rossman et al.,
1999)
4. Associated with Chaetopsina anamorphs ................................5
4. Associated with other anamorphs, not Chaetopsina ...............9
5. Ascospores more than 20 µm in length ....................................................6
5. Ascospores less than 20 µm in length.......................................................7
6. Ascospores 36–41.5 ¥ 6–7 µm, smooth; anamorph
Chaetopsina sp. ...........Cosmospora macrochaetopsinae (Samuels)
Rossman & Samuels (Samuels et al., 1990 as Nectria
macrochaetopsinae)
6. Ascospores 25–42 ¥ 6–10 µm, striate; anamorph Chaetopsina
penicillata...........Cosmospora chaetopsinae-penicillatae (Samuels)
Rossman & Samuels (Samuels and Brayford, 1994 as N.
chaetopsinae-penicillatae)
7. Ascospores 8–9.5 ¥ 2–3 µm, smooth; anamorph Chaetopsina cf. fulva.......
Cosmospora chaetopsinae (Samuels) Rossman & Samuels (Samuels, 1985
as N. chaetopsinae)
7. Ascospores more than 10 µm in length ....................................................8
8. Ascospores 11–15 ¥ 3–3.5 µm; anamorph Chaetopsina poly-
blastia, conidiogenous cells proliferating percurrently; conidia
11–15 ¥ 3–3.5 µm, borne in slimy heads ..............Cosmospora
chaetopsinae-polyblastiae (Samuels) Rossman & Samuels

(Samuels, 1985 as N. chaetopsinae-polyblastiae)
8. Ascospores 11–13 ¥ 3.5–4.5 µm; anamorph Chaetopsina
catenulata, conidiogenous cells determinate; conidia 10–19.5
¥ 1.9–3.5 µm, borne in chains ..........Cosmospora chaetopsinae-
catenulatae (Samuels) Rossman & Samuels (Samuels, 1985 as
N. chaetopsinae-catenulatae)
9 (4). Ascospores non-septate .....................................................................10
9. Ascospores with one septum or more .....................................................12
10. Ascospores 5.5–7 ¥ 1.5–2 µm, smooth; on decaying plant
parts of Agavaceae.....................Nectria miltina (Mont.) Mont.
10. Ascospores more than 7 µm in length, smooth
or rugose .............................................................................11
11. Associated with decaying leaves and fruits of Clusia; ascospores 9–11 ¥
3–4 µm, ellipsoid, smooth; anamorph Gliocephalotrichum bulbillium........
Leuconectria clusiae (Samuels & Rogerson) Rossman et al. (Rossman et
al., 1993, 1999)
11. Primarily isolated from soil, causing fruit- and root-rots and seedling
damping-off; ascospores 10–15.5 ¥ 7.5–12 µm, subglobose, rugose;
anamorph acremonium-like ................ Neocosmospora vasinfecta E.F. Sm.
12 (9). Ascospores with more than 1 septum .........................13
12. Ascospores with 1 septum..................................................30
13. Ascospores dictyosporous, 17–31 ¥ 6–15 µm; anamorph synnematous;
perithecia on recently dead or decaying woody substrata.............Nectria
pseudotrichia Berk. & M.A. Curtis (Samuels et al., 1990)
13. Ascospores phragmosporous.................................................................14
14. Ascospores 13–25-septate, 150–270 ¥ 8–12 µm, faintly
striate; perithecia solitary, warty; on decaying woody sub-
strata ............Ophionectria trichospora (Berk. & Broome) Sacc.
14. Ascospores 3–5-septate ......................................................15
15. Ascospores mainly 3-septate .................................................................16
15. Ascospores mainly 5-septate .................................................................29
16. Perithecia dark brick-red to dark blue or purple, appearing
black en masse .....................................................................17
16. Perithecia yellow-orange to red or dark-red ......................21
17. On stromata of Phyllachora on living leaves; perithecia dark brick-red;
ascospores 26–35 ¥ 8–10 µm, smooth..................Allonectella guaranitica
(Speg.) Rossman (Rossman et al., 1999)
17. On decaying herbaceous and woody substrata ....................................18
18. Ascospores averaging more than 20 µm in length ...........20
19. Ascospores smooth, 13–18 ¥ 5–8 µm; anamorph Fusarium

lateritium ...........................Gibberella baccata (Wallr.) Sacc. (Booth, 1971)
19. Ascospores roughened unequally over upper cell, 15–20 ¥ 5–6.5 µm;
anamorph Fusarium xylarioides...............Gibberella xylarioides R. Heim &
Saccas (Booth, 1971)
20. Ascospores 22–27 ¥ 5.5–7 µm, 3-septate; anamorph
Fusarium sambucinum ................Gibberella pulicaris (Fr.) Sacc.
20. Ascospores 19–24 ¥ 3–4 µm, 0–1-, eventually 3-septate;
anamorph Fusarium graminearum; primarily on grasses........
Gibberella zeae (Schwein.) Petch (Booth, 1971)
21 (16). On scale insects; ascospores 25–35 ¥ 6–8 µm, smooth ...................
Cosmospora diploa (Berk. & M.A.Curtis) Rossman (Rossman, 1983
as N. diploa)
21. On decaying woody or herbaceous substrata or on ascomycetous stro-
mata or rust cankers .............................................................................22
22. On ascomycetous stromata or rust cankers of
Gymnosporangium................................................................23
22. On decaying woody or herbaceous substrata ....................24
23. On cankers of Gymnosporangium on Cupressaceae; ascospores 23–32 ¥
7–10 µm, finely spinulose................‘Nectria’ (Neonectria) gymnosporangii
(Jaap) Rossman (Rossman, 1983)
23. On stromata of Botryosphaeria and Valsa on decaying wood; ascospores
25–39 ¥ 8.5–14 µm, striate..........Cosmospora diminuta (Berk.) Rossman
& Samuels (Rossman et al., 1999)
24. Perithecia with a dark apical disc, smooth; ascospores
50–67 ¥ 10–14 µm, smooth..................‘Nectria’ (Neonectria)
phaeodisca Rossman (Samuels and Brayford, 1993)
24. Perithecia smooth, scaly or warted; ostiolar region concol-
orous ...................................................................................25
25. Perithecia smooth, apex acute, lacking a disc; ascospores 24–40 ¥
8–12 µm, smooth .....................Cosmospora glabra (Rossman) Rossman &
Samuels (Rossman, 1983 as N. glabra)
25. Perithecia roughened to warty or scaly with or without a flattened api-
cal disc ...................................................................................................26
26. Perithecia scaly, with a flattened apical disc; ascospores
42–65 ¥ 7–11 µm, smooth to slightly roughened;
anamorph Cylindrocarpon.......................‘Nectria’ (Neonectria)
microdisca Rossman (Rossman, 1983)
26. Perithecia warty, without an apical disc; anamorph syn-
nematous or Cylindrocladium ..............................................27
27. Ascospores 14–20 ¥ 4–6 µm, striate; anamorph synnematous; on
recently dead wood............Nectria lateritia (P. Karst.) Rossman (Samuels
and Brayford, 1994)
27. Ascospores smooth; anamorph Cylindrocladium; on decaying leaves,

stems and other herbaceous debris.......................................................28
28. Perithecia orange-yellow with blood-red base; ascospores
30–70 ¥ 4.5–8 µm, 3-septate; anamorph Cylindrocladium
colhounii...................Calonectria colhounii Peerally (Crous and
Wingfield, 1994)
28. Perithecia red to dark red; ascospores 40–70 ¥ 4–7 µm,
smooth, 1–3-septate; anamorph Cylindrocladium ilicicola.......
Calonectria pyrochroa (Desm.) Sacc. (Rossman et al., 1999)
29 (15). Perithecia scaly, with a well-developed apical disc, on a well-devel-
oped stroma with erect hyphae protruding from the surface; ascospores
65–98 ¥ 8–15 µm; lignicolous.....................‘Nectria’ (Neonectria) fusispora
Rossman (Samuels and Brayford, 1993)
29. Perithecia smooth, lacking an apical disc, not obviously stromatic;
ascospores 54–65 ¥ 7–10 µm; on bamboo.........‘Nectria’ (Neonectria) lae
tidisca Rossman (Samuels and Brayford, 1993)
30 (12). Ascospores averaging less than 20 µm in length .....31
30. Ascospores averaging 20 µm or more in length................59
31. Ascospores green, 13–17 ¥ 5–7 µm, smooth; anamorph Penicillifer
bipapillatus.......................Viridispora alata (Samuels) Samuels & Rossman
31. Ascospores hyaline or yellow-brown.....................................................32
32. Perithecia forming on insects, old termite nests or
ascomycetous stromata ......................................................33
32. Not obviously associated with insects or ascomycetous
stromata ..............................................................................40
33. On scale insects or adelgids or old termite nests ..................................34
33. On stromal surface of ascomycetes or their anamorphs......................36
34. On old termite nests; ascospores disarticulating into broadly
ellipsoid part-spores, 3–4 ¥ 3–3.5 µm, yellow-brown,
densely spinulose..............................Haematonectria termitum
(Höhn.) Samuels & Rossman (Rossman et al., 1999)
34. On scale insects or adelgids; ascospores not
disarticulating.....................................................................35
35. Ascospores broadly fusiform, 12–15 ¥ 5.5–6.5 µm................Cosmospora
aurantiicola (Berk. & Broome) Rossman & Samuels (Booth, 1981a as
N. aurantiicola)
35. Ascospores ovoid to ellipsoid, 16–20 ¥ 7.5–10 µm ................Cosmospora
flammea (Tul. & C. Tul.) Rossman & Samuels (Booth, 1981b as
N. flammea)
36. On Parodiella sp. on living leaves; ascospores 14–19 ¥ 5.5–7
µm, smooth....................Cosmospora papilionacearum (Seaver)
Rossman & Samuels (Samuels et al., 1991 as N. papilionacearum)
36. On diverse ascomycetes ......................................................37
37. Red hyphal hairs arising from the perithecial surface; ascospores
12–13 ¥ 5–7 µm, spinulose to verrucose; on Cucurbitaria .......................
Cosmospora rubrosetosa (Samuels) Rossman & Samuels (Samuels et al.,
1990 as Nectria rubrosetosa)
37. Perithecia glabrous ................................................................................38
38. Ascospores 16–17 ¥ 7–9 µm, spinulose; anamorph
acremonium-like; on Pseudovalsa berkeleyi............Cosmospora
wegeliana (Rehm) Rossman & Samuels (Samuels et al., 1991
as N. wegeliana)
38. Ascospores less than 16 µm in length ...............................39
39. Ascospores 8–11 ¥ 4–5.5 µm, tuberculate; anamorph Acremonium
butyri with green conidia, common .............Cosmospora vilior (Starbäck)
Rossman & Samuels (Samuels et al., 1990 as N. vilior)
39. Ascospores 11–13 ¥ 6–7.5 µm, smooth to finely tuberculate;
anamorph acremonium-like.........Cosmospora pseudepisphaeria (Samuels)
Rossman & Samuels (Samuels et al., 1991 as N. pseudepisphaeria)
40 (32). Perithecia completely immersed in a compact stroma;
ascospores 15.5–19 ¥ 5–6.5 µm, smooth or striate.................
‘Nectria’ paraguyensis Speg. (Samuels and Brayford, 1994)
40. Perithecia not completely immersed in a stroma; ascospores
striate, smooth or verrucose...............................................41
41. Perithecia smooth, often reflecting light, apex darker than the body,
sometimes with a flattened disc; cells at the surface of the perithecial
wall difficult to discern; anamorph Cylindrocarpon ..............................42
41. Perithecia warted, hirsute or smooth but not glistening; cells at
perithecial surface easily discerned; anamorphs Cylindrocarpon,
Fusarium or others.................................................................................43
42. Ascospores 11.5–17 ¥ 5–7.5 µm, spinulose; cultures purple
...................Neonectria discophora (Mont.) Mantiri & Samuels
(Samuels et al., 1990 as N. discophora)
42. Ascospores 12.5–15 ¥ 5.5–6.5 µm, finely spinulose; cul-
tures white to tan..............‘Nectria’ (Neonectria) lucida Höhn.
(Samuels et al., 1990)
43. Perithecia with red hyphal hairs arising from surface; ascospores 7–8.5
¥ 2–3.5 µm, smooth to spinulose .........Cosmospora geastroides (Samuels)
Rossman & Samuels (Samuels et al., 1991 as N. geastroides)
43. Perithecia glabrous, scaly or warted, without red hyphal hairs;
ascospores averaging greater than 8 µm in length ..............................44
44. Ascospores smooth, spinulose, verrucose, warted or tuber-
culate...................................................................................49
45. Ascospores golden-brown, coarsely striate, 10–17 ¥ 5–8 µm; perithecia
with greenish warts ..................Rubrinectria olivacea (Seaver) Rossman &
Samuels (Rossman et al., 1999)
45. Ascospores at most pale yellow-brown, striations fine, sometimes diffi-

cult to see; perithecia with concolorous reddish warts ........................46
46. Cells at the surface of the perithecial wall typically less
than 15 µm diam. and their walls typically less than 2 µm
thick; perithecia cupulate when dry ..................................47
46. Cells at the surface of the perithecial wall typically more
than 20 µm diam. and their walls typically more than 2 µm
thick; perithecia cupulate when dry or not .......................48
47. Ascospores 8.5–13.5 ¥ 4–5 µm, smooth, becoming striate; perithecia
typically dark red.........Nectria pseudocinnabarina Rossman (Samuels and
Brayford, 1994)
47. Ascospores 14.5–16.5 ¥ 6.5–8 µm; perithecia typically red-orange........
Nectria guarapiensis Speg. (Samuels and Brayford, 1994)
48. Perithecia warted, collapsing by lateral pinching when dry;
cells at the perithecial surface angular, 20–25 µm diam.;
ascospores yellow-brown, finely striate, 13–16 ¥ 6–8 µm;
associated with Fusarium anamorph ..............Haematonectria
haematococca (Berk. & Broome) Samuels & Nirenberg
48. Perithecia at most scaly, cupulate when dry; cells at the
perithecial surface circular, 15–25 µm diam.; ascospores
hyaline, finely but conspicuously striate, 13.5–18 ¥ 4.5–6.5
µm; anamorph Cylindrocarpon rugulosum ...............Neonectria
rugulosa (Pat. & Gaillard) Mantiri & Samuels (Samuels and
Brayford, 1994 as Nectria rugulosa)
49 (44). Ascospores tuberculate, rarely smooth ........................................50
49. Ascospores smooth, spinulose or striate ...............................................55
50. Perithecia obpyriform with conical papilla; ascospores 8–12
¥ 4–8 µm ............Cosmospora obscura Lowen (Rossman et al.,
1999)
50. Perithecia globose to broadly pyriform or obpyriform with
flattened apex; ascospores averaging more than 10 µm in
length ..................................................................................51
51. Perithecia obpyriform, sometimes flattened apically, but without an api-
cal disc; solitary or caespitose, often forming laterally and terminally
on orange mycelial cords; ascospores 14–21 ¥ 5–9 µm, yellow-brown,
tuberculate ..........Corallomycetella repens (Berk. & M.A. Curtis) Rossman
& Samuels (Rossman et al., 1999)
51. Perithecia globose to broadly pyriform, smooth to scaly with a conspic-
uous apical disc or with concolorous or paler warts; solitary to densely
gregarious; ascospores hyaline, smooth, roughened or warted ...........52
52. Perithecia smooth to scaly with a conspicuous apical
disc ......................................................................................53
52. Perithecia warted with concolorous or paler warts...........54
53. Ascospores 15–19.5 ¥ 6.5–8 µm, roughened, rarely smooth;

anamorph Cylindrocarpon candidulum .......................‘Nectria’ (Neonectria)
veuillotiana Sacc. & Roum. (Brayford and Samuels, 1993 as Nectria
veuillotiana)
53. Ascospores 9.5–11 ¥ 3.5–4.5 µm, smooth or warted; anamorph
Cylindrocarpon permirum.......................‘Nectria’ (Neonectria) platycephala
Brayford & Samuels (Brayford and Samuels, 1993)
54. Ascospores 11.5–13.5 ¥ 4.5–5.5 µm, warted; anamorph
Cylindrocarpon arcuatum ........‘Nectria’ (Neonectria) rubrococca
Brayford & Samuels (Brayford and Samuels, 1993)
54. Ascospores 16.5–19 ¥ 6–7 µm, warted; anamorph
Cylindrocarpon torpidum ..........................‘Nectria’ (Neonectria)
verrucospora Brayford & Samuels (Brayford and Samuels,
1993)
55 (49). Perithecia scaly to warted, surface cells distinctive, circular, thin-
walled; perithecial wall more than 20 µm in width ....................56
55. Perithecia smooth, surface cells lacking a distinctive outline; perithecial
wall less than 20 µm in width ..............................................................57
56. Perithecia with an apical disc; ascospores 15–19.5 ¥ 6.5–8
µm, smooth or warted; anamorph Cylindrocarpon candidu-
lum ...............‘Nectria’ (Neonectria) veuillotiana Sacc. & Roum.
(Brayford and Samuels, 1993 as Nectria veuillotiana)
56. Perithecia without an apical disc; ascospores 10–13 ¥ 3–5
µm, smooth; anamorph Cylindrocarpon destructans ................
Neonectria radicicola (Gerlach & Nilsson) Mantiri & Samuels
(Samuels and Brayford, 1990 as Nectria radicicola)
57. Perithecia densely gregarious on immersed, orange stroma; ascospores
12–14.5 ¥ 5.5–7.5 µm, smooth to minutely spinulose; anamorph acre-
monium-like ..........Cosmospora meliopsicola (Henn.) Rossman & Samuels
(Samuels et al., 1991 as Nectria meliopsicola)
57. Perithecia solitary to aggregated in groups of a few; ascospores 10–12
¥ 2.5–4 µm, smooth; anamorph Stilbella or Volutella...........................58
58. Anamorph Volutella; ascospores 10–11 ¥ 3–4 µm.................
Cosmospora consors (Ellis & Everh.) Rossman & Samuels
(Samuels, 1977 as N. consors)
58. Anamorph Stilbella; ascospores 10–12 ¥ 2.5–3 µm...............
Cosmospora stilbellae (Samuels & Seifert) Rossman & Samuels
(Samuels et al., 1991 as N. stilbellae)
59 (30). Ascospores less than 25 µm in length .........................................60
59. Ascospores more than 25 µm in length ...............................................65
60. Ascospores 19–26 ¥ 3.5–4.5 µm; on stromata of
Phyllachora ....................‘Nectria’ (Cosmospora) prodigiosa Syd.
60. Ascospores more than 6 µm in width, not obviously fungi-
colous ..................................................................................61
61. Ascospores yellow-brown, smooth, 18–21 ¥ 6.5–7.5 µm; perithecia

forming on a rust gall ................. ‘Nectria’ (Cosmospora) uredinaecola Pat.
61. Ascospores hyaline, lignicolous.............................................................62
62. Perithecia minute, with conspicuous saccate cells around
the apex; ascospores 16–24 ¥ 5.5–8.5 µm, striate, some-
times appearing spinulose or smooth .......Neonectria coronata
(Penz. & Sacc.) Mantiri & Samuels (Samuels and Brayford,
1994 as Nectria coronata)
62. Saccate cells not formed .....................................................63
63. Ascospores smooth to finely punctate, 19.5–24.5 ¥ 7–8.5 µm; perithecia
caespitose, red-orange, deeply cupulate when dry ..........‘Nectria’ noackiana
Syd.
63. Ascospores striate; perithecia collapsing laterally pinched or not at all
when dry................................................................................................64
64. Ascospores 17.5–22 ¥ 6.5–8 µm, finely striate; perithecia
smooth, not collapsed when dry; anamorph not known .......
‘Nectria’ seriata Rehm (Samuels and Brayford, 1994)
64. Ascospores 19–27 ¥ 8–10.5 µm, striate; perithecia conspic-
uously warted, collapsing laterally pinched or not collapsed
when dry; anamorph Fusarium......‘Nectria’ (Haematonectria)
borneensis Petr. (Samuels and Brayford, 1994)
65 (59). Perithecia on Dothideales on bamboo leaves; ascospores 28–37 ¥
8–13.5 µm, smooth, pale brown ..............Cosmospora tungurahuana (Petr.)
Rossman & Samuels (Samuels et al., 1991 as Nectria tunguarhuana)
65. Not on bamboo leaves and not obviously fungicolous .........................66
66. Ascospores smooth or warted ............................................71
67. Ascospores 35–52 ¥ 11–14 µm, coarsely striate; perithecia with a felty
white matrix covering the surface...................Calostilbe striispora (Ellis &
Everh.) Seaver (Rossman et al., 1999)
67. Ascospores averaging less than 35 µm in length .................................68
68. Ascospores 26–35 ¥ 10–13 µm, finely striate; perithecia
caespitose, immersed in Hypocrea-like stroma ...........‘Nectria’
balansae Speg. (Samuels and Brayford, 1994)
69. Perithecia pyriform, dark red, with a knobby, often dark red apex, smooth,
often glistening; ascospores 22–29 ¥ 9–10 µm, coarsely striate..............
Neonectria jungneri (Samuels and Brayford, 1994 as Nectria jungneri)
69. Perithecia subglobose, orange-red to red, warted or roughened .........70
70. Ascospores 25–30 ¥ c. 10 µm, smooth to finely striate .........
‘Nectria’ (Haematonectria) subsequens Rehm
70. Ascospores 22.5–32 ¥ 9.5–13 µm, smooth to coarsely
striate......................‘Nectria’ (Neonectria) pulcherrima Berk. &
Broome (Samuels and Brayford, 1994)
71 (66). Perithecia typically formed at the base of red synnemata, superfi-

cial on an erumpent stroma, caespitose, with white to pale yellow fur-
furaceous coating on the lower third; ascospores 29–35 ¥ 9–11 µm,
yellow-brown, smooth.......................Corallomycetella jatrophae (A. Möller)
Rossman & Samuels (Rossman et al., 1999)
71. Ascospores hyaline ................................................................................72
72. On decaying leaves and roots; perithecia solitary, concolor-
ous, not collapsed when dry; ascospores 18–48 ¥ 4–7 µm,
smooth; anamorph Cylindrocladium floridanum ......................
Calonectria kyotensis Terashita (Crous and Wingfield, 1994)
72. On decaying wood; perithecia caespitose, red with a darker
red ostiolar area, cupulate or not collapsed when dry;
ascospores 28.5–37 ¥ 7.5–9.5 µm anamorph not known ....
‘Nectria’ pseudadelphica Rehm
Acknowledgements
The authors acknowledge a great debt to our teacher and friend, Clark T.
Rogerson, for having laid the groundwork for and supporting our studies of
the Hypocreales. In addition, various agencies have provided financial support
to GJS over the years that it took to accumulate the information summarized
herein, most notably the American Philosophical Society, the National
Geographic Society, and the US National Science Foundation (BSR 8500236,
8721877 and ‘PEET’ grant, BSR 9712308, ‘Monographic Studies of
Hypocrealean Fungi: Hypocrea and Hypomyces’).
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A Reassessment of the Taxonomy 3
of Some Tropical Sooty Moulds
J.L. FAULL,1 I. OLEJNIK,1 M. INGROUILLE1 AND D. REYNOLDS2
1
School of Biological and Chemical Sciences, Birkbeck College,
University of London, Malet Street, London WC1E 7HX, UK;
2
Botany Section, Natural History Museum, 900 Exposition
Boulevard, Los Angeles, CA 90007, USA
Introduction
The sooty moulds are a group of tropical and sub-tropical fungi, numbering
over 200 species, that live on plant surfaces forming dense, dark mats of
hyphae so that the leaves appear to be covered with soot-like material. The
general term sooty mould was first used by Berkeley and Desmazières (1849)
in their detailed study of some members of Fumago Pers. on herbarium
material, including specimens on coffee leaves from Ceylon, on citrus leaves
in the Azores and Madeira, and on citrus species in their own conservatories
in France. The term was popularized in US plant pathology literature starting
with Weber’s paper in 1897. The presence of sooty moulds is often associated
with insect infestations of plants, the honeydew serving as a nutrient
source for the saprobic community (Hughes, 1976). Included within this
community are species which are commercially and agriculturally important.
For example, Caldariomyces fumago Woron. is used to produce the enzyme
chloroperoxidase for industrial purposes (Pickard et al., 1991) and ‘Capnodium
citri’ Pers. is a saprobic association with sucking insect infestations on Citrus
spp. and a wide variety of ornamental plants (Reynolds, 1999).
To date, the taxonomy of this group of saprobic phylloplane fungi has
been very unsatisfactory. The current confusion is caused by some basic prob-
lems pertaining to the sooty moulds. They exist as a community on plant sur-
faces, comprising many different mitosporic and ascosporic states. Their
mycelium is uniformly darkly pigmented with melanin, and they share spore
dispersal strategies adapted to wet leaf surfaces (Hughes, 1976; Reynolds,
1999). They share many other physical features that are adaptations to the

34 J.L. Faull et al.
phylloplane habitat, and this creates superficial morphological similarities,

especially in the mitosporic structures. The morphological characters com-
mon to the sooty moulds have been used by workers in the 1800s and 1900s
to establish taxonomic groupings, and names given to families of sooty moulds
include the Capnodiaceae Sacc. and Chaetothyriaceae Hans. This has given
rise to the ‘polymorphic’ species concepts that have confused sooty mould
taxonomy (McAlpine, 1896). The first descriptions of sooty moulds from
herbarium specimens, named as Fumago species by Persoon (1822), recorded
great variability in morphological structures including the presence of ‘small
tubes’ on the leaf surfaces of several deciduous tree species (Tulasne and
Tulasne, 1863; Zopf, 1878). The descriptions of the specimens we now would
recognize as Caldariomyces were from Citrus medica L. leaves from southern
Europe. In a recent study of over 270 herbarium specimens of the
Caldariomyces fumago and Leptoxyphium graminum Speg. sooty moulds, over
54% of the collections came from India, Malaysia, Cuba, New Borneo and the
Philippines. Of the remaining specimens studied, all but four came from other
tropical or sub-tropical regions, and the four collected in temperate regions
were actually isolated from hothouse environments or seed, confirming this
group to be Mediterranean to tropical in distribution (Olejnik, 1999).
Some of the known mitosporic structures ascribed to members of the
sooty moulds are illustrated in Fig. 3.1 and a summary of their interpreta-
tion is shown in Fig. 3.2a–f. Figure 3.1a illustrates the cover of dense sooty
mould that citrus trees may support. Individual leaves may be partly or com-
pletely covered (Fig. 3.1b). The mitosporic structure may be more or less
fringed by elongate hairs (Fig. 3.1c,d) and may be tightly or loosely confined
(Fig. 3.1d,e). The centrum, the mitospore production site (sensu Luttrell in
Reynolds, 1981), varies in location between the different species. In
Leptoxyphium, Caldariomyces (Fig. 3.2a) and Ciferrioxyphium Bat. & H. Maia
(Fig. 3.2d), the centrum is at the apex of an elongated stipe, although in
Caldariomyces and Leptoxyphium the structure is interpreted as an elongated
pycnidium. The mitosporic centrum in Polychaeton Pers. (Fig. 3.2c) is at the
base or in the mid-section of the stipe, whilst in Conidiocarpus Woron. (Fig.
3.2b) the centrum is in the mid-section and the stipe produces percurrent pro-
liferations to form a series of mitosporic structures, again interpreted as pyc-
nidia. The commonest mitosporic centrum seen in Aithaloderma H. & P. Syd.,
shown in Fig. 3.2e, is a stalkless pycnidium. Other species of sooty moulds
have long, thin mitosporic structures where the location of the centrum is not
characterized by a swelling, e.g. Scolecoxyphium Cif. & Bat. (Fig. 3.2f). The dif-
ferences between these structures are ill-defined and contribute to the taxo-
nomic confusion.
The phylogenetic and subsequent taxonomic relationships between
mitosporic and ascosporic species are also confused and incomplete. The asco-
mata in the sooty mould group are black, ovoid to ellipsoidal or dome-shaped
perithecia that may have setae. They contain a type of bitunicate ascus with
Taxonomy of Some Tropical Sooty Moulds 35
Fig. 3.1. Light and electron micrographs of mitosporic centra of sooty moulds.
a, Sooty mould on leaves in a citrus plantation; b, sooty mould on citrus leaf;
c, Leptoxyphium graminum Jena 886, bar = 10 µm; d, Leptoxyphium graminum
DAOM 137632, bar = 10 µm; e, Caldariomyces fumago ATCC32110, bar =
40 µm; f, Leptoxyphium graminum DAOM 149569, bar = 40 µm.
eight hyaline to brown septate spores (Fig. 3.2g,h). Associated with these
ascosporic structures are mitosporic structures, some of which are clearly
recognizable as pycnidia (e.g. Aithaloderma, Fig. 3.2e), whilst others are
Ciferrioxyphium-like structures (Fig. 3.2d), interpreted as synnemata.
The need to clarify the taxonomy of this group is now pressing. Attempts
to improve on the commercial production of chloroperoxidase by screening
related strains have proved unpromising. Species of Scorias Fr., Ciferrioxyphium
Fig. 3.2. Mitosporic and ascomatic centra of the sooty moulds (after Hughes,
1976). Mitosporic centra: a, Leptoxyphium/Caldariomyces; b, Conidiocarpus;
c, Polychaeton; d, Ciferrioxyphium (occasional mitosporic state of Aithaloderma);
e, common mitosporic state of Aithaloderma; f, Scolecoxyphium; g, ascoma
centrum of a sooty mould, Aithaloderma sp. with setae (after Hughes, 1976);
h, septate ascospore. * — * denotes location of mitotic centrum.
and Polychaeton have been tested, but none produced the enzyme (Hashimoto
and Pickard, 1984). The results of a taxonomic study of the Caldariomyces/
Leptoxyphium/Aithaloderma group are reported here and are integrated into
the wider concept of a revised taxonomy of the sooty moulds.
Methods
A total of 272 isolates, including herbarium specimens and living cultures,

was studied and used as taxonomic ‘units’. Herbarium specimens were char-
acterized on the basis of micro-morphological data. Living cultures were coded
on growth and morphology, substrate utilization and enzyme assays, creating
a series of 168 binary variables. Macro- and microscopic structures were also
coded for all samples as a series of 34 binary variables. Continuous charac-
ters were encoded as a series of additive binary variables. Variable codings
have been listed by Olejnik et al. (1999).
Principal components analysis, hierarchical clustering, with average link-
age as a clustering method, and discriminant analysis were carried out using
an SPSS PC package (SPSS Inc., 1993).
Results
Initial principal components analysis for the living isolates failed to show
any clear differences between isolates of Caldariomyces and Leptoxyphium (Olejnik
et al., 1999). Accepting the pre-existing identifications, combined data from all
herbarium specimens again failed to separate Leptoxyphium and Caldariomyces.
Furthermore, inclusion of the herbarium specimens of the mitosporic
Ciferrioxyphium and the putative ascosporic state of Leptoxyphium, Aithaloderma
(Hughes, 1976) created three clear groupings: one was a combination of
Leptoxyphium and Caldariomyces, the second contained only Aithaloderma, and the
third was a mixture of Leptoxyphium and Aithaloderma isolates (Olejnik et al., 1999).
Re-examination of the herbarium specimens revealed that, of samples
identified originally as belonging to the form genera Caldariomyces or
Ciferrioxyphium, over 40% appeared to have been misidentified and should
have been placed within the form genera Polychaeton or Conidiocarpus. Of
the herbarium specimens named as Leptoxyphium, fewer than 20% had been
correctly assigned. Many of the samples misidentified as Leptoxyphium
had mitosporic structures more closely resembling Polychaeton specimens.
Re-analysis of the data using principal components analysis, based on
more accurate identifications, further strengthened the congeneric nature of
Leptoxyphium and Caldariomyces, and separated off most Polychaeton speci-
mens (Fig. 3.3). However, Conidiocarpus was less well resolved and further
work is needed, especially on Polychaeton and Conidiocarpus specimens.
A preliminary examination of the herbarium specimens of Aithaloderma
spp. also revealed a substantial level of misidentification of the ascosporic
structures. However, removing the misidentified Leptoxyphium and
Caldariomyces specimens from the analysis and reassigning the mitosporic
herbarium specimens to their correct identifications, reinforced the difference
between Aithaloderma and the Caldariomyces/Leptoxyphium group (Fig. 3.4).
Fig. 3.3. Three-dimensional scatter plot of the first three factor scores provided
by a principal components analysis of re-identified herbarium specimens.
◆ Caldariomyces; ■ Leptoxyphium; 䉯 Polychaeton; 䉰 Conidiocarpus; * unknown.
Fig. 3.4. Two-dimensional scatter plot of discriminant scores of 272 samples of

Aithaloderma, Caldariomyces/Leptoxyphium, Polychaeton and Conidioxyphium.
◆ Caldariomyces; ■ Leptoxyphium; 䉯 Polychaeton; 䉰 Conidiocarpus;
* unknown; ⵧ Aithaloderma; + Ciferrioxyphium.
Discussion
These re-analysed results concur with the opinion of Hughes (1976)

and Olejnik et al. (1999) that Caldariomyces and Leptoxyphium are poorly
differentiated and likely to be congeneric. It is of some concern that there were
many misidentifications of the herbarium specimens, particularly among
those assigned to Leptoxyphium, which have added to the confusion that exists
in the taxonomy of the sooty moulds. While it appears that three of the
mitosporic taxa, Leptoxyphium, Caldariomyces and Ciferrioxyphium, are con-
generic, specimens described as Polychaeton or Conidiocarpus are extremely
variable and need further study.
Hughes (1976) stated that the mitosporic state of Aithaloderma,
Ciferrioxyphium, shares many features with Leptoxyphium. However, the
mitosporic structure of Ciferrioxyphium is termed a synnema with a mitosporic
locus at the apex, whereas the mitosporic structures of Leptoxyphium are
termed elongate pycnidia with a mitosporic locus either at the base or placed
sub-apically within the structure. The author’s studies strongly suggest that
the structure seen in Caldariomyces/Leptoxyphium is a synnema rather than a
pycnidium, in agreement with the work of Roquebert and Bury (1988).
Although a semantic argument, defining the mitotic structure of
Caldariomyces/Leptoxyphium as a synnema would strengthen the affiliation of
Aithaloderma to this group. However, all our analyses to date indicate clear dif-
ferences between the Caldariomyces/Leptoxyphium group and the Aithaloderma
specimens. The latter must remain separated from the mitosporic clades until
further research work shows that the two groups can be linked.
References
Berkeley, M.J. and Desmazières, J.B.H.J. (1849) On some moulds referred by authors to
Fumago, and on certain allied or analagous forms. Journal of the Horticultural
Society of London 4, 244–260.
Hashimoto, A. and Pickard, M. (1984) Chloroperoxidase from Caldariomyces
(= Leptoxyphium) cultures. Journal of General Microbiology 130, 2051–2058.
Hughes, S.J. (1976) Sooty moulds. Mycologia 68, 693–820.
McAlpine, D. (1896) The sooty mould of citrus trees: a study in polymorphism.
Proceedings of the Linnean Society of New South Wales 21, 469–499.
Olejnik, I. (1999) Caldariomyces/Leptoxyphium, a taxonomic study of a mould of indus-
trial importance. PhD thesis, University of London, London, UK.
Olejnik, I.M., Ingrouille, M. and Faull, J.L. (1999) Numerical taxonomy of the sooty
moulds Leptoxyphium, Caldariomyces and Aithaloderma based on micromorphol-
ogy and physiology. Mycological Research 103, 333–346.
Persoon, C.H. (1822) Mycologia Europeae. Sectio prima. Completa Omnium Fungorum in
Variis Europae Regionibus Detectorum Enumeratio. Erlangen, Germany.
Pickard, A., Kadima, T. and Carmichael, R. (1991) Chloroperoxidase – a peroxidase
with potential. Journal of Industrial Microbiology 7, 235–242.
Reynolds, D.R. (1981) Ascomycete Systematics: the Luttrellian Concept. Springer Verlag,
New York.
Reynolds, D.R. (1999) Capnodium citri: the sooty mould fungi comprising the taxon
concept. Mycopathologia 148, 141–147.
Roquebert, M.F. and Bury, E. (1988) Leptoxyphium: pycnide ou synnèma? Canadian
Journal of Botany 66, 2265–2272.
SPSS Inc. (1993) SPSS, Statistical Package for Social Scientists. Release 6, June 1992.
Chicago, Illinois, USA.
Tulasne, L.-R. and Tulasne, C. (1863) Selecta Fungorum Carpologia II, IX Fumago,
Imperiali Typograph, Paris, pp. 279–285.
Weber, H.J. (1897) Sooty mold of orange and its treatment. USDA Division of Vegetable
Physiology Pathology Bulletin 13.
Zopf, W. (1878) Die Konidien Fruchts von Fumago. Nova Acta Academie Caesaraeae
Leopoldina Carolinea German Naturae Curiosum 40, 255–329.
Lignicolous Freshwater Higher 4
Fungi with Reference to their
Teleomorph and Anamorph Stages
S. SIVICHAI, E.B. GARETH JONES AND N.L. HYWEL-JONES
BIOTEC–Mycology, Yothi Laboratories, 73/1 Rama 6 Road,

Introduction
This chapter reviews our knowledge of filamentous, tropical freshwater fungi

with special reference to those growing on submerged wood. There is a rich
and diverse range of tropical freshwater fungi and the observed mycota
depends on the substrata examined. Senescent and fallen leaves support
mainly Ingoldian fungi, while woody substrata support a different range of
species, with many ascomycetes. These differences also extend to the teleo-
morph/anamorph relations, with discomycetes forming the majority of the
teleomorphs of the Ingoldian fungi. However, teleomorphs of lignicolous fungi
are generally pyrenomycetes.
Webster (1992) reviewed the teleomorph/anamorph relationships of
Ingoldian fungi, listing 22 Ascomycota, five Basidiomycota and one tetraradi-
ate conidial zygomycete (Entomophthorales). Shearer (1993) also listed the con-
nections for freshwater ascomycetes, which included 33 for Ingoldian fungi,
while Wong (1996) compiled and added a further nine. Most of these were
reported from temperate regions with only four connections known
from tropical zones: Corynascus sepedonium (Emmons) v. Arx / Sepedonium-like
sp.; Gnomonia papuana Sivan. & D. Shaw / Sesquicillium sp.; Hymenoscyphus
malawiensis P.J. Fisher & Spooner / unnamed hyphomycete, and Microascus
lunasporus Jones / Scopulariopsis lunaspora Jones (Hyde et al., 1997).
Shearer (1989) reported five new species of Pseudohalonectria of which
P. phialidica Shearer is the only species that yields an unnamed hyphomycete
in culture. However, this connection was not listed by Shearer (1993). Webster
et al. (1995) collected and isolated Tricladium indicum Sati & Tiwari from a

42 S. Sivichai et al.
foam sample in South Africa and this gave rise to Cudoniella indica Webster,
Eicker & Spooner (teleomorph) when incubated in sterile water. This is the first
reported connection of a Tricladium with this teleomorph genus. Furthermore,
T. indicum developed from cultures of Cudoniella ascospores. Hyde and Goh
(1998) reported a new connection between Anthostomella aquatica K.D. Hyde
& Goh and Geniculosporium sporodochiale K.D. Hyde & Goh from tropical fresh-
water habitats, with both stages being found on submerged wood, and also
confirmed in culture studies. Sivichai et al. (1998b) were the first to link a
Monotosporella anamorph with Ascotaiwania sawada H.S. Chang & S.-Y. Hsieh,
while Ranghoo and Hyde (1998) also described a Monotosporella anamorph
for Ascotaiwania mitriformis Ranghoo & K.D. Hyde. Ranghoo and Hyde (1998)
described the new genus Ascolacicola and linked the type species Ascolacicola
aquatica Ranghoo & K.D. Hyde with Trichocladium uniseptatum Berk. &
Broome. In 1998, Descals et al. reported two teleomorph links within the
genus Anguillospora, namely Pezoloma sp. (Leotiales) as the teleomorph of
Anguillospora furtiva Webster & Descals and an Orbilia sp. (Leotiales) as the
teleomorph of A. rosea.
Goh (1997) noted that, in a number of teleomorph–anamorph connec-
tions, many of the anamorphic genera are heterogeneous. Some anamorphic
genera have teleomorphs in various genera: e.g. Tricladium spp. with teleo-
morphs in Cudoniella, Hydrocina and Hymenoscyphus. Conversely, some teleo-
morph genera have a range of anamorph stages: e.g. Massarina spp. with
anamorphs in Anguillospora, Clavariopsis and Tumularia.
Many related fungal states found in tropical freshwater habitats are poorly
reported while the number of new species of teleomorphs and anamorphs is
increasing dramatically. Fifty-six teleomorph/anamorph relationships have
been recorded, of which 26 are discomycetes, 13 have been assigned to the
Loculoascomycetes and 12 belong to unitunicate genera, while five anamorph
species have basidiomycete teleomorphs (Sivichai, 2000).
In this survey of the lignicolous freshwater fungi of Thailand, > 236
species were observed (108 ascomycetes, 8 basidiomycetes, 120 anamorphic
fungi). Three hundred and fourteen sporulated on cornmeal agar medium
(CMA) in a cooled illuminated cabinet and 21 teleomorph–anamorph con-
nections were established.
Materials and Methods
Fungi were isolated from submerged wood collected from freshwater streams
at various sites in Thailand, and from four timber species (Alstonia scholaris
R. Br., Anisoptera oblonga Dyer., Dipterocarpus alatus Roxb. and Xylia dolabri-
formis Benth.) exposed at Khao Yai National Park. The methods used have
been described previously (Sivichai et al., 1998a,b, 2000; Sivichai and Hywel-
Jones, 1999).
Lignicolous Freshwater Higher Fungi 43
Results
Of the 236 freshwater fungi collected, 343 different strains were established
(from 2400 single-spore isolates), of which 206 sporulated on CMA. Six of
these have known connections with four previously reported from terrestrial
habitats: Melanochaeta hemipsila (Berk. & Broome) E. Müller (Fig. 4.3) with
Sporoschisma saccardoi Mason & S. Hughes (Goh et al., 1997) (Fig. 4.3); Nectria
chaetopsinae Samuels with Chaetopsina fulva Samuels; Nectria chaetopsinae-poly-
blastiae Samuels with Chaetopsina polyblastiae Samuels (Samuels, 1985) and
Tubeufia cylindrothecia (Seaver) Höhnel with Helicomyces roseus Link (Barr,
1980) and two from freshwater habitats: Ascotaiwania sawada with a
Monotosporella anamorph stage (Sivichai et al., 1998b) and Hymenoscyphus
varicosporoides Tubaki with a Varicosporium anamorph stage (Tubaki, 1966).
A further 14 connections were made with teleomorphs in 14 pyreno-
mycetes, four loculoascomycetes and three discomycetes. Most of the
anamorph stages can be referred to typical terrestrial genera, with only two
Ingoldian freshwater species. Of the 21 connections listed in Table 4.1, 15 are
referred to named taxa, while for four species only one stage can be referred
to a named taxon.
Discussion
Webster (1992) listed 27 teleomorph/anamorph connections for freshwater

fungi of which 15 (c. 55%) had a discomycete teleomorph. These were pre-
dominantly from temperate regions and had Ingoldian fungi as the anamorph.
Similarly, of the 288 freshwater ascomycetes, predominantly temperate,
reported by Shearer (1993), 112 belonged to the discomycetes and 176 were
assigned to other ascomycete groups. Teleomorph/anamorph connections
were reported for 33 (c. 11%) of the 288 species. However, significantly, of
these 33, 20 (c. 60%) were from discomycetes while 13 (40%) were from other
ascomycete groups.
Of the discomycete connections reported from temperate areas (Webster,
1992; Shearer, 1993), only three (Hymenoscyphus malawiensis; Pezoloma
rhodocarpa P.J. Fisher & Spooner and Cudoniella indica), have been recorded
from tropical regions. Hyde et al. (1997) also noted that they found only two
discomycetes in 5 years of collecting freshwater lignicolous fungi. This sug-
gests that discomycetes are probably more common in temperate regions or
are favoured by lower temperatures for sporulation, and agrees with data from
this study showing that discomycetes are not the most frequently isolated
group in freshwater habitats in the tropics. In the insect-associated fungi,
Evans (1982) noted that by far the greater majority of entomogenous fungal
species in tropical forests belong to the genus Cordyceps (Clavicipitales;
Ascomycotina) in contrast to temperate habitats where Entomophthora species
Table 4.1. Teleomorph/anamorph connections established in this study and

means of confirmation.
Connections Confirmation
Teleomorph Anamorph Ascospores Conidia Both
Anthostomella taiwanensis Geniculosporium-like* + – –

Ascotaiwania sawada Monotosporella sp.* + – –
Delitschia sp. 1 Phomopsis sp. 1-like + – –
Diaporthe sp. 1 Phomopsis sp. 2-like* + – –
Hymenoscyphus Tricladium anamorph of
varicosporoides H. varicosporoides + – –
Hysterographium sp. 1 Phomopsis sp. 3-like + – –
Melanochaeta garethjonesii Sporoschisma uniseptatum* + – –
Melanochaeta hemipsila Sporoschisma saccardoi + – –
Microascus sp.1-like Unidentified hyphomycete* + – –
Nectria chaetopsinae Chaetopsina fulva + + +
Nectria chaetopsinae- Chaetopsina polyblastiae + – –
polyblastiae
Nectria sp. 1 Cylindrocarpon sp. 1* + – –
Nectria sp. 2 Chaetopsina sp. 2* + + +
Oxydothis sp.1-like Phialophora cf. cyclaminis* + – –
Tubeufia cylindrothecia Helicomyces roseus + – –
Tubeufia sp. 1 Aquaphila albicans + – –
Teleomorph unidentified Dactylaria sp. 2* – + –
Teleomorph unidentified Ellisembia brachypus* + – –
Teleomorph unidentified Phaeoisaria clematidis* + + +
Unidentified discomycete sp. 7 Unidentified hyphomycete + – –
Unidentified discomycete sp. 9 Sporodochial sp. 1 + – –
* Teleomorph–anamorph connections reported for the first time.
(Entomophthorales; Zygomycota) predominate. However, the Entomophthorales

were also found in the tropics but more frequently in the winter (November
to February) and when temperatures are lower, e.g. 12–15°C. This is a topic
worthy of further research in the current evaluation of freshwater fungal
diversity. Consideration must be given to the mode of dispersal of ascospores.
Discomycetes may be better adapted to terrestrial or amphibious/marginal
aquatic habitats for ascospore release.
In contrast to the above, most of the teleomorph/anamorph connections
in this study involved pyrenomycete ascomycetes (14; 67%), with only four
loculoascomycetes (19%) and three discomycetes (14%). The four loculo-
ascomycete connections included: one Delitschia, one Hysterographium and two
Tubeufia species. All of these were new connections, apart from Tubeufia cylin-
drothecia (Fig. 4.1), which was already linked with Helicomyces roseus. The
anamorphs connected with Delitschia and Hysterographium were the first
reports of anamorphs for these genera.
Three discomycete connections were made, two reported for the first time
(Figs 4.5–4.7). Hymenoscyphus varicosporoides was first reported from Japan
(Tubaki, 1966) with a ‘Varicosporium’ anamorph. However, Tubaki also indi-
cated that the anamorph stage could be assigned equally well to Tricladium or
Polycladium. Then, Webster et al. (1995) established the connection between
Cudoniella indica with Tricladium indicum as the anamorph. The anamorph of
Hymenoscyphus varicosporoides is also similar to T. indicum, but this was not
discussed by Webster et al. (1995). Thus it is important to establish whether
the apical ring of the teleomorph’s asci gives a positive or negative reaction
with I/KI. Further studies are in progress by the authors to determine the cor-
rect assignment of the teleomorph.
Most of the teleomorph/anamorph connections established were with
pyrenomycetes and isolations were made from ascospores which yielded the
anamorph stage in culture. Of the 21 connections in Table 4.1, many are
linked to known anamorphic genera, although a Microascus sp. was linked to
an unidentified hyphomycete. Of the 11 pyrenomycete connections, only two
can be assigned to species level (Sporoschisma uniseptatum, Phialophora cf.
cyclaminis). The others await further study for complete identification. Of the
11 connections, four can be assigned to species level (Anthostomella taiwa-
nensis, Ascotaiwania sawada, Melanochaeta garethjonesii (Fig. 4.4), Phaeoisaria
clematidis (Fig. 4.2)), five to generic level, while two remain to be identified.
Future work will be directed to the full identification of these taxa and their
description as new taxa.
Only two teleomorph/anamorph connections of Ingoldian hyphomycetes
were recorded: Hymenoscyphus varicosporoides with a ‘Tricladium anamorph of
H. varicosporoides’ and Nectria sp. with a Cylindrocarpon sp., with teleomorphs
belonging to discomycetes and pyrenomycetes, respectively. The ratio of
Ingoldian fungi connections in this study was low (0.09) and this may be due
to the examination of woody substrata (as most occur on senescent, fallen
leaves) and the temperature used for their incubation (20°C).
In conclusion, the current study has produced many teleomorph/
anamorph connections. This can be accounted for by: (i) the diversity of fungi
collected, a total of 236 species; (ii) the large number of samples/collections
studied (638 herbarium specimens were deposited); (iii) the high number of
isolations made (from c. 2400 single spore-isolates), of which 206 sporulated
in culture; (iv) previous studies of freshwater fungi being in temperate regions,
where fungal diversity is lower compared with tropical regions; and (v) sub-
strata being retrieved throughout the year at regular intervals, thus enabling
sampling of early to late colonizers. This material was incubated routinely
under laboratory conditions for 3 months, and often up to 12 months. Lamore
and Goos (1978) showed that species richness of freshwater fungi was higher
when collected in the rainy season. Two obvious examples in the present study
are Tubeufia cylindrothecia and Helicomyces roseus. The latter is a common
species which occurred over the whole period of the study, but T. cylindrothecia
Figs 4.1–4.8. Examples of teleomorph–anamorph connections on test blocks

exposed at Khao Yai National Park, Thailand.
Continued
was mostly found in the dry season (April and May) when the water at the
29.2 km test site, in Khao Yai National Park, Thailand, dried out. Previous
investigations focused on the isolation of anamorph conidia, whereas, in the
present study, most of the connections were obtained from ascospores and
nearly 60% of the cultures obtained sporulated, yielding 51% anamorphs, 8%
teleomorphs, and 26% teleomorph/anamorphs. It can be argued that dis-
comycete teleomorphs are less frequent in the tropics than in temperate
waters, while pyrenomycetes are more frequent.
This study has also shown that teleomorphs usually require prolonged
incubation before they develop fully and mature on the substrata. Of the ster-
ile strains obtained, some may be Ingoldian fungi that require aeration, or
submergence in water, before they produce conidia. All the strains isolated
appeared to be homothallic, thus ensuring sporulation of the ascomycetes (e.g.
Ascotaiwania sawada, Nectria chaetopsinae and Nectria sp. 2 (Fig. 4.8)). However,
some of the non-sporulating cultures may be heterothallic and this aspect
warrants further study.
Acknowledgements
This work was supported by the TRF/BIOTEC special Programme for

Biodiversity Research and Training grant BRT 143016. The British
Mycological Society is thanked for supporting the attendance of S. Sivichai at
the British Mycological Society Millennium Meeting, Tropical Mycology 2000,
25–29 April 2000, at Liverpool John Moores University.
Figs 4.1–4.8. Opposite
Fig. 4.1. Tubeufia cylindrothecia and its anamorph Helicomyces roseus with
white ascomata and coiled conidia (arrowed). Scale bar = 500 µm.
Fig. 4.2. Unidentified teleomorph and its anamorph Phaeoisaria clematidis with
colony of the teleomorph (arrowed t) and anamorph (arrowed a). Scale bar =
1000 µm.
Fig. 4.3. Melanochaeta hemipsila (arrowed t) and its anamorph Sporoschisma
saccardoi (arrowed a). Scale bar = 200 µm.
Fig. 4.4. Melanochaeta garethjonesii (arrowed t) and its anamorph Sporoschisma
uniseptatum (arrowed a). Scale bar = 200 µm.
Figs 4.5–4.7. Unidentified discomycete sp. 9 and its anamorph sporodochial
sp. 1.
Fig. 4.5. White apothecia of discomycete with discrete masses of brown conidia
(arrowed). Scale bar = 500 µm.
Fig. 4.6. Section through an apothecium which developed from an acervulus.
Scale bar = 80 µm.
Fig. 4.7. Conidiophores on hyaline hyphae with developing conida. Scale bar =
10 µm.
Fig. 4.8. Nectria sp. 2 and its anamorph Chaetopsina sp. 2 with a pyriform
ascoma and conidiophore. Scale bar = 50 µm.
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Descals, E., Marvanová, L. and Webster, J. (1998) New taxa and combinations of
aquatic hyphomycetes. Canadian Journal of Botany 76, 1647–1659.
Evans, H.C. (1982) Entomogenous fungi in tropical forest ecosystems: an appraisal.
Ecological Entomology 7, 47–60.
Goh, T.K. (1997) Tropical freshwater Hyphomycetes. In: Hyde, K.D. (ed.)
Biodiversity of Tropical Microfungi. Hong Kong University Press, Hong Kong, pp.
189–227.
Goh, T.K., Ho, W.H., Hyde, K.D. and Umali, T.E. (1997) New records and species of
Sporoschisma and Sporoschismopsis from submerged wood in the tropics.
Mycological Research 101, 1295–1307.
Hyde, K.D. and Goh, T.K. (1998) Tropical Australian freshwater fungi XIII. A new
species of Anthostomella and its sporodochial Geniculosporium anamorph. Nova
Hedwigia 67, 225–233.
Hyde, K.D., Wong, S.W. and Jones, E.B.G. (1997) Freshwater ascomycetes. In: Hyde,
K.D. (ed.) Biodiversity of Tropical Microfungi. Hong Kong University Press, Hong
Kong, pp. 179–187.
Lamore, B.J. and Goos, R.D. (1978) Wood-inhabiting fungi of a freshwater stream in
Rhode Island. Mycologia 70, 1025–1034.
Ranghoo, V.M. and Hyde, K.D. (1998) Ascomycetes from freshwater habitats: Ascocicola
aquatica gen. et sp. nov. and a new species of Ascotaiwania from wood submerged
in a reservoir in Hong Kong. Mycologia 90, 1055–1062.
Samuels, G.J. (1985) Four new species of Nectria and their Chaetopsina anamorphs.
Shearer, C.A. (1989) Pseudohalonectria (Lasiosphaeriaceae), an antagonistic genus from
wood in freshwater. Canadian Journal of Botany 67, 1944–1955.
Shearer, C.A. (1993) The freshwater Ascomycetes. Nova Hedwigia 56, 1–33.
Sivichai, S. (2000) Tropical freshwater fungi: their taxonomy and ecology. PhD thesis,
Portsmouth University, Portsmouth, UK.
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Sivichai, S., Hywel-Jones, N.L. and Jones, E.B.G. (1998b) Lignicolous freshwater
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The Pandanaceae – Does it Have 5
a Diverse and Unique Fungal
Biota?
E.H.C. MCKENZIE,1 S.R. WHITTON1 AND K.D. HYDE2
1
Landcare Research, Private Bag 92170, Auckland, New Zealand;
2
Centre for Research in Fungal Diversity, Department of Ecology
and Biodiversity, The University of Hong Kong, Pokfulam Road,
Hong Kong S.A.R, China
Introduction
During the past 10 years several studies have been undertaken to discover and
describe the diversity of microfungi associated with the Pandanaceae. This
chapter provides a summary of current knowledge and attempts to answer
the question of whether or not the Pandanaceae supports a diverse and unique
fungal biota.
The Pandanaceae
The Pandanaceae is a monocotyledon family, predominantly tropical but

extending as far south as New Zealand, and as far north as Nepal. The family
occurs throughout the Pacific, South-East Asia, and South Asia to western
Africa. It contains only three genera but 800–900 species: Freycinetia Gaud.,
about 200 species, Pandanus Parkins., 600–700 species, and Sararanga Hemsl.,
two species.
Freycinetia species are usually lianes with scrambling or climbing stems
and adventitious, adhesive roots clasping around trunks. The leaves are
usually linear, rarely over 2 m long, and dead leaves often remain attached.
The genus is distributed from Sri Lanka, throughout South-East Asia and
Malesia to Taiwan, New Zealand, and the Pacific islands in the east. Towards
its geographical limits in the east and south of the Pacific, there is often only

52 E.H.C. McKenzie et al.
a single species in each country. For example, in New Zealand there is only
F. baueriana ssp. banksii (Cunn.) Stone, with the other subspecies, baueriana,
endemic to Norfolk Island. In the Cook Islands, F. wilderi Martelli ex. Wilder
is the only species, and in Hawaii it is F. arborea Gaudich.
Pandanus species or screwpines are usually trees or shrubs with adventi-
tious prop-roots, never clasping but produced from stems and stem bases.
The leaves are usually linear, often more than 2 m in length; they may remain
attached for a long time following leaf death, and large amounts of dead leaves
may accumulate under plants. The genus is characteristically found on the
coast, sometimes in thick groves, but extending to montane forests. Pandanus
is distributed throughout the range of the family but with only 24 species in
Africa.
Sararanga species are trees growing to 20 m, producing adventitious prop-
roots. The genus, which is typically found in open lowland swamps, is
restricted to the Philippines, Papua New Guinea and the Solomon Islands.
The green leaf fibre of Freycinetia and Pandanus is sometimes used for plait-
ing into mats, baskets, fans, sails, and garments or, where abundant, for house
thatch. Stems and aerial roots are used as close-fitting coverings for imple-
ments, for making fish traps, and for tying thatch onto house roofs. Many
species have edible flowers or edible leafy flower bracts. Some species of
Pandanus (e.g. P. veitchii Hort., or the widespread maritime species, P. tectorius
Parkins.) are cultivated for edible fruit or as ornamentals; some of these have
variegated or striped leaves.
Fungi described from the Pandanaceae
Prior to 1996, 169 species of fungi, mainly ascomycetes and mitosporic fungi
(Table 5.1), had been described from the Pandanaceae (McKenzie and Hyde,
1996). However, there had been no systematic study of fungi on this family
of plants. Subsequently, a further 18 ascomycetes and 14 mitosporic fungi
have been described on Pandanus, and three ascomycetes and one
hyphomycete on Freycinetia (Table 5.2).
Table 5.1. Number of fungi described from the

Pandanaceae, prior to McKenzie and Hyde (1996).
Number of fungal species

Freycinetia Pandanus
Ascomycetes 15 59
Basidiomycetes 4 15
Hyphomycetes 13 25
Coelomycetes 3 35
Total 35 134
The Pandanaceae 53
Table 5.2. Fungi described from the Pandanaceae since McKenzie and Hyde
(1996).
Ascomycetes on Pandanus
Anthostomella minutoides K.D. Hyde, Nova Hedwigia 62, 315 (1996) – Java.
A. petrinensis Dulym., P.F. Cannon & Peerally, Mycological Research 102, 1319
(1998) – Mauritius.
A. theobromina Dulym., P.F. Cannon & Peerally, Mycological Research 102,
1322 (1998) – Mauritius.
Astrocystis cepiformis Dulym., P.F. Cannon & Peerally, Mycological Research
102, 1328 (1998) – Mauritius.
A. fimbriata Dulym., P.F. Cannon & Peerally, Mycological Research 102, 1326
A. rarissima Dulym., P.F. Cannon & Peerally, Mycological Research 102, 1327
Fasciatispora pandanicola K.D. Hyde, Nova Hedwigia 61, 261 (1995) – Java.
Linocarpon appendisporum K.D. Hyde, Botanical Journal of the Linnean Society
123, 116 (1997) – Irian Jaya.
L. breve K.D. Hyde, Botanical Journal of the Linnean Society 123, 119 (1997) –
Irian Jaya.
L. falciformisporum K.D. Hyde, Botanical Journal of the Linnean Society 123,
123 (1997) – Irian Jaya.
L. fasciatum Dulym., P.F. Cannon & Peerally, Mycological Research 102, 1332
L. pandanicola K.D. Hyde, Botanical Journal of the Linnean Society 123, 129
(1997) – Irian Jaya.
L. spathulatum Dulym., P.F. Cannon & Peerally, Mycological Research 102, 1333
L. sulcatum Dulym., P.F. Cannon & Peerally, Mycological Research 102, 1336
Meliola kapoorii Hosag. & Raghu, Meliolales of India, 229 (1996) – India.
M. pandanacearum Hosag. & T.K. Abraham, Indian Phytopathology 51, 303
(1999) – India.
Nipicola pandani K.D. Hyde, Nova Hedwigia 63, 419 (1996) – Hong Kong.
Stictis pandani Whitton, K.D. Hyde & McKenzie, Fungal Diversity 2, 172 (1999)
– Australia.
Hyphomycetes on Pandanus
Acrodictys lamma Whitton, McKenzie & K.D. Hyde, Fungal Diversity 4, 163
(2000) – Hong Kong.
A. triarmatus Whitton, McKenzie & K.D. Hyde, Fungal Diversity 4, 166 (2000) –
Mauritius.
Bahusutrabeeja dubhashii Bhat, Indian Journal of Forestry 17, 129 (1994) – India.
Cryptophiale pandanicola Dulym., P.M. Kirk & Peerally, Mycotaxon 73, 313
Dictyochaeta fimbriaspora Whitton, McKenzie & K.D. Hyde, Fungal Diversity 4,
138 (2000) – Philippines.
D. microcylindrospora Whitton, McKenzie & K.D. Hyde, Fungal Diversity 4, 141
(2000) – Hong Kong.
Continued
Table 5.2. Continued
D. multisetula Whitton, McKenzie & K.D. Hyde, Fungal Diversity 4, 143 (2000) –
Australia.
D. seychellensa Whitton, McKenzie & K.D. Hyde, Fungal Diversity 4, 148 (2000)
– Seychelles.
Fuscophialis suttonii Dulym., W.P. Wu & Peerally, Mycoscience 39, 285 (1998) –
Mauritius.
Paraceratocladium triseptata Dulym., W.P. Wu & Peerally, Mycoscience 39, 288
Spadicoides mauritiana Dulym., P.M. Kirk & Peerally, Mycotaxon 73, 319 (1999)
– Mauritius.
Sporidesmium paradecorosum Dulym., W.P. Wu & Peerally, Mycoscience 39,
290 (1998) – Mauritius.
Troposporopsis atroapicis Whitton, McKenzie & K.D. Hyde, Fungal Diversity 3,
176 (1999) – Hong Kong (+ Australia, Mauritius, Philippines).
Coelomycete on Pandanus
Rubikia splendida Dulym., Minter & Peerally, Mycological Research 102, 1242
Ascomycetes on Freycinetia
Anthostomella kapiti Whitton, K.D. Hyde & McKenzie, in Lu & Hyde, A World
Monograph of Anthostomella, 102 (2000) – New Zealand.
A. manawatu Whitton, K.D. Hyde & McKenzie, in Lu & Hyde, A World
Monograph of Anthostomella, 119 (2000) – New Zealand.
A. okatina Whitton, K.D. Hyde & McKenzie, in Lu & Hyde, A World Monograph
of Anthostomella, 138 (2000) – New Zealand.
Hyphomycete on Freycinetia
Dictyochaeta renispora Whitton, McKenzie & K.D. Hyde, Fungal Diversity 4, 146
(2000) – Philippines (+ Australia, Brunei).
McKenzie (1991a–c, 1995) and McKenzie and Kuthubutheen (1993)

carried out an intensive study of hyphomycetes associated with 12 species of
Freycinetia from 11 countries. Close attention was paid to F. baueriana ssp.
banksii in New Zealand, with 39 collections examined for hyphomycetes.
Dulymamode et al. (1998a–e, 1999) have recently described several new
species of ascomycetes and mitosporic fungi, found in Mauritius, on one or
other of the 14 endemic species of Pandanus. Whitton (1999) studied micro-
fungi associated with members of the Pandanaceae. He examined nine species
of Freycinetia, 23 species of Pandanus, and one species of Sararanga from 11
countries. Some new species have been described from this study (Whitton et
al., 1999a,b, 2000a,b; Lu and Hyde, 2000).
If the purported ratio of 5.7 species of fungi for every vascular plant
species is correct (Hawksworth, 1991), then the Pandanaceae should support
5000 or so unique species of fungi. Is this possible? The family does not
The Pandanaceae 55
form ectomycorrhizas, and monocotyledonous plants, in general, support few

basidiomycetes. Obvious pathogenic associations appear to be rare; there are
relatively few leaf spots, and only two rusts are known on the Pandanaceae
(McKenzie and Hyde, 1997). Endophytic fungi associated with the Pandanaceae
have not been studied. Of the 205 fungal species described to date on mem-
bers of the Pandanaceae, less than 10% are macroscopic. Thus, most of the
known mycota on the Pandanaceae is microscopic and saprotrophic, and it is
found mainly on dead leaves, or sometimes on dead ‘bark’. Approximately 450
species of fungi are now known to occur on the Pandanaceae (Whitton, 1999).
How many of these species are unique to the Pandanaceae or are restricted to
only one or a few species of Freycinetia or Pandanus? As soon as a fungal species
is found on a second member of the Pandanaceae, or on a different host sub-
stratum, then it can count as only 0.5 of a fungus towards the 5.7 figure of
Hawksworth (1991). While many of the microfungi may be restricted to the
Pandanaceae, it is hard to imagine that they would be restricted to only a single
species of Freycinetia or Pandanus.
How many species of fungi are unique to this plant family? Matsushima,
for instance, recorded five hyphomycetes on Pandanus tectorius from Taiwan
(Matsushima, 1980) and one from Japan (Matsushima, 1987), and two on
Pandanus sp. from Australia (Matsushima, 1989). None of these species are
restricted to Pandanus, and some (Bipolaris australiensis (M.B. Ellis) Tsuda &
Ueyama, B. hawaiiensis (M.B. Ellis) J.Y. Uchida & Aragaki, Curvularia lunata
(Wakker) Boedijn, and Nigrospora sphaerica (Sacc.) E.W. Mason) are common
and widespread on a broad range of substrata.
Fungi on Freycinetia baueriana ssp. banksii in

New Zealand, a Temperate Area
In New Zealand, the only representative of the Pandanaceae is the endemic
Freycinetia baueriana ssp. banksii. Approximately 150 species of fungi, of which
about 75 are ascomycetes and 65 are hyphomycetes, have been collected on
this plant. Approximately two-thirds of these have been determined to species
(53 ascomycetes, 10 basidiomycetes, 37 hyphomycetes). F. baueriana ssp.
banksii is the host plant from which 14 fungal species have been described. Of
these, five species are known from only the type collections (Anthostomella
manawatu Whitton, K.D. Hyde & McKenzie, A. okatina Whitton, K.D. Hyde &
McKenzie, Crepidotus parietalis E. Horak, Odontia flexibilis G. Cunn., Puccinia
freycinetiae McKenzie). Only three species occurring in more than one collec-
tion (Anthostomella kapiti Whitton, K.D. Hyde & McKenzie – 3 specimens,
Chalarodes bisetis McKenzie – 11 specimens, and Stachybotrys freycinetiae
McKenzie – 35 specimens) have been found exclusively on F. baueriana ssp.
banksii. Two species, Chaetosphaeria aotearoae S. Hughes (Melanochaeta
aotearoae (S. Hughes) E. Müll., Harr & Sulmont) and Stictis subiculata
P.R. Johnst., are known on other hosts in New Zealand and elsewhere. S. subic-
ulata also occurs on Pandanus in Australia, while the anamorph of M. aotearoae
is the widespread Sporoschisma mirabile Berk. & Broome. Two species described
from F. baueriana ssp. banksii also occur on other species of Freycinetia in some
Pacific island countries (Stachybotrys breviuscula McKenzie – New Caledonia,
and Zebrospora bicolor McKenzie – Cook Islands, New Caledonia, Samoa), while
Guedea novaezelandiae S. Hughes occurs on Pandanus in Brunei, and Nectria
freycinetiae Samuels on Pandanus in Hong Kong. One other fungus, Coronospora
novaezelandiae Matsush., which is commonly found on F. baueriana ssp. banksii
(31 collections), was described by Matsushima (1985) from a single collection
on the New Zealand nikau palm (Rhopalostylis sapida H. Wend. & Drude). Of
the other fungi found on F. baueriana ssp. banksii and determined to species
level, three were described elsewhere on the Pandanaceae – Clypeosphaeria
stevensii Syd. was described from Freycinetia sp. in Hawaii, Echidnodes pandani
(Rostr.) Hansf. (Asterina pandani Rostr.) from Pandanus sp. in Thailand, and
Linocarpon pandani (Syd. & P. Syd.) Syd. & P. Syd. (Linospora pandani Syd. &
P. Syd.) in the Philippines. Another seven species of ascomycetes and five
species of hyphomycetes are awaiting description as new species. The other
approximately 125 species occur on a broad range of plants, either in
New Zealand or overseas. Thus, ten described species of fungi are known
so far to be restricted to F. baueriana ssp. banksii, along with 12 as yet
undescribed species. It is probable that more intensive collecting will lead
to the discovery of some of these fungi on other plant species, or perhaps
on other species of the Pandanaceae in other countries. Detailed examination
of fungi collected on F. baueriana ssp. banksii, but determined only to
genus, may also reveal new species. However, the situation in New Zealand
is perhaps unique. New Zealand is very isolated and it has only a single
member of the Pandanaceae. It could be expected that this isolation has
led to the evolution of fungi which are restricted to this one species of
Freycinetia.
Fungi on Pandanaceae in the Tropics
In a further study of hyphomycetes on Freycinetia, McKenzie (unpublished

data) examined 26 collections of Freycinetia from Indonesia, Malaysia, and
nine Pacific island countries. He found 64 genera of hyphomycetes; 23 of
these had not been recorded on F. baueriana ssp. banksii in New Zealand, but
only eight of the genera were unknown in New Zealand. It is, perhaps, notable
that Chalarodes bisetis, Coronospora novaezelandiae, and Stachybotrys freycinetiae,
three species which are commonly found in New Zealand on Freycinetia, were
not found in collections from these tropical countries. Conversely, Chalarodes
obconica McKenzie, Stachybotrys nephrodes McKenzie, S. parvispora S. Hughes
and Sporidesmium freycinetiae McKenzie are widespread in the tropical Pacific,
The Pandanaceae 57
but were not found in New Zealand. Two fungi, Stachybotrys breviuscula and
Zebrospora bicolor, were found both in New Zealand and elsewhere.
Whitton (1999) made a widespread study of microfungi on the
Pandanaceae, also including ascomycetes. He found 225 species of fungi (149
mitosporic in 78 genera, and 76 ascomycetes in 27 genera) on 33 species of
the Pandanaceae, an average of 6.8 species of fungi per species of host plant.
He found five new genera of mitosporic fungi (Dichotophora, Nakatopsis,
Ramocapitis, Sporotretophora and Troposporopsis (Whitton, 1999; Whitton et al.,
1999b)), two new ascomycete genera (Callerascus and Flexuoniesslia), and 54
new species of fungi. Thus, about 25% of all species identified were new to
science, and there was a ratio of 1.6 new species per host species studied.
There are published records of at least 65 species of mitosporic fungi on
Freycinetia spp. (Whitton, 1999). Of these, 17 species have been described with
Freycinetia as the type substratum, a further ten are putatively new species
(Whitton, 1999), and 38 were originally described from other substrata,
including one described from Pandanus. Of the 27 species described or cur-
rently awaiting description from Freycinetia, eight are known from more than
one species of Pandanaceae, leaving 19 so-called ‘unique species’. Similarly, 61
ascomycetes have been recorded on Freycinetia, 18 with Freycinetia as the type
substratum, eight putatively new species, and 35 described from other sub-
strata, including four described from Pandanus. Of the 26 species described or
to be described from Freycinetia, eight are known from more than one species
of Pandanaceae, leaving 18 ‘unique species’ (Table 5.3).
Table 5.3. Ascomycetes and mitosporic fungi recorded from the Pandanaceae.
Type on
other
Total no. Type on plant Undescribed ‘Unique’
of species Pandanaceae species species species*
On Freycinetia
Mitosporic 65 17 38 10 19
Ascomycetes 61 18 35 8 18
On Pandanus
Mitosporic 198 66 106 26 76
On Sararanga
Mitosporic 5 0 4 1 1
Total 449 171 220 58 175
* Those species known only on the Pandanaceae, and on only one member
(species) of the Pandanaceae.
Corresponding figures for mitosporic fungi on Pandanus are: 198 species

known on Pandanus, 66 described from Pandanus, 26 putatively new species,
and 106 described from other substrata. Of the 92 species so far described or
to be described from Pandanus, 16 are known from more than one species of
Pandanaceae, leaving 76 ‘unique species’. For ascomycetes on Pandanus: 118
species are known, 70 have been described from Pandanus, 13 are putatively
new species, and 35 were described from other substrata (including two
described on Freycinetia). Of the 83 species described or to be described from
Pandanus, 22 are known from more than one species of Pandanaceae, leaving
61 ‘unique species’.
Only two species of ascomycetes and five mitosporic fungi have been
recorded on Sararanga, all from the Philippines. The two ascomycetes were
originally described from palms in Australia. Four of the mitosporic fungi were
described from other substrata, but one species, Cryptophiale hamulata ined.,
is to be described as a new species, presently unique to Sararanga.
The diversity of fungi known from Freycinetia appears to differ from that
inhabiting Pandanus. Of the 133 species of fungi known from Freycinetia (Table
5.4), 44 species are known also to inhabit Pandanus, giving a species compo-
sition overlap of 33%. Of the overlapping species, 36 are known from other
substrata besides the Pandanaceae, suggesting that they are the less fastidious,
more ubiquitous species. It can only be surmised whether the apparent
difference in fungal species colonizing Freycinetia is due to morphological,
ecological, or geographical differences. Differences in distribution of
Freycinetia and Pandanus exist, but to a large extent wherever Freycinetia is
found so is Pandanus. It is unlikely, therefore, that geography plays a major
role in fungal species composition differences between Freycinetia and
Pandanus. Morphologically there are some differences; generally the leaves of
Freycinetia are smaller than those of Pandanus, and the leaves of the latter are
often in contact with the soil and with other decaying plant litter.
Two species of Pandanus (P. furcatus and P. tectorius) occur in Hong Kong.
Whitton (1999) found a difference in overall fungal species composition
Table 5.4. Number of fungal species and myxomycetes published as occurring

on the Pandanaceae.
Overlap between
Freycinetia
Freycinetia Pandanus Sararanga and Pandanus
Ascomycetes 61 118 2 20
Basidiomycetes 6 17 0 0
Mitosporic fungi 65 198 5 23
Oomycetes 1 1 0 1
Myxomycetes 0 2 0 0
Total 133 336 7 44
The Pandanaceae 59
between these two species of Pandanus. P. furcatus had 45 species of micro-

fungi associated with its decaying plant parts, whilst P. tectorius had 35 species,
with only ten species in common. Reasons for the apparent disparity in species
composition between these two host substrata include:
1. Morphological differences – P. furcatus produces long leaves, and has a rel-
atively thin cuticle and no trunk or branches. The leaves typically remain
attached for some time following senescence, and are often in contact with
the soil as soon as they die, if not before. P. tectorius has a distinct trunk, many
branches, and much shorter leaves with a thicker cuticle. Because of the short
leaves and trunk-forming habit, the leaves, which also remain attached for
some time following senescence, typically do not come in direct contact with
the soil until they fall from the plant.
2. Environmental differences – P. furcatus is restricted to southern China, and
is typically found along the edges of streams in areas covered by forest. It tol-
erates low light levels and encounters high humidity, better developed soils,
very little direct wind, and no salt spray. P. tectorius is found along coastlines
throughout South-East Asia, southern China, northern Australia, the Pacific,
and Japan. It is subjected to direct sunlight, salt spray, coastal storm and tidal
events, desiccation from coastal winds, and shallow, poorly developed soils.
3. Ecological/varietal differences – P. furcatus shows very little morphologi-
cal variation throughout its geographical range, whereas P. tectorius is divided
into many varieties based on morphological variations (Stone, 1982, 1988).
When fungi known on P. furcatus and on P. tectorius from other parts of
the world are combined with those known on these hosts in Hong Kong, there
was still a difference in the number of fungal species recorded on these two
hosts, and there was still only a small number of overlapping species. However,
P. tectorius now has the higher number of fungal species – 88 species, com-
pared to 45 on P. furcatus, with only 15 species in common. The higher num-
ber of species recorded on P. tectorius presumably reflects its broader
geographical range, with more samples having been investigated, and the
morphological/ varietal differences in this species.
Of the 35 species of fungi known from P. tectorius in Hong Kong, and the
61 species reported from this host in other parts of the world, there is an over-
lap of only eight species. More collections from the whole geographical (and vari-
etal) range of P. tectorius are needed to ascertain whether or not the difference
in fungal diversity is a true phenomenon, or if it is due to insufficient sampling.
Conclusions
The Pandanaceae certainly supports a diverse fungal biota, with approximately

450 species known on this family. However, less than 40% of these fungi are
‘unique’ to a single member of the Pandanaceae, and with time it is likely that
many of these will be found on other members of the Pandanaceae, or on other

host families. The 175 ‘unique’ fungi provide a ratio of only 0.2 species of
fungi per host species, a long way short of the 5000 species which would be
required by the 5.7:1 ratio suggested by Hawksworth (1991) in a calculation
of possible world fungal diversity. However, every collection taken during the
investigation by Whitton (1999) revealed at least some fungi that previously
had not been known from the Pandanaceae. This suggests that the species accu-
mulation curve has not flattened out and, therefore, indicates that current
knowledge of microfungi on the Pandanaceae is incomplete. While the likeli-
hood of any one type of substratum being a true indicator of overall biodi-
versity is slim, and an integrated approach using various different substrata
and habitats is perhaps preferable (Hawksworth et al., 1997), there is still a
challenge to find, delimit, and describe the as yet undiscovered fungal treas-
ures associated with the Pandanaceae.
References
Dulymamode, R., Cannon, P.F. and Peerally, A. (1998a) Fungi from Mauritius:
Anthostomella species on Pandanaceae. Mycological Research 102, 1319–1324.
Dulymamode, R., Cannon, P.F. and Peerally, A. (1998b) Fungi from Mauritius: three
Astrocystis species from Pandanus. Mycological Research 102, 1325–1330.
Dulymamode, R., Cannon, P.F. and Peerally, A. (1998c) Fungi from Mauritius:
Linocarpon species on Pandanus. Mycological Research 102, 1331–1337.
Dulymamode, R., Minter, D.W. and Peerally, A. (1998d) Fungi from Mauritius: Rubikia
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Pandanus leaves from Mauritius. Mycoscience 39, 285–291.
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hyphomycete species on endemic plants. Mycotaxon 73, 313–323.
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1. Stachybotrys. Mycotaxon 41, 179–188.
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McKenzie, E.H.C. (1991b) Dematiaceous hyphomycetes on Freycinetia (Pandanaceae).

2. Zebrospora gen. nov. Mycotaxon 41, 189–193.
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Aspects of Graminicolous Downy 6
Mildew Biology: Perspectives for
Tropical Plant Pathology and
Peronosporomycetes Phylogeny
M.A. SPENCER AND M.W. DICK
Centre for Plant Diversity and Systematics, School of Plant Sciences,

The University of Reading, Whiteknights, Reading RG6 6AS, UK
Introduction
The Peronosporomycetes (Oomycetes) include important tropical plant

pathogens. Several genera have a profound effect on local and regional
economies due to their effect on gramineous crops. The graminicolous downy
mildews (GDMs) cause extensive losses to tropical crops such as maize,
sorghum, pearl millet and sugar cane (Shaw, 1981 and references therein).
Knowledge of their biology and taxonomy is essential for the control of these
pathogens within agricultural systems. This chapter will review aspects of
tropical GDM biology, and consider their probable phylogenetic position and
importance in tropical mycology and plant pathology. Compilation of data for
this review is constrained by deficiencies in both host and pathogen nomen-
clature; thus difficulties occur when evaluating the identity of potential hosts,
the geographic ranges of the pathogens and whether the pathogens affect
hosts throughout their natural and cultivated distributions. The host family
Poaceae has a complicated taxonomic and nomenclatural history, resulting in
a proliferation of binomials, many of which are illegitimate. Host binomials
have been corrected to current usage in this chapter. Many of the pathologi-
cal data have been derived from crop plants. This has resulted in a serious
imbalance of information on disease incidence. The frequent omission of
epidemiological data (as opposed to occurrence) from wild-type infections is
lamentable. Renfro and Bhat (1981) commented on this and the situation has
not improved since then. Such data could have considerable impact on our
understanding of pathogen taxonomy and the potential for future economi-
cally damaging epiphytotics. The obligate parasitism of the majority of the

64 M.A. Spencer and M.W. Dick
GDMs (Shaw, 1981; Dick, 2001b and references therein) makes study ex situ
either problematic or impossible, depending upon the nature of the study.
Taxonomy of the Graminicolous Downy Mildews
The GDMs (Sclerosporales) were traditionally placed within the subclass

Peronosporomycetidae as ‘sister’ to the Peronosporales (Shaw, 1978, 1981). The
present authors believe this placement is incorrect and place them within the
Saprolegniomycetidae as ‘sister’ to the Leptolegniaceae (Dick et al., 1999) based
on a suite of morphological, epidemiological, ultrastructural and biogeo-
graphical characters. This assertion is supported by analysis of rDNA restric-
tion sites (Klassen et al., 1988), 28S rDNA molecular data (Riethmüller et al.,
1999) as well as currently unpublished molecular data of the authors. The
Sclerosporales (Dick et al., 1984) contain two families (Sclerosporaceae and
Verrucalvaceae) comprising 24 species and one variety. Revision may establish
some synonymy, especially within the genus Peronosclerospora. A complete list
of currently accepted or used binomials is provided in Table 6.1.
Aspects of Graminicolous Downy Mildew Biology
Host preference and range
The majority of hosts of GDMs are found within the primarily tropical sub-
families Chloridoideae and Panicoideae of the Poaceae. Differences in core host
range between Peronosporales and Sclerosporales strongly indicate the deep
phylogenetic divergence between the orders (contra Shaw, 1981). A summary
of the known host ranges of the GDMs is shown in Fig. 6.1. The Peronosporales
are mainly pathogens of dicotyledonous herbs of temperate regions (Dick,
2001c). Notable exceptions (Table 6.1), parasitizing the subfamily Panicoideae
of the Poaceae, are: Albugo ipomoeae-panduranae, Bremia graminicola and B.
graminicola var. indica on species of Arthraxon (Naomoff, 1913; Patel, 1948;
Tai, 1979); Plasmopara oplismeni on Oplismenus compositus (Viennot-Bourgin,
1959) and Plasmopara penniseti on Pennisetum glaucum (Kenneth and Kranz,
1973 as Pennisetum typhoides). Additionally, Peronospora destructor and P. fugi-
tai infest various Liliaceae s.l. (Constantinescu, 1991). These host–pathogen
relationships are derived and should not be considered ancestral. It is notable
that there are no known host transferrals by the GDMs from the Poaceae to
the dicotyledons; indeed, they are highly restricted in their host range with
only a few reports of Sclerophthora macrospora parasitizing Cyperaceae (Erwin
and Ribiero, 1996, including references on Cyperus spp.).
Graminicolous Downy Mildew Biology
Table 6.1. Species of the Sclerosporales (types in bold print) and graminicolous Peronosporomycetidae (Peronospora and gramini-
colous Pythium species excluded). Authors, publication dates and type hosts are provided; synonyms not included. * Indicates non-
type host.
SCLEROSPORACEAE M.W. Dick 1984; in Dick et al., 1984

Sclerospora J. Schröt. 1879
Sclerospora butleri W. Weston 1933 Eragrostis aspera (Jacq.) T. Nees
Sclerospora graminicola (Sacc.) J. Schröt. 1886 Setaria viridis (L.) P. Beauv.
Sclerospora iseilematis Thirum. & Naras. 1949 Iseilema laxum Hackel ex Duthie
Sclerospora northii W. Weston 1929 Saccharum maximum Trin.
Sclerospora secalina Naumov 1949 Secale cereale L.
Peronosclerospora (S. Ito) Hara 1927; in Shirai, 1927
Peronosclerospora dichanthiicola (Thirum. & Naras.) C.G. Shaw 1978 Dichanthium annulatum (Forrsk.) Stapf
Peronosclerospora globosa Kubicek & R.G. Kenneth 1984 Eriochloa contracta Hitchc.
Peronosclerospora heteropogonis Siradhana et al. 1980 Hetropogon contortus (L.) Roemer & Schultes
Peronosclerospora maydis (Racib.) C.G. Shaw 1978 Zea mays L.
Peronosclerospora miscanthi (T. Miyake) C.G. Shaw 1978 Miscanthus japonicus Anderss.
Peronosclerospora noblei (W. Weston) C.G. Shaw 1980 Andropogon australis Spreng.
Peronosclerospora philippinensis (W. Weston) C.G. Shaw 1978 Zea mays L.
Peronosclerospora sacchari (T. Miyake) Hara; in Shirai, 1927 Saccharum. sp.
Peronosclerospora sorghi (W. Weston & Uppal) C.G. Shaw 1978 Sorghum arundinaceum (Desv.) Stapf
Peronosclerospora spontanea (W. Weston) C.G. Shaw 1978 Zea mays L.
Peronosclerospora westonii (M.C. Sriniv. et al.) C.G. Shaw 1978. Iseilema laxum Hackel ex Duthie
Peronosclerospora zeae Yao 1991 Zea mays L.
VERRUCALVACEAL M.W. Dick 1984; in Dick et al., 1984
Pachymetra B.J. Croft & M.W. Dick 1989; in Dick et al., 1989
Pachymetra chaunorhiza B.J. Croft & M.W. Dick 1989; in Dick et al., 1989 Saccharum officinarum L.
65
Continued
66
Table 6.1. Continued
Sclerophthora Thirum., Shaw, C.G. & Naras. 1953
Sclerophthora cryophila W. Jones 1955 Dactylis glomerata L.
Sclerophthora farlowii (Griffiths) R.G. Kenneth 1979 Chloris virgata Sw.
Sclerophthora lolii R.G. Kenneth 1963 Lolium rigidum Gaudin
Sclerophthora macrospora (Sacc.) Thirum. et al., 1953 Alopecurus sp.
Sclerophthora rayssiae R.G. Kenneth et al., var. rayssiae 1964 Hordeum vulgare L.
Sclerophthora rayssiae R.G. Kenneth et al. var. zeae Payak & Renfro 1967 Zea mays L.
Verrucalvus P. Wong & M.W. Dick 1984; in Dick et al., 1984
Verrucalvus flavofaciens P. Wong & M.W. Dick 1984; in Dick et al., 1984 Pennisetum clandestinum Chiov.
PERONOSPOROMYCETIDAE Dick 1984; in Dick et al., 1984
Albugo ipomoeae-panduranae (L.) G. Meyer *Arthraxon hispidus (Thunb.) Makino
Bremia graminicola Naumov var. graminicola 1913 Arthraxon ciliaris P. Beauv.
Bremia graminicola Naumov var. indica M.K. Patel 1948 Arthraxon lancifolius (Trin.) Hochst.
Peronospora destructor (Berk.) Casp. *Liliaceae s.l.
Peronospora fugitai S. Ito & Tokun. *Liliaceae s.l.
Phytophthora cyperi (Ideta) S. Ito 1935; in Ito and Tokunaga, 1935 Cyperus malaccensis Lam.
Phytophthora cyper-bulbosi Seethal. & K. Ramakr. 1953 Cyperus bulbosus Vahl
Phytophthora fragrariae Hickman var. oryzobladis J.S. Wang & J.Y. Lu 1978 Oryza sativa L.
M.A. Spencer and M.W. Dick

Phytophthora japonica Waterhouse 1974 Oryza sativa L.
Phytophthora leersiae H.H. Ho & H.S. Chang 1992 Leersia hexandra Sw.
Phytophthora lepironiae Sawada 1919 Lepironia mucronata Rich.
Plasmopara oplismeni Vienn.-Bourg. 1959 Oplismenus compositus (L.) P. Beauv.
Plasmopara penniseti R.G. Kenneth & J. Kranz 1973 Pennisetum glaucum (L.) R. Br.
Pythiogeton zeae H.J. Jee et al. 2000 Zea mays L.
Bromopsis Agropyron Dactyloctenium Andropogon Axonopus
Bromus Elymus Eleusine Apluda Beckmannia
Hordeum Eragrostis Chionachne Brachiaria
Bromeae Secale Muhlenbergia Dichanthium Digitaria
Triticum Sporobolus Heteropogon Echinochloa
Iseilema Eriochloa
Glyceria
Triticeae Eragrostideae Miscanthus Panicum
Melica
Rottboellia Paspalum
Dactylis Saccharum Pennisetum
Meliceae
Festuca Chloris Sorghastrum Sacciolepis
Lolium Cynodon Sorghum Setaria
Agrostis
Poa Schedonnardus Tripsacum Stenotaphrum
Alopecurus
Zea
Avena
Poeae Cynodonteae Paniceae
Holcus
Andropogoneae
Polypogon

Phalaris Chloridoideae
Phleum
Rostraria
Elytrophorus Panicoideae
Aveneae Phragmites
Pooideae
Arundinoideae
Oryza
Zizania
Bambusoideae Ancestral Poaceae

Fig. 6.1. Host genera of graminicolous downy mildews (records of artificial inoculation excluded). All subfamilies and tribes of
the Poaceae detailed are parasitized by Sclerophthora; ✚ tribe parasitized by taxa assigned to Sclerospora; ✖ tribe or genus para-
sitized by Peronosclerospora; ............... subfamilies with C4 photosynthetic pathways.
67
Morphology and ultrastructure
The obligate biotrophic parasitism of Peronosporales and Sclerosporales

differs widely not only in host selection but also in the morphology and
morphogenesis of the pathogens. The asexual conidiosporangiophores of
Sclerosporales and Peronosporomycetidae are alike in gross morphology, reflect-
ing similarities in propagule dispersal. However, there are considerable
differences in detailed morphology and morphogenesis. Peronospora coni-
diosporangiophores are dichotomous, isodiametric and persistent, unlike the
pseudo-dichotomous (or simple), inflated and evanescent conidiosporangio-
phore of the Sclerosporales. These differences are indicative of convergent
evolution rather than phylogenetic radiation (Dick et al., 1984; Dick,
2001c). Sexual reproductive organs (antheridia and oogonia) of the
Peronosporomycetes are highly important for evaluation of phylogenetic rela-
tionships (Dick, 1995, 2001a,b). Details of oosporogenesis have been impor-
tant in establishing differences between the two main subclasses of the
Peronosporomycetes (Dick et al., 1984, 1989). Oospores of the Peronosporo-
mycetidae develop centripetally whereas those of the Saprolegniomycetidae
develop centrifugally (Dick, 2001a). Safeeulla and Thirumalachar (1955)
demonstrated centrifugal oospore nuclear development in Peronosclerospora
sorghi (described as Sclerospora andropogonis-sorghi (Kulkarni) Kulkarni).
Centrifugal oospore nuclear development, characteristic of the Saprolegnio-
mycetidae, has also been observed in Sclerospora graminicola (McDonough,
1937) and Sclerophthora macrospora (McDonough, 1947). This contrasts with
oosporogenesis in Albugo (Safeeulla and Thirumalachar, 1955) and
Phytophthora infestans (Graham, 1954), in which oospores develop cen-
tripetally. The matrix of the oospore ooplast is highly distinctive within the
Sclerosporales and is comparable to the granular fluid ooplast of the
Saprolegniales, unlike the solid translucent ooplast of the Peronosporomycetidae
(Dick et al., 1984). The oogonia of the Pythiales and Peronosporales are thin-
walled, unlike those of the Sclerosporales, which are thick-walled and similar
to those of the Saprolegniales (Dick et al., 1989).
Epidemiology, physiology and ecology
There is considerable functional convergence of asexual propagules of the

Sclerosporales, Pythiales and Peronosporales, reflecting environmental selection
pressures. The conidia and conidiosporangiophores of Peronospora and
Plasmopara are not truly homologous with those of Peronosclerospora. The coni-
diosporangiophores of Peronosclerospora are evanescent while those of
Peronosporales are highly persistent. Similarities in epidemiological function
are convergences; dissimilarities in function, supporting ultrastructural
and morphological data, indicate a deep phylogenetic divide between the
Graminicolous Downy Mildew Biology 69
Sclerosporales and the Peronosporomycetidae (Dick et al., 1989; Riethmüller et

al., 1999). The dispersal and germination of conidia of Peronospora and
Peronosclerospora display considerable differences in epidemiology; the conidia
of Peronospora may remain viable for several weeks (Pegg and Mence, 1970)
and are light-sensitive (Fried and Stutville, 1977). Conversely those of
Peronosclerospora are viable for and germinate in only a few hours (Dogma,
1975), are non-reactive to light, nocturnally dispersed and germination is
dependent on high relative humidity (Dogma, 1975; Bock et al., 1998). While
both genera have a wide range of temperature tolerance, conidial formation
is usually inhibited by temperatures over 27–30°C; Peronospora species may
successfully sporulate and germinate at lower temperatures (Pegg and Mence,
1970), 0°C compared to a minimum of 10°C for Peronosclerospora (Bock et al.,
1998).
Losses of anabolic sterol metabolic pathways have been reported within
the Peronosporomycetes (Warner et al., 1983) and the GDMs may require exoge-
nous sources of sterols. Hosts growing in areas of high solar incidence fre-
quently exhibit raised carbohydrate production via photosynthesis. This is
conducive to the production of secondary metabolites (flavonoids and
sulphonated flavonoids from phenylalanine, sterols, essential oils and
alkaloids from carotenoid precursors). The loss of ability (and subsequent
unlikelihood of restoration) to utilize certain inorganic forms of nitrogen
and sulphur by certain groups of Peronosporomycetes is still accepted
(Cantino, 1955). The Peronosporomycetidae are able to use SO42–, and their ability
to metabolize different inorganic NO3– sources is variable. The
Saprolegniomycetidae, however, are unable to utilize SO42–- or NO3–-containing
substrates. While the Sclerosporales have not been experimentally demonstrated
to be unable to synthesize SO42–, their association with grasses that retain sub-
stantial photosynthetic ‘sinks’ of organic sulphonated flavonoids is notable.
The Verrucalvaceae are intermediate between the Peronosporomycetidae and
the Saprolegniomycetidae in terms of response to the fungicidal isoxazoles
(Hymexazol) and phenylamides (Metalaxyl) (Kato et al., 1990). The
Verrucalvaceae are relatively resistant to Metalaxyl, as are the Saprolegniaceae
and Leptomitaceae, unlike the susceptible Phytophthora (although resistance is
known to occur in some populations of P. infestans; see Erwin and Ribiero,
1996). It is noteworthy that Kato et al. (1990) observed similarities of response
in axenic culture between Verrucalvus and Pachymetra and that of Leptolegnia
caudata. This close relationship is supported by unpublished 18S rDNA data
of the authors and Riethmüller et al. (1999). However, Kang and Lee (1987)
noted a 133% yield increase and reduced disease incidence in a rice crop
infested with Sclerophthora macrospora following application of Metalaxyl.
Similarly, others (Eastwood and Malein, 1998; Panicker and Gangadharan,
1999) found that Peronosclerospora was also susceptible to Metalaxyl. These
apparently contradictory results are unexplained but may be due to differing
methodologies or extrinsic factors; it is possible that the noted responses may
have been affected by synergistic effects on mycopathogens within soil or plant

tissue. With respect to Hymexazol, Sapromyces and Phytophthora behaved
similarly, whereas the Verrucalvaceae had responses more similar to those of
Pythium (Kato et al., 1990). Peronosporales are known to be susceptible to
phosphonate fungicides (including fosetyl-Al) (Cohen and Coffey, 1986),
unlike Peronosclerospora sacchari, where resistance to fosetyl-Al is known
(Eastwood and Malein, 1998). In Peronosclerospora sorghi, reduction in sporu-
lation but not germination has been observed (Panicker and Gangadharan,
1999) following treatment with fosetyl-Al.
While most downy mildews are unculturable, a few species have been
grown in biphasic systems using host tissue culture (Dick, 2001a). Others,
such as Verrucalvus and Pachymetra, are culturable with difficulty (Dick et al.,
1984, 1989). Claims that Sclerospora graminicola (Tiwari and Arya, 1969) and
Sclerophthora macrospora (Tokura, 1975) have been maintained axenically
require careful reinvestigation as the original cultures do not appear to be
extant. The Pythiogetonaceae (Peronosporomycetidae) are similar to sclerospo-
raleans in being either highly fastidious (Pythiogeton) or unculturable
(Medusoides) (Voglmayr et al., 1999). Similarity of culturability of organisms
should not be considered a reflection of phylogenetic relatedness, but poten-
tially a reflection of independent substrate specialization; therefore, the obli-
gate nature of the Peronosporales should not necessarily be considered as a
synapomorphic trait with the Sclerosporales.
Evolution of the Poaceae
Clayton and Renvoize (1986) suggested that the origins of the grasses
may have been in Gondwana on what is now northern South America and
western Africa with the subfamily Bambusoideae evolving first; this is
supported by molecular data that place the herbaceous neotropical tribes
Streptochaeteae and Anomochloeae as basal to the Bambusiodeae (Clark et al.,
1995) and Poaceae as a whole. The first radiation of the Poaceae possibly
occurred during the late Cretaceous and early Tertiary (Maastrichtian – mid-
Eocene, 70–40 m.y. BP) (Jacobs et al., 1999) although clear fossil evidence is
not present until the Palaeocene (65–57 m.y. BP) (Dick, 1988, 2001c). This
early radiation of primarily C3 photosynthetic pathway-utilizing bambusoid,
oryzoid and primitive pooid clades (Kellogg, 1998) was predominantly in
mesic forest/savannah ecotones with relatively low levels of insolation
(Clayton and Renvoize, 1986; Jacobs et al., 1999).
South American Oligocene (35–23 m.y. BP) fossils of grinding hypsodont
mammalian teeth imply the formation of early grasslands and the spread of
Poaceae out of the forest/savannah ecotone (Dick, 1988, 2001c; Jacobs et al.,
1999). Clear vertebrate and palaeobotanical evidence of widespread grass-
dominated ecosystems in northern continents did not occur until the mid- to
late Miocene (9–5 m.y. BP) (Clayton and Renvoize, 1986; Jacobs et al., 1999).
The late Miocene spread of C4 grasses possibly involved a decrease in atmos-
pheric CO2 and heralded the establishment of modern seasonality and rain-
fall patterns. The Himalayan uplift (20 m.y. BP), which drained the Tethys
Ocean remnants and resulted in colder montane and Siberian steppe climates
to the north, would have forced the early C4 tropical grasses southwards into
Africa, India, and South-East Asia. C4 grasses are abundant from approxi-
mately 15 m.y. BP, undergoing a dramatic expansion in the lower latitudes
of North America, South America, East Africa, and Pakistan between 9 and
4 m.y. BP. The modern temperate pasture grasses may have radiated from
tropical and warm temperate regions as the climate cooled and habitats were
opened up by the grazing mammals (Dick, 1988, 2001c). The later radiation
of mainly C4 photosynthetic pathway-utilizing arundinoids, chlorids,
centothecoids and panicoids (Kellogg, 1998) displaced many of the genera of
the earlier radiation to the forest understorey/margins and cooler temperate
regions. Throughout this period Australasia was isolated, drifting northwards
with a previously temperate-adapted biota. From the Oligocene, land bridges
permitted tropical panicoid grasses a southward migration into Australia,
which continued through the Quaternary.
Coevolution of the Sclerosporales and Poaceae
There are no known fossils of sclerosporaleans. Although both GDMs and

downy mildews of dicotyledonous plants are of relatively recent origin, the
antecedents of the latter are probably more ancient (Dick, 1988, 2001a,b).
Comparison of the present host ranges of the downy mildews and the rela-
tive antiquity of their hosts, together with what is known of host ranges for
Pythium and Phytophthora, presents some problems. The affinity of
Phytophthora for woody dicotyledonous hosts and the distinct, later origin of
the pasture grasses makes it possible to suggest that, contrary to accepted phy-
logenies (Shaw, 1978, 1981; Barr, 1983), some sections of Pythium (with
many taxa associated with grasses) may have evolved later than Phytophthora.
This receives some support from mitochondrial genome analyses (Belkhiri and
Dick, 1988), which indicated that Phytophthora lacks the inverted repeat char-
acteristic of Pythium (Klassen et al., 1987). While it is realistic to propose a
late Cretaceous origin for Phytophthora, the origin of some sections of Pythium
s.l. may have been with the pasture grasses in the Tertiary. It is probable that
GDMs evolved during the mid- to late Miocene (9–5 m.y. BP) at the same time
as the second main radiation of the Poaceae. This may be inferred by the
predominance of hosts from within the subfamily Panicoideae (Fig. 6.1).
Notably the paucity of hosts from the pooids and the oryzoids indicate that
these particular host–pathogen relationships developed relatively late
in Poaceae evolution (probably secondarily derived following anthropogenic
disturbance). The probable inability of the GDMs to synthesize inorganic sul-

phur suggests that they require organic sulphur substrates; these are partic-
ularly abundant as sulphonated flavonoids in the C4 pathway-utilizing grasses
(but not the non-sulphonated flavonoids of the dicotyledons). Reliance on
other aspects of host metabolism, such as sterol pathways and secondary
metabolites other than sulphonated flavonoids, may play an essential role in
GDM physiology and coevolution with the grasses. As yet there are no
data to indicate such metabolic relationships. Investigation of these bio-
chemical pathways may prove informative for both systematists and crop
protectionists.
The GDMs are primarily associated with the highly evolved tropical
grazing grasses of the subfamilies Chloridoideae and Panicoideae (Fig. 6.2). The
hypothesis that the putative ancestral hosts were within the bambusoids, ory-
zoids, and south hemisphere grasses can be dismissed. The subfamily
Panicoideae has tropical evolutionary origins and is currently centred in South-
East Asia (Clayton and Renvoize, 1986). The Panicoideae contains species with
leaf anatomy of both kranz (C4 photosynthesis) and non-kranz kinds. The
kranz anatomy can be of both MS and PS kinds (for details see Clayton and
Renvoize, 1986). Kranz MS species have evolved metabolic pathways that
‘sink’ high levels of carbohydrates arising from their habitats, including
marshes, ruderal and arid habitats. These are all habitats with high insola-
tion. The occurrence of Sclerospora and Sclerophthora in the non-kranz genus
Oryza is apparently anomalous on presently available information, but future
studies may reveal metabolic similarities between the wetland Oryza and the
Panicoideae.
Present-day geographical distributions disguise evolutionary origins and
may merely confirm the opportunism of these parasites. The recent transcon-
tinental host–pathogen interaction of Peronosclerospora with Zea is one
example of this. The relationship between Peronosclerospora and the tribe
Andropogoneae in Melanesia (Figs 6.1, 6.2) is probably of a primary nature
(Weltzein, 1981). Saccharum (including Erianthus) consists of 35–40 Old
World warm temperate and tropical species; the sugar cane species have their
centre of diversity in Melanesia (Clayton and Renvoize, 1986). Wild sugar
cane species and other Andropogoneae are often infected with Peronosclerospora
in habitats with minimal anthropogenic disturbance (Renfro and Bhat, 1981).
The possibility that a high diversity of pathogen taxa infecting Saccharum and
other Andropogoneae in wild-type habitats is secondary must be considered to
be minimal. The tribe Paniceae is probably primarily C3 (Clayton and Renvoize,
1986) and the high prevalence of these taxa as hosts for Peronosclerospora
cannot be discounted. It may be that Peronosclerospora coevolved with ances-
tral C3 Paniceae during the mid- to late Miocene (9–5 m.y. BP) in the region now
occupied by the Himalayas. Subsequent cooling of this region (Dick, 1988,
2001c) and the evolution of C4 Panicoideae may have forced Peronosclerospora
southward into Melanesia. It is notable that there are two apparent centres of
I
L
G
M
L L%
L
2 G
G
G

L
G
GL
!I
Fig. 6.2. Known distribution of Peronosclerospora species: ✣ P. dichanthiicola; ■ P. globosa; ▼ P. heteropogonis; ● P. maydis;
✦ P. noblei, ✙ P. philippinensis; ▲ P. sacchari; — P. sorghi; ✪ P. spontanea; ✖ P. westonii; ✳ P. zeae.
73
diversity for Peronosclerospora, one in subcontinental India (P. dichanthiicola,

P. heteropogonis and P. westonii) and the other in eastern Melanesia and
Australasia (P. globosa, P. maydis, P. miscanthi, P. noblei, P. sacchari and P. spon-
tanea). The remaining species (P. philippinensis and P. sorghii) are of widespread
distribution and their origins are conjectural. The suggestion that
Peronosclerospora may have originated at the site of the Himalayan orogeny
allows for a sympathetic explanation of the origin of Sclerospora. Whilst the
tropical conidial Peronosclerospora migrated southwards the temperate zoospo-
rangial Sclerospora may have evolved to the north and west of the Himalayan
orogeny and the Indian subcontinent (Fig. 6.3). Although Sclerospora gramini-
cola is well characterized, other members of the genus are poorly known.
S. iseilematis, S. northii and S. secalina are oogonial and have no known asexual
phase; therefore evaluation of their relationship to the other genera is prob-
lematic other than defining them as GDMs. Sclerospora butleri has previously
been assigned to Basidiophora (B. butleri (Weston) Thirumalachar and
Whitehead, 1952). This placement has been questioned by some later authors
(Kenneth and Kranz, 1973; Dick et al., 1984) and later rejected from
Basidiophora (Barreto and Dick, 1991).
The current known distribution of Pachymetra and Verrucalvus in
Australia (Dick et al., 1984, 1989) presents problems for a sclerosporalean
origin (but not a later diversification) on the Indian/Asian convergence zone.
It is possible that they may have migrated south at the same time as
Peronosclerospora, but their poor dispersal capacity (airborne conidia are
unknown in either taxon) allows only a most remote possibility that they were
able to cross Wallasia during the Miocene. Pachymetra is known from native
Blady Grass, Imperata cylindrica (R.C. Magarey, 1989, Queensland, Australia,
personal communication), a widespread Old World tropical panicoid grass, as
well as sugar cane. It is possible that either Pachymetra or Verrucalvus may be
present in wild-type habitats in South-East Asia; collections from this region
as well as further sampling in eastern Australia are desirable. If either genus
is to be found in South-East Asia the possibility arises that both are of recent
anthropogenic origin in Australian agricultural systems. This is an essential
element for future research to provide an understanding of the pathogenicity
of these potentially globally damaging organisms.
The origins of Sclerophthora are possibly distinct (but not independent)
from those of Sclerospora and Peronosclerospora. Unlike the latter genera,
Sclerophthora has no predisposition to grasses of the Panicoideae or Chloridoideae
(Fig. 6.1) and has been noted from over 140 taxa throughout the Poaceae (see
Kenneth, 1981, including references). This may reflect cryptic speciation
within the current species concept and further investigation may enable pat-
terns of host–pathogen relations to be evaluated (Shaw, 1981). Sclerophthora
macrospora is found in most warm temperate regions of the world. The others
(S. cryophila, S. lolii, S. rayssiae and S. rayssiae var. zeae) are centred on the
Middle East and drier regions of India (Fig. 6.3). S. cryophila, on Dactylis, an
∗
G
G % 2
I%
M
I !∗
I
!
I !
Fig. 6.3. Known distribution of graminicolous downy mildews of the genera Sclerospora and Sclerophthora: ■ Sclerospora butleri;
●●●●●
Sclerospora graminicola; ✣ Sclerospora iseilematis; ✦ Sclerospora northii; ✳ Sclerospora secalina; ▼ Sclerophthora cryophila;
● Sclerophthora farlowii; ✙ Sclerophthora lolii; — Sclerophthora macrospora; ✪ Sclerophthora rayssiae.
75
introduced Old World genus, has outliers of anthropogenic origin in North

America. Sclerophthora farlowii (Griffiths) R.G. Kenneth in Arizona may be a
remnant of the circum-tethyan flora, but it is more likely that it is indigenous
to the Old World, its presence in America being of anthropogenic origin. It is
also possible that S. farlowii is not a Sclerophthora; Shaw (1981) noted that
R.G. Kenneth ‘by careful re-examination of the original collections of
S. farlowii has found the anamorph thereof ’ (Kenneth, 1979, 1981). This is
considered unlikely by the authors as the asexual organs of Sclerosporales are
evanescent and impermanent in herbarium exsiccatae. Conidiosporangio-
phores are therefore unlikely to have survived adequately to allow description
for approximately 70 years since Griffiths’ original description.
Graminicolous Downy Mildews and Phytophthora

Several taxa ascribed to Phytophthora s.l. are probably misplaced and a critical
reappraisal of all aspects of their biology may reveal alternative relationships.
P. verrucosa was described from glasshouse crops of Meconopsis and
Lycopersicon (Foister, 1940). While this host range is clearly anomalous for
GDMs, similarities in oogonial morphology suggest that P. verrucosa may be
more appropriately associated with the GDMs (Dick et al., 1984, 1989).
Phytophthora cyperi, P. cyper-bulbosii and P. lepironiae, pathogens of tropical
Cyperaceae, are likewise probably not Phytophthora s.s.; each has sexual
reproductive organs similar to those of Sclerophthora (Dick et al., 1984, 1989).
Like GDMs, they are apparently unculturable axenically (Erwin and Ribiero,
1996). Their host range on tropical monocotyledons is also atypical of many
Phytophthora species. Great emphasis is often placed upon the presence of
amphigynous antheridia as a defining character of Phytophthora. The presence
of amphigyny in several of the preceding species has been considered suffi-
cient for inclusion within Phytophthora. However, there are two other genera
where amphigyny has been observed, Trachysphaera and Peronophythora. The
possibility that differing morphogenetic events in amphigynous antheridial
maturation occur within Phytophthora species suggests that the condition may
not be homologous (Stevenson and Erwin, 1972; Hemmes and Ribeiro, 1977).
Therefore the inclusion of taxa within Phytophthora (as currently circum-
scribed) solely because amphigynous antheridia are present is inappropriate.
It is possible that, following critical evaluation of biochemical, epidemiologi-
cal, ultrastructural and molecular data in conjunction with a wide range of
Peronosporales and other Pythiales, Phytophthora s.l. could be untenable in its
current form (Dick et al., 1999; Dick, 2001a,b).
Conclusion
GDMs are not closely related to the Pythiales or the Peronosporales but to the
water moulds of the Saprolegniomycetidae, especially the Leptolegniaceae.
The warm temperate and tropical distribution and the phytogeography of their
hosts is incompatible with an evolutionary relationship with the cool tem-
perate Peronosporales. The absence of a similarity in core host range between
the Peronosporales and Sclerosporales suggests differing origins. The large suite
of fine morphological and ultrastructural differences indicate convergent
adaptation to similar ecological niches. This is derived from a presumed need
to access substrates rich in secondary metabolites, including sterols or
sulphonated flavonoids. Considerable differences in epidemiology and
fungicidal response occur, which arise from constraints of biochemistry.
Investigation of all aspects of tropical Peronosporomycetes biology will further
resolve relationships between GDMs and the Peronosporomycetidae. It is espe-
cially important that published molecular data are complemented by other
gene loci and wider taxonomic sampling. Resolution of these relationships will
enable directed strategies of control to be developed for these important
tropical plant pathogens.
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Invasive Neotropical Pathogens 7
of Tree Crops
H.C. EVANS
CABI Bioscience, Silwood Park, Ascot, Berkshire SL5 7TA, UK
Introduction
Since the early 1990s increasing attention has been drawn towards the
environmental and socio-economic problems resulting from biological inva-
sions on a global scale (Cronk and Fuller, 1995; Kaiser, 1999; Mack et al.,
2000; Pimentel et al., 2000). In such invasions, alien or exotic, non-
indigenous organisms, either deliberately or accidentally introduced, become
adapted to and subsequently dominate these new habitats, with inevitable and
often profound detrimental impacts on the flora and fauna of both natural
and agricultural ecosystems. Most of the available literature and popular
reviews on the subject pertain to biological invasions involving either plants
(D’Antonio and Vitousek, 1992; Lonsdale, 1999), invertebrates (Howarth,
1985; Porter and Savignano, 1990), vertebrates (Maciolek, 1986; Dobson,
1988) or a combination of these (Mooney and Drake, 1986; Simberloff et al.,
1997). In particular, in the general overviews of invasive species little mention
has been made of the actual and potential importance of alien fungal plant
pathogens, despite recent high-profile examples, such as Phytophthora
cinnamomi Rands in Australia (Weste and Marks, 1987); Phytophthora infes-
tans (Mont.) de Bary in Europe (Fry et al., 1992); Cryphonectria parasitica
(Murr.) Barr (chestnut blight) and Discula destructiva Red. (dogwood anthrac-
nose) in the USA (Anagnostakis, 1987; Daughtrey and Hibben, 1994).
This chapter focuses on neotropical fungal pathogens of two of the major
commodity crops, rubber and cacao, at least one of which has the potential
to change ‘the political history of the world’ (Disraeli’s view of potato blight;
in Ramsbottom, 1953), tracing their past and present impacts on agriculture

84 H.C. Evans
in the region and highlighting the continuing threat they pose to other Latin
American countries, as well as to other continents. In essence, it is revisiting
the subject of catastrophic or threatening plant diseases (the term invasive
being of relatively recent coinage), so admirably reviewed by Klinkowski
(1970) and Thurston (1973). Following the example set by the latter author,
the chapter covers plant diseases of ‘potential international importance’ but
those which are ‘at present limited to a few countries or a continent’ and
which ‘are important in developing countries’ (Thurston, 1973).
South American Leaf Blight of Rubber
Historical
All the natural rubber of commerce is now derived from the neotropical
euphorbiaceous tree, Hevea brasiliensis (A. Juss.) Muell. Arg., which is culti-
vated predominantly in the Palaeotropics, far removed from its native range
(Purseglove, 1968). H. brasiliensis is indigenous to the tropical rainforests of
the Amazon basin, occurring only south of the river and extending to Peru
and Bolivia (Schultes, 1956). The intriguing history of the early rubber
germplasm collections, their establishment in the Far East and the eventual
creation of the Asian plantation industry in the early 1900s, was admirably
synthesized by Purseglove (1968), and was retold in a more dramatic and
expansive form by Dean (1987) and Davis (1997). The descendants of these
earlier Brazilian collections were returned in the late 1800s to the Neotropics
as part of a colonial programme to develop rubber plantations, particularly
in north-east South America, Central America and the Caribbean. It was from
the Guianas and Trinidad that the first indications that a serious disease was
affecting the crop began to surface; and, in the period 1910–1917, a number
of colonial pathologists investigated the problem, which resulted in the
identification of the causal agent and an insight into its biology (Petch, 1914;
Stahel, 1917, 1927). Indeed, Holliday (1970a) described the publication by
Stahel (1917) as being still the original source of much of our current
knowledge of the disease. Within a relatively short time, it was realized that
the disease posed a serious threat to the booming Asian rubber industry
(Belgrave, 1922).
The pathogen had been collected earlier by E. Ule in western Amazonia
(Brazil and Peru) on wild Hevea spp., and the teleomorph was subsequently
described by P. Hennings, as Dothidella ulei P.Henn., in 1904 (see Holliday,
1970a). However, the hyphomycete anamorph was not described until 1912
by J. Kuyper in Surinam, and the two were not linked until several years later
(Petch, 1914; Stahel, 1917). Stahel (1917) erected the new genus
Melanopsammopsis to accommodate the teleomorph, but von Arx subsequently
transferred this to the genus Microcyclus Sacc. (von Arx and Müller, 1975).
Invasive Neotropical Pathogens of Tree Crops 85
The nascent rubber industries in both Guyana and Surinam were literally
nipped in the bud by the new disease as the early yield data starkly revealed,
with rubber production falling in Guyana alone from over 20,000 lb in 1920
to less than 2000 lb in the following year (Holliday, 1970a). As Maclaren
(1924) commented, South American leaf blight, which first appeared in the
Colony in 1909, ‘reduced the vitality of the trees to a low ebb’.
Causal agent
Microcyclus ulei (P. Henn.) von Arx (Dothideales, Mycosphaerellaceae)

Anamorph: Fusicladium macrosporum Kuyper
A pycnidial form, Aposphaeria ulei P. Henn., has also been described but it
appears to play no role in the infection cycle (Chee, 1978), and it is assumed,
but not confirmed, that the pycnospores function as spermatia and that the
fungus is heterothallic (Holliday, 1970a; Cannon et al., 1995).
The superficial, black, globose ascostromata form mainly on the upper
leaf surface, often crowded and associated with shot-hole symptoms. The ellip-
soidal, hyaline, 1-septate ascospores, produced in clavate, bitunicate asci, are
typical of the genus Mycosphaerella Johanson. As discussed by von Arx (1983)
and Cannon et al. (1995), only the excessive development of stromatic tissue
in Microcyclus, resulting in erumpent ascostromata, separates it from
Mycosphaerella. Indeed, some of the Mycosphaerella spp. associated with pine
trees have similar prominent ascostromata (Evans, 1984).
The Fusicladium anamorph appears as olive-grey to greenish black,
powdery, velvet-like masses on the lower leaf surface, which consist of simple
conidiophores producing predominantly 1-septate, cylindrical, irregular and
twisted conidia singly from the tip. Von Arx (1983) remarked that Fusicladium
is ‘much alike’ Passalora, the genus assigned to it by G. Massee in 1913
(Holliday, 1970a), and, furthermore, he considered that Fusicladium was more
referable to anamorphs of Venturiaceae than Mycosphaerellaceae. Clearly, a re-
examination of its taxonomic position would be justified.
Both the conidia and ascospores are infective and penetrate directly
through the leaf cuticle following formation of appressoria (Stahel, 1917),
and foliage up to 10 days old is especially susceptible. Conidia may be pro-
duced on the leaf within a week of infection, but the fungus can also infect
and sporulate on petioles, green stems, inflorescences and fruits. The palisade
layer of infected leaves may be 3–4 times thicker than that of healthy leaves,
whilst petioles and green stems can be up to twice the normal size due to the
overproduction of cortical cells (hyperplasia), which often results in growth
distortions (hypertrophy) (Stahel, 1917; Fig. 7.1). Repeated infection results
in severe defoliation, canopy dieback and death, even of mature trees
(Holliday, 1970a). Ascospores survive in the leaf litter and provide the main
inoculum source to renew disease cycles at the beginning of the rainy season
86 H.C. Evans
Fig. 7.1. Transverse section through a healthy petiole (a) of Hevea brasiliensis
and one infected with Microcyclus ulei (b), showing the tissue disorganization
and gross increase in petiole diameter following infection. Mycelium of M. ulei
colonizes the upper and lower cortex (arrows), grows intercellularly (c) and
appears to disrupt the cambium, leading to overproduction of cells (hyperplasia),
of larger size than normal (hypertrophy), compared to a healthy petiole (d). After
Stahel (1917).
(Chee, 1976), although their precise role and overall importance are still
uncertain.
A number of races, as well as two morphological types, of the pathogen
have been identified in Brazil (Langford and Townsend, 1953; Chee et al.,
1986; Hashim and Almeida, 1987).
Distribution and impact: actual and potential
The pathogen is specific to Hevea and attacks at least four of the 12 species in
the genus: H. brasiliensis, H. benthamiana Muell. Arg., H. guianensis Auld. and
H. spruceana (Benth.) Muell. Arg. Between them, these species cover the whole
of the Amazon and Orinoco river basins, extending from Guyana in the north
to Bolivia in the south, and from the Atlantic coast to the eastern Andes
(Fig. 7.2). The spread of the disease from 1908 onwards into emerging rubber
plantations in Bolivia, Brazil, Colombia, Ecuador, Guyana, Peru, Surinam and
Venezuela has been attributed to infection from wild Hevea trees in the sur-
rounding forests (Hilton, 1955; Holliday, 1970a). Prior to this, most of the
world’s rubber was collected from scattered forest trees in the Brazilian
Amazon, which made fortunes for a few and kept many in virtual slavery
(Dean, 1987). It has been speculated that these sparse forest populations were
the resistant survivors of much more extensive original populations. However,
this sporadic distribution (estimated at six trees per acre; Thurston, 1973) is
typical of tropical trees in primary forest ecosystems, be it rubber, cacao or
Brazil nut. As Hilton (1955) correctly deduced, ‘the immunity of many of the
jungle trees [Hevea] is due to the fact that being isolated from their neighbours
they have never been exposed to high concentrations of infective material’.
The pathogen spread rapidly from these forest foci, with a dramatic impact on
the monocultures of H. brasiliensis. In Guyana, for example, the disease was
first reported in 1909 (Rands, 1924), and within a decade led to the aban-
donment of plantation rubber in that country. Rands (1924) also noted that
the disease impact in Surinam was even greater, probably due to the planta-
tions being closer to the forest zone, and thus nearer to natural infection foci
on H. guianensis, as well as being away from the drying coastal winds. As
Stahel (1917) reported, by 1916 trees of all ages were dying from the disease,
and the destruction of infected wild Hevea populations, as well as chemical
spraying, proved to be both ineffective and uneconomic.
Leaf blight was first recorded outside its natural range in 1916 when J.B.
Rorer identified M. ulei in Trinidad (in Lamont et al., 1917). Much later, the
pathogen reached and invaded Central America, appearing first in Costa Rica
(Stevenson, 1935), and shortly after in Panama (Hilton, 1955), and some 10
years later in Mexico (Martin, 1948). Subsequently, M. ulei was reported in
Honduras (Waite and Dunlap, 1952) and Guatemala in 1955 (Holliday,
1970a). The pathogen has purportedly been present in the Brazilian State of
88 H.C. Evans
Fig. 7.2. Distribution of Microcyclus ulei on wild and plantation rubber (as
shown by dotted line). The important collecting sites of wild, resistant Hevea
germplasm (Leticia, Iquitos, Acre, Madre de Dios), and the research bases where
rubber clonal gardens were established (Belém, Tingo Maria, Turrialba, Villa
Arteaga), are included in the map. In addition, the failed, abandoned Ford plan-
tations on the Rio Tapajoz (Belterra and Fordlandia) are also marked. The wild
hosts in the Amazon–Orinoco basins are: H. benthamiana (northerly); H.
brasiliensis (southerly); H. guianensis (throughout); H. spruceana (lower and mid-
dle Amazon). After Holliday (1970a).
Bahia since the late 1930s and reached its most southernly distribution in
São Paulo State in the 1960s (Holliday, 1970a). Thus, there were successive
invasions or disease fronts in Latin America: in the Guianas (1914–1923);
Brazil (1930–1943); and Central America (1935–1955), with serious socio-
economic impacts, all of which strongly indicated that any attempts at estab-
lishing a plantation industry in the Neotropics, and especially within the
native range of Hevea, would be fraught with difficulties. Henry Ford ignored
these early warnings, probably because, as Holliday (1989) sardonically
commented, he considered that ‘history is bunk’. However, his efforts at plant-
ing rubber on a huge scale in the Brazilian Amazon from 1927 to 1943, firstly
at Fordlandia (> 8000 ha) and then at Belterra (> 12,000 ha), near to the
original collecting sites of the Asian rubber material in the previous century
(Fig. 7.2), proved to be one of the most costly failures of any agricultural
project. Despite the importation of selected, high-yielding stock from Sumatra
and Malaysia, and the use of new grafting techniques which involved
top-budding with disease-resistant H. guianensis and H. spruceanum, the
plantations succumbed to disease (Dean, 1987; Hecht and Cockburn, 1990).
Indeed, a significant part of the US$20 million investment was used in top-
budding more than 2 million trees, occupying 600 workers over a 4-year
period (Davis, 1997).
The pathogen has now reached its invasive limit in the Neotropics; wher-
ever rubber has been planted M. ulei has caught up with its host, either
naturally by airborne inoculum from indigenous forest trees, or, in the case
of exotic plantations, by human agency (Martin, 1948; Altson, 1955; Hilton,
1955). As discussed earlier, the threat posed by M. ulei to plantations in the
Old World was recognized at an early stage when Belgrave (1922) speculated
on the possible arrival and the potential impact of the pathogen in Malaysia.
Due to the short-lived nature of the main infective propagules (conidia), it was
considered highly unlikely that the fungus could reach palaeotropic planta-
tions, either through natural or human means. However, the extremely short
survival period (15 h) originally proposed (Stahel, 1917) has now been shown
to be erroneous and, in low light intensity, the conidia can survive up to 2
weeks (Holliday, 1969). Later, Hilton (1955) analysed climatic data and con-
cluded that conditions in the Far East rubber-growing regions were suitable
for pathogen development and, therefore, that, given the proven susceptibil-
ity and narrow genetic base of the material, epiphytotics would be inevitable.
As Altson (1955) put it: ‘the not improbable sequel might be the ruin of the
present Malayan rubber industry’.
The fact that M. ulei has not reached African and Asian plantations has
been attributed to the few importations of rubber germplasm which have been
made directly from the Neotropics. Fortuitously, the original introductions had
gone through a third country quarantine at Kew Gardens, at a time, of course,
when leaf blight was unknown. Nevertheless, the chances of its accidental
introduction have vastly increased in the last 20–30 years in line with increas-
ing international trade and communications (Rao, 1973; Edathil, 1986).
Control
Prevention
Obviously, the best form of control is prevention, and Malaysia, in particular,
has had long-established legislation in place, designed both to prevent or
reduce the chances of importation of the pathogen through stringent quar-
antine, and to empower its eradication should the disease appear (Altson,
1955; Hilton, 1955). Pamphlets for early recognition of the disease were
issued in 1953 and an action plan was developed to deal with any outbreak
90 H.C. Evans
area, including a guard belt extending to 400 m. This involved defoliation of

the trees with aerial spraying of 2,4,5-T in oil, followed by felling and burn-
ing. Use of military-style flamethrowers was also seriously considered (Altson,
1955). These earlier pamphlets have been replaced by a colour brochure
detailing the biology and aetiology of the fungus, as well as quarantine
measures, advice for travellers and the phytosanitary treatments necessary to
eradicate the pathogen from Hevea germplasm (Anon., 1986). Most of the new
information in this document resulted from the work of Malaysian scientists
seconded to the Neotropics. Earlier, Berg (1970) had summarized the
quarantine measures in force and the legislation enacted by the various coun-
tries belonging to the Inter-African Phytosanitary Commission and the Plant
Protection Committee for the Southeast Asia and the Pacific Region, in order
to keep out South American leaf blight.
Chemical
Work in Trinidad identified several fungicides, including triadimefon,
benomyl, mancozeb and chlorothalonil, which showed protectant as well as
suppressant activity against M. ulei (Chee, 1978). Large-scale application of
some of these compounds, using fogging and helicopter spraying, has been
practised in both the Amazonian and Bahian regions of Brazil, often govern-
ment-subsidized (Chee and Wastie, 1980). However, these authors recom-
mended more basic research to resolve technical problems, especially relating
to application. It is still not clear whether or not the use of fungicides is eco-
nomically sustainable.
Biological
Stahel (1917) described a white fungal overgrowth on the ascostromata of
M. ulei and assigned it to the genus Botrytis. However, an illustration depicts
Hansfordia-like conidiophores, while others show clear mycoparasitism of the
anamorph, with the ‘Botrytis’ hyphae entwining and apparently penetrating
the Fusicladium conidiophores. Hilton (1955) seems to have been the first to
suggest that biotic factors could have an influence on the disease and that
‘indigenous parasites’ may be involved in its control, although he dismissed
any role in this for the Botrytis sp. It is possible that this fungus is a specific
mycoparasite of M. ulei and, therefore, that it merits evaluation as a potential
biofungicide. Mycological surveys in the forest populations of Hevea may yield
other exploitable biocontrol agents.
Resistance
The English botanist Richard Spruce has been credited with first drawing
attention to the economic possibilities of rubber as a result of his work in
Amazonia in the 1850s (Dickenson, 1996; Schultes, 1996), and, on the basis
of his collections, six new species of Hevea were described (Smith, 1996).
Smith (1996) reflected that, if M. ulei should ever reach the Old World, ‘a
veritable scramble for resistance genes would ensue, and that the early work
of Spruce on the taxonomy and distribution of the genus Hevea would become
critical’. During the Second World War, the USA became acutely aware of the
necessity to prospect in South America not only for alternative sources of rub-
ber but also for higher-yielding and disease-resistant Hevea material. To this
end, a series of experimental stations and survey bases were established in
the Neotropics (Langford and Townsend, 1953). The myco-botanist R.E.
Schultes undertook the surveys for Hevea germplasm, which ultimately
spanned a 12-year period. Morphologically distinct, high-yielding and resist-
ant ecotypes of H. brasiliensis were identified in the Upper Amazon, around
Leticia in Colombia, and Madre de Dios in south-east Peru (Fig. 7.2). Rubber
from the latter region, known as Acre fino, was also far superior in quality to
that of commercial rubber from the Old World plantations (Schultes, 1970).
These collections, including over 350 clonal selections made by R. Seibert in
the forests of Madre de Dios, were established at the Turrialba Research Station
in Costa Rica. However, in the 1950s, the US State Department decided that
the rubber programme was superfluous to requirements, particularly since
synthetic rubber was at that time replacing natural rubber, and, as a result,
most of the Costa Rican collections were destroyed. As Davis (1997)
eloquently and pithily phrased it: ‘The clonal garden that had once served as
a repository for germplasm of an entire continent was replaced by a field of
sugarcane. The last of the Schultes and Seibert collection . . . was destroyed
by a forgettable botanist from Scotland. As an act of folly it has few equals in
botanical history.’
Witches’ Broom Disease of Cacao
Historical
Cacao (Theobroma cacao L., Sterculiaceae) is endemic to the Amazon basin and,
once again, the Upper Amazon region has been identified as its probable centre
of origin or genetic diversity (Cheeseman, 1944). However, unlike rubber,
there is a clear distinction between the botanical birthplace of cacao and its
region of earliest use or domestication, which lies in Mesoamerica
(Cuatrecasas, 1964; Schultes, 1984). Shortly after the conquest of Mexico,
cacao was being exported to Spain and the foundations of a chocolate industry
were laid. By the 17th century, chocolate houses were a popular feature in
many European countries. During the 19th century, techniques were devel-
oped in The Netherlands and Switzerland to make the product more palatable,
particularly by removing excess fat (butter) and adding milk, and thus even
more popular. From the 1850s onwards, cacao was extensively planted in the
Guianas, as well as in Ecuador, to supplement those plantations already well
92 H.C. Evans
established in Venezuela and in the Caribbean islands, especially Trinidad

(Purseglove, 1968), and considerable wealth was generated from the crop on
both sides of northern South America. The first documented record of a
witches’ broom disease attacking cacao was from Surinam in 1895 (Stahel,
1919; Baker and Holliday, 1957), although there is anecdotal evidence that
witches’ broom symptoms were described by early explorers in the Brazilian
Amazon many years before (Silva, 1987).
What followed in Surinam, as the disease swept through the plantations,
was a catalogue of misidentifications, detailed by Stahel (1915): firstly, as
Exoascus (Taphrina) theobromae Ritzema Bos., G. Massee initially agreed with
this finding but others suggested Fusarium and then Lasiodiplodia, although it
should be remembered that these mycologists were dealing with only alcohol-
preserved specimens. In 1908, the name Colletotrichum luxificum van Hall &
Drost was proposed, which was finally accepted as the causal pathogen in the
book by Massee (1910), Diseases of Cultivated Plants and Trees. This gave a
thorough description of the disease symptoms, and of the ‘Colletotrichum’
causal agent. Incredibly, as it seems now, relatively sophisticated and expensive
control measures, including spraying with Bordeaux mixture and pollarding,
were in operation throughout the Colony before the aetiology and epidemiol-
ogy of the disease were known. Later, Rorer (1913) concluded that the causal
agent was a basidiomycete fungus, after observing clamp connections in cul-
tures from diseased tissues. Shortly afterwards, Stahel (1915) linked the small,
pinkish-red mushrooms appearing on old brooms with the disease, and named
the fungus Marasmius perniciosus Stahel. Once again, the names of Rorer and
Stahel figured prominently in this pioneering work, and, as with South
American leaf blight, Stahel’s monograph on witches’ broom disease (Stahel,
1919) remained the standard reference for many years (Holliday, 1989).
Subsequently, the disease was reported in Guyana (1906), Colombia
(1917), Ecuador (1918), Trinidad (1928), Tobago (1939), Grenada (Holliday,
1952), and, most recently, in the Bahia region of Brazil (1989) (Pereira et al.,
1990). Almost 50 years previously, in an obscure taxonomic monograph, Singer
(1942) had transferred M. perniciosus to the genus Crinipellis Pat. Although this
transfer was acknowledged by Baker and Holliday (1957) in their treatise on
witches’ broom disease, they argued that the name Marasmius perniciosus was
so well known that it should be conserved. For this reason, the adoption and
common use of the correct binomial is of relatively recent origin and, indeed,
some still appear to be unaware of the name change (Willson, 1999).
Causal agent
Crinipellis perniciosa (Stahel) Singer (Agaricales, Tricholomataceae)

C. perniciosa var. ecuadoriensis (Stahel) Pegler
C. perniciosa var. citriniceps Pegler
Pegler (1978) revised the species concept based on new collections on both
cacao and liana from Ecuador (Evans, 1978), as well as on original material
from Surinam, and recognized three varieties. However, this interpretation has
been questioned recently (Griffith et al., 1994), and morphological differences
may warrant separation at the subspecies level. Earlier, Dennis (1951)
redescribed the fungus (C. ‘perniciosus’), based on Trinidadian material, and
he also included as a synonym an unpublished species, Marasmius scalptura-
tus Berk. & Curt., from Berkeley’s collection in Herb. K, originally obtained
from Cuba on ‘dead twigs’ in 1858. This specimen was re-examined by the
present author and the twigs appeared to be malformed. Tissues from this
material were later analysed at Kew and the wood structure was found to be
characteristic of the genus Theobroma (Pegler, 1978). However, witches’
broom disease has never been reported from Cuba, but it is possible that this
specimen came from long-abandoned and ancient Spanish colonial planta-
tions and that the fungus had been inadvertently imported with planting
material, probably from Venezuela, the origin of the favoured Trinitario selec-
tions (Purseglove, 1968; Thorold, 1975). Lack of interest in cacao, and of
mycologists in Cuba, may explain why the disease has not been recognized
since and reported.
Basidiospores are the only infective propagules of C. perniciosa. These are
liberated during the hours of darkness as the temperature drops and the
humidity rises, and they appear to penetrate directly through the cuticle, or
via stomata (Stahel, 1919). Infection of unhardened flushes (shoots), flowers
and young pods (cherelles) results in hypertrophy and hyperplasia in these
actively growing, meristematic tissues, with severe growth abnormalities:
vegetative brooms (terminal and lateral); flower or cushion brooms; partheno-
carpic and immature pods (strawberry- and carrot-shaped); and distorted or
indurated, mature pods (Stahel, 1919; Holliday, 1952; Baker and Holliday,
1957; Thorold, 1975; Evans, 1978; Rudgard, 1989).
The mycelium of C. perniciosa initially grows intercellularly, and visible
symptoms may not appear until 5–6 weeks after infection; some 6 weeks later,
the host tissues die as they become invaded intracellularly. Evans (1980) pro-
posed that the life cycle could be divided into two well-defined, genetically and
physiologically independent phases: a biotrophic phase represented by a thick,
convoluted or irregular mycelium, without clamp connections, only found in
planta or in in vivo callus tissues, considered to be monokaryotic; and a
necrotrophic phase, in which the dying host tissues are invaded by a vigor-
ous, thin, regular mycelium with clamps, considered to represent the dikary-
otic condition. Stahel (1915) illustrated both these mycelial forms. On average,
basidiomata are produced 5–6 months after tissue death, but this will vary
between climatic zones and, typically, in western Ecuador up to a year may
elapse between infection and sporulation (Evans, 1981a). It is not surprising,
therefore, that opportunistic fungi colonizing the necrotic brooms and pods
were originally thought to be the causal agents of witches’ broom disease.
94 H.C. Evans
In addition to the morphological forms or varieties described above, a com-

plex of physiological races or pathotypes has been identified from liana
(Bignoniaceae) and solanaceous hosts, and from other Theobroma species
(Evans, 1978, 1981a; Bastos and Evans, 1985; Bastos and Andebrahn, 1986;
Wheeler and Mepstead, 1988; Griffith and Hedger, 1994).
Crinipellis perniciosa has a strikingly similar natural range to rubber blight,

occurring on wild cacao and other Theobroma spp. throughout the Amazon
and Orinoco river basins (Fig. 7.3). Almost certainly, the invasions of cacao
plantations in the Guianas were initiated from inoculum sources within this
lowland forest ecosystem. Baker and Holliday (1957) described and illustrated
the symptoms on a range of Theobroma hosts. However, it is rare to see con-
spicuous symptoms, such as vegetative brooms, on wild cacao trees, and
Fig. 7.3. Distribution of Crinipellis perniciosa on cacao (as shown by dotted

line). The 19th-century record of C. perniciosa from Cuba is not included and
requires verification. The area between Leticia and Iquitos in the Upper Amazon
is the purported centre of origin or diversification of cacao, where resistance to
C. perniciosa has been reported (Pound, 1943).
parthenocarpic pods (chirimoyas) are the most common and obvious evidence
of infection.
In germplasm collections in both Ecuador and Brazil, closely related
Herrania spp. have also been shown to be susceptible (Evans and Barreto,
1996). In Pará State (Brazil), Theobroma grandiflorum (Spreng.) Schum., or
cupuaçu, is commonly cultivated and highly prized for its aromatic pulp,
which is used in a variety of beverages and dishes, but trees are invariably
laden with conspicuously large brooms, justifying its local name of ‘mãe da
vassoura de bruxa’ (mother of witches’ broom).
From its appearance in the Guianas just over a century ago, witches’
broom disease decimated the cacao plantations in both Surinam and Guyana,
and it has been cited as the sole cause of the decline and subsequent aban-
donment of the once flourishing cacao industry in these countries (Stahel,
1919; Thorold, 1975). For example, Padwick (1956) showed that there was
a catastrophic drop in cacao exports in Guyana, from over 70,000 t in 1906,
when the disease was first reported, to almost zero by 1923. Witches’ broom
did not reach Trinidad until 1928, when exports of cacao stood at nearly 60
million lb (30 million kg, Padwick, 1956), and, although its initial impact was
relatively slow, with a lag phase of 5–6 years, by the end of the 1930s the
disease was significantly affecting cacao production and exports fell to less
than 17 million lb (8 million kg) in 1939. During the war years, exports aver-
aged less than 10 million lb (5 million kg) per annum, although additional
factors compounded this decline (Thorold, 1975). A similar story emerges
from Ecuador, as the vast cacao haciendas of the Pacific region, which pro-
duced over 40,000 t of high-quality Arriba cacao in 1915, were invaded by
several diseases, including witches’ broom, from the 1920s onwards. A decade
later, yields amounted to less than 15,000 t (Evans et al., 1977). As Baker and
Holliday (1957) pointed out, the Andes proved to be an effective barrier to
natural dispersal of the pathogen from its Amazonian range, thereby allow-
ing the cacao industry of western Ecuador to prosper for almost 80 years,
based on the susceptible ‘Nacional’ variety. Its arrival was probably aided by
man, as also seems to have been the case in Trinidad (Baker and Holliday,
1957).
Cacao has been cultivated in the Brazilian Amazon for centuries (Silva,
1987) but not on a large scale, although it was apparently the dominant
species in floodplain forest of the lower Amazon and is considered to repre-
sent the remnants of old plantings (Smith, 1996). C. perniciosa is also endemic
in the forests of the Amazon and Orinoco river basins of Colombia, Peru and
Venezuela, and the disease seems to have progressed and invaded from these
relatively isolated foci to wherever cacao plantations were established.
During the 1970s, Brazil opened up the Amazon basin for colonization
with the installation of the Trans-Amazonian highway. Cacao was considered
to be a priority crop because of its high market value, ease of transportation,
ecological benefits and sustainability. The government empowered the
96 H.C. Evans
Brazilian cacao organization (CEPLAC) to develop cacao centres (‘polos

cacaueiras’), from Pará State in the east to Rondonia in the west. With new
technology and ‘resistant’ varieties, the ravages of witches’ broom disease
were judged to be past history and, as a result of this Amazonian initiative
(potentially > 300,000 ha of additional cacao), Brazil would soon take the
lead as the world’s major cacao producer. This scheme failed badly when the
extremely productive Rondonian plantations in particular succumbed rapidly
and heavily to witches’ broom disease (90–100% pod losses were not uncom-
mon), mimicking the situation in the ‘new age’ plantations of coastal Ecuador
established in the 1960s and subsequently abandoned in the 1970s (Evans,
1981a). Whether this catastrophic loss of resistance was due to environ-
mental factors, virulent pathotypes or inoculum pressure has never been
resolved satisfactorily.
The Amazonian venture caused disquiet in Bahia, which was also voiced
by the cacao industry in general, since it was argued that increased cacao pro-
duction in Amazonia would inevitably result in the pathogen reaching the
principal cacao-growing region of the Neotropics. ‘If this disease would ever
hit Bahia’s cocoa area its production would decline severely in the following
5 years. This forecast might well cause the market to explode’ (IOCC, 1984).
From epidemiological data, it was concluded that C. perniciosa would not be
capable of reaching Bahia through natural dissemination due to ecological
barriers (Evans, 1981a), and that the real threat was accidental introduction
of planting material, either cacao or the increasingly popular cupuaçu
(T. grandiflorum). A public awareness campaign was initiated and a cordon
sanitaire was established in 1978, covering all the major airports and roads
out of Amazonia (Pereira et al., 1997). However, in 1989, after more than
200 years of escape, the pathogen caught up with its coevolved host and, as
predicted by IOCC (1984), its impact was swift and dramatic, as it spread
through the 600,000 ha of almost contiguous cacao. This was despite the
bimodal rainfall pattern in Bahia, rather than the unimodal Amazon type,
which some observers had claimed would not favour disease development.
Initial attempts to eradicate the disease through mechanical removal of
brooms, fungicide application (from air and ground), and the killing and burn-
ing of infected trees all proved to be unsuccessful and the strategy turned to
containment (Pereira et al., 1997). Despite elaborate and costly control meas-
ures, C. perniciosa has continued its rampage through Bahian plantations,
even achieving notoriety in a national soap opera, surely a mycological
first! Pre-disease annual yields of over 400,000 t have now been reduced to
less than 150,000 t, discouraging farmers and threatening the long-term
future of this region as a major cacao producer. The pathogen has almost
reached the limits of its neotropical invasion, although the recent arrival of
C. perniciosa in Panama poses a threat to those cacao-growing countries (Costa
Rica, Honduras and Mexico) lying to the north.
Control
Prevention
There can be no doubt that the arrival of witches’ broom disease was human-
assisted. Whether it was an accidental introduction on infected planting
material, and this is feasible given that seed transmission can occur, albeit at
an extremely low frequency (Cronshaw and Evans, 1978), or deliberate, as
popular belief would have it, will always remain a mystery. Through efficient
quarantine procedures and adherence to well-documented technical guide-
lines (Frison and Feliu, 1989), the need for which was recognized many
decades ago when cacao germplasm (especially Pound’s Upper Amazon
material) was being moved through Kew Gardens on its way to Africa and
Asia, movement of C. perniciosa to the Palaeotropics is certainly preventable.
Cultural
As described by Evans (1981a), and even more recently by Pereira et al. (1997)
in Bahia, the difficulties of removing all diseased tissues, and therefore infec-
tion foci (‘hidden inoculum’), are an impossible task, particularly in mature
cacao plantations containing highly susceptible hybrids. However, if tailored
to or synchronized with the disease cycle in each climatic region, and carried
out judiciously, with the destruction of all prunings, then cultural control can
be extremely valuable by helping to reduce inoculum potential and, thereby,
increasing the effectiveness of other control measures (see Resistance).
Chemical
Past experiences, notably in Trinidad (Baker and Holliday, 1957) and most
recently in Bahia (Pereira et al., 1997), demonstrate that, although certain
fungicides are highly active against C. perniciosa, the economics and mechan-
ics (particularly timing) of spraying are too challenging for effective and sus-
tainable control. However, it may be economical to limit the target spray to
the trunk region in those areas where yields are high and pod production is,
or can be, concentrated on the lower trunk (Evans et al., 1977).
Biological
Specific mycoparasites, Cladobotryum amazonense Bastos, Evans & Samson and
Lecanicillium acerosum Gams, Evans & Zare, have been described from basid-
iomata of C. perniciosa collected in the Brazilian Amazon (Bastos et al., 1981;
Zare and Gams, 2001). In addition to physically overgrowing the pileus and
preventing spore release, C. amazonense also produces an extracellular, heat-
stable toxin, which lyses the basidiospores and which, potentially, could be
exploited as a mycochemical fungicide (Simmonds et al., 1992). However, fur-
ther progress has been halted in the mire of patenting rights, and interest has
now switched to another, newly described, Amazonian fungus, Trichoderma
98 H.C. Evans
stromaticum Samuels & Pardo-Schultheiss, isolated from cacao brooms

(Samuels et al., 2000). This mycoparasite actively colonizes the mycelium of
C. perniciosa within the broom tissues and inhibits basidioma production.
Currently, it is being mass-produced and marketed in Bahia as Tricovab® by
the Ministry of Agriculture. This fungus is particularly promising since it is a
coevolved parasite of C. perniciosa and, as a consequence, is adapted to invad-
ing broomed tissues. If problems relating to product quality, formulation and
spray techniques can be addressed, there is cause for optimism concerning the
future role of T. stromaticum as a biocontrol agent of witches’ broom disease.
Resistance
The pioneering surveys of wild and semi-domesticated cacao populations by
F.J. Pound in the Upper Amazon demonstrated that, as with rubber, there are
good sources of disease resistance in this region (Pound, 1943). The resultant
clones and their hybrids from these collections formed the basis of new plant-
ing in Trinidad, Ecuador and Brazil (Baker and Holliday, 1957; Evans, 1981a),
and, at least in the former two countries, may have contributed to a gradual
lowering of inoculum potential and thus to reduced disease incidence over the
intervening years. Nevertheless, as previously discussed, in certain regions,
such as Rondonia, this material has not performed too well. It may, however,
still prove to be useful in the long term, especially if highly susceptible clones
and their hybrids are identified and removed from future breeding programmes
and, ideally, from existing plantations, in order to provide a firm foundation for
a broader integrated pest management approach (Purdy and Schmidt, 1996).
Frosty (Monilia) Pod Rot of Cacao

Historical
The early history of this disease is still somewhat anecdotal, with reports as
long ago as 1851 from the Antioquia region of Colombia of pods being covered
by a powdery, velvet-like fungus (Baker et al., 1954; Thorold, 1975). Jorgensen
(1970) quotes from the diary of one of the cacao hacienda owners in western
Ecuador in 1895 describing similar frosty pod rot-like symptoms – ‘pods become
white while maturing on the trees . . . inside is watery . . . . The beans are rot-
ten and useless’. At this time, Ecuador was by far the biggest cacao producer
and alarm bells must have sounded since one of the foremost cacao experts,
C.J.J. Van Hall, toured the cacao-growing region, centred in Los Rios Province,
seemingly to report on the disease situation. He delimited two pod disease types:
‘mancha’ (rot), causing decay of the whole pod; and ‘helada’ (frost), with
abnormal growth of both pods and beans (Van Hall, 1914). Both these condi-
tions can be induced by frosty pod rot and it can be concluded that he was
describing different stages of the same disease, and that this was the first sci-
entific report of the ‘new’ pod disease. However, he failed to identify the causal
agent and subsequent attempts are cited by Jorgensen (1970) from two unpub-
lished 1916 reports to the Ecuadorian agricultural board: one identifying it as
of cryptogamic origin (a common belief was held that the condition was abi-
otic, because of a sudden drop in temperature) and the other actually uses the
name Monilia but determined the cause as Phytophthora. Finally, the experi-
enced J.B. Rorer (see previously) was contracted by concerned plantation own-
ers in 1917 to investigate the problem. Following surveys in coastal Ecuador,
he forwarded specimens of the pod fungus to R.E. Smith (University of
California), who identified it as a species of Monilia, close to M. fructicola (Wint.)
Honey, a serious disease of stone fruit in the USA. Over the next 8 years, Rorer
made periodic visits to Ecuador, working around the Quevedo region of Los
Rios Province, to monitor the disease and to undertake fungicide trials (Rorer,
1926). For this reason, Quevedo disease, along with watery pod rot
(‘podredumbre-acuosa’), was one of the first common English names for the
disease. No further identification of the causal agent was attempted until spec-
imens were sent from Ecuador by E. Parodi to R. Ciferri – ‘a versatile and pro-
lific mycologist and plant pathologist’ (Holliday, 1989) – in Italy, who
considered it to be an undescribed Monilia species, close to M. seaveri Reade,
which he named M. roreri Cif. in recognition of Rorer’s pioneering work (Ciferri
and Parodi, 1933). He suspected this to represent the anamorph of an
unknown Sclerotinia species, and, indeed, Wellman (1972) later included it
within a section on sclerotial diseases in his treatise on neotropical plant dis-
eases. Investigations in Ecuador during the 1970s led to the hypothesis that M.
roreri was an anamorphic basidiomycete, based on comparative symptomatol-
ogy with C. perniciosa and the mushroom-like odour of the white pseudostroma
formed on and within pods. Subsequent SEM studies of conidiogenesis and TEM
sections of hyphae proved this to be the case and a new genus was proposed
(Evans et al., 1978; Evans, 1981b). As with witches’ broom disease, however,
some have shown a reluctance to adopt the new generic name, preferring to
keep Monilia for the scientific as well as the common name.
Causal agent
Moniliophthora roreri Evans, Stalpers, Samson & Benny

The presence of dolipore septa in the hyphae and the retrogressive develop-
ment of basipetally maturing chains of mitospores readily separate this fun-
gus from Monilia species – ascomycete anamorphs with acropetalous conidial
chains – as well as from other genera of mitosporic fungi. The theory that
M. roreri and C. perniciosa are closely related, if not actually two evolutionary
branches of the same taxon (Evans, 1981b), is nearer to being proved; pre-
liminary molecular data show them to be very similar, based on comparisons
of their rRNA (G.W. Griffith, Ascot, UK, 2001, personal communication).
100 H.C. Evans
Conidia are the only infective propagules and penetrate either directly
through the epidermis or via stomata. In the field, only pod tissues appear to
be susceptible, although, under high inoculum potential in greenhouse con-
ditions, it has been found that germinating beans and shoots can become
infected, leading to growth abnormalities (Evans, 1981b). In addition to its
ability to cause hormonal imbalances in the host, another characteristic
which M. roreri shares with C. perniciosa is the long incubation period from
initial penetration to external symptom appearance, in the form of chlorosis
(premature ripening) and necrosis. This ranges c. 40–90 days but is depend-
ent on a complex of factors, including: pod age; host cultivar; inoculum
density; and even climate. It also explains why Rorer in Ecuador and various
investigators in Colombia, even as late as the 1950s, failed to obtain infection
from inoculation experiments using detached pods in the laboratory and only
short observation periods in the field.
Pods infected at an early (cherelle) stage frequently develop lateral
swellings and in extreme cases are totally distorted, symptoms which are indis-
tinguishable from C. perniciosa infection. Moreover, internal development of
both pathogens follows a similar pattern. In cherelle infections, the beans may
fail to differentiate and the pod contents are replaced by gelatinous substances,
hence the watery pod rot symptom, while in older pod infections there is over-
production of bean tissue leading to compaction and a denser, heavier pod,
which becomes dry or indurated with age. As the infected pods ripen prema-
turely, the first signs to the farmer that the apparently ‘healthy’ pod is not as
it seems, chocolate brown, irregular lesions appear on the pod surface, rap-
idly followed by a white pseudostroma, on which develops a cream-coloured,
spore bloom, which becomes greyish-tan and powdery with age, imparting a
frosted (‘helada’) appearance.
The period from chlorosis to sporulation is 3–8 days and it is only as the
pseudostroma emerges that a positive identification can be made of the causal
agent. Up to this point, C. perniciosa and M. roreri infections are indistin-
guishable, which may explain previous erroneous records of M. roreri (Evans,
1986). The dry, powdery spores are readily dislodged and freely carried in the
convection currents and by wind, probably over considerable distances.
Conidia have been detected in quantity, particularly during the day
(11.00–18.00 h) in volumetric spore traps situated c. 1 km from the nearest
inoculum source (Evans, 1981b). The spore wall thickens with age and this
may account for spore longevity, since viable conidia have been collected from
mummified pods in the cacao canopy up to 9 months after infection.
During his surveys in Ecuador, Rorer (1926) also recorded this disease on
Theobroma bicolor Humb. & Bonpl. and Herrania balaoensis Preuss. The fungus
had also been found on T. gileri Cuatr. in north-west Colombia (Baker et

al.,1954), which led Holliday (1970b) to speculate that this was the original
wild host. T. gileri was originally described from north-west Ecuador (in
Cuatrecasas, 1964), and a disease very similar to M. roreri was described from
the pods. The type locality was visited recently by the present author and M.
roreri-infected pods, showing severe mycoparasitism, were collected. It would
seem probable, therefore, that T. gileri does represent the coevolutionary host
of M. roreri, with its endemic range extending from western Ecuador to north-
west Colombia. It is likely that most species of Theobroma and Herrania are
susceptible, judging from the results of a survey in a comprehensive
germplasm collection in western Ecuador (Evans, 1981b). Almost certainly,
M. roreri invaded the burgeoning cacao plantations of coastal Ecuador from
these forest foci, on the lower, western slopes of the Andean cordillera, some-
time during the 19th century. However, the Andes proved to be a powerful
barrier to eastward movement and Amazonian Ecuador remained free of the
disease until relatively recently (Pound, 1943; Evans et al., 1998). The devel-
opment of a trans-Andean oil pipeline in the 1970s opened up the previously
remote eastern region of Ecuador to colonists and cacao cultivars were
imported from western Ecuador; the subsequent arrival of M. roreri was
inevitable and predictable (Evans, 1981b). Evans (1986) postulated that it
was only a matter of time before the pathogen reached the Brazilian Amazon,
although progress could be slow due to the scattered distribution of wild and
planted cacao from the Napo to the Amazon river. In fact, the pathogen has
moved rapidly southwards along the eastern slopes of the Andes to reach
northern Peru in 1989 and, by stepwise movements through the isolated
cacao-growing valleys of the Huallaga, Apurimac and Ene rivers, it arrived in
the Cuzco region in 1996 (Evans et al., 1998). If it moves across the Sierra
Madre de Dios, the pathogen then poses a direct threat to the Brazilian plan-
tations in Rondonia, as well as to Bolivian cacao (Fig. 7.4). Previous records
in the 1950s of M. roreri in both Peru and Bolivia can be discounted, and pod
infections were almost certainly due to C. perniciosa (Evans, 1981b).
Movement of M. roreri from its possible western Colombian forest habitats
was much earlier: firstly, eastwards to Zulia State of western Venezuela, prob-
ably in the 1940s, and northwards to eastern Panama in the 1950s (Orellana,
1956) and Costa Rica in the 1970s (Enriquez and Suarez, 1978).
Dissemination may have been by airborne conidia, but the pathogen was prob-
ably introduced accidentally by man into Panama (Orellana, 1956). Thus,
M. roreri is still on a invasive front, with recent reports of its arrival in
Guatemala and Honduras (W. Phillips, Reading, UK, 2000, personal commu-
nication), and it now threatens not only Mexico to the north but also Bolivia
in the south, as well as the much more extensive and economically important
plantations in the Brazilian Amazon and Bahia.
Wherever M. roreri has invaded, crop production has been severely
affected. From its appearance in epiphytotic proportions in Ecuador (c. 1916)
102 H.C. Evans
Fig. 7.4. Distribution of Moniliophthora roreri on cacao (as shown by dotted

line). The records from Nicaragua, Honduras and Guatemala are recent (W.
Phillips, Reading, UK, 2000, personal communication). A purported centre of ori-
gin of the pathogen on its forest host, Theobroma gileri, is marked T.g. on the
western slopes of the Ecuadorian Andes.
until the arrival of witches’ broom disease (after 1920), annual losses have
been estimated at 10,000 t or 20–30% of the total exports. More recent data
from several of the principal cacao-growing zones put pod infection due to M.
roreri at 20–43%, with similar figures being quoted for Colombia, with an
annual revenue loss of US$21 million (Evans, 1981b). From its detection in
Costa Rica in 1978, cacao production decreased rapidly, with up to 60–90%
pod loss (Evans, 1986). Until 1990, the major cacao disease in Peru was
witches’ broom, with a complex of Phytophthora diseases a poor second. The
situation changed rapidly and dramatically thereafter, with an overall fall in
production of 40–50%, and even total crop loss in some areas, leading to the
abandonment of farms (Evans et al., 1998).
There is ample evidence from the eastern Andes and Peru that the
pathogen once free of natural barriers moves quickly and efficiently by
airborne conidia. Evans et al. (1998) estimated that it took 5–7 years to
reach north-east Peru from the Napo region of Ecuador, a distance of some
500–600 km. It is not too dissimilar a distance to the Rondonian plantations
from south-east Peru, and, surely, it must be only a matter of time before it
invades plantations in the south-east of Mexico from Guatemala. The critical
ecological situation in Central America has also been highlighted recently,
following a multidisciplinary workshop in Panama. The concern is that cacao
growers on the Caribbean coast of both Panama and Costa Rica will abandon
their farms due to increasing disease and uneconomic returns. The predicted
land use change, from a forest-shaded, perennial crop to annual cash or sub-
sistence crops could have a dramatic impact on the biodiversity of the region.
An even darker scenario threatens Peru. If the alternative crops programme
fails, and here cacao takes a leading role, then farmers will abandon com-
modity crops and return to cocao-growing. If M. roreri cannot be contained
or controlled, then the economic returns from cacao will provide little incen-
tive to continue with this crop.
Control
Prevention
The talcum powder-like qualities of the spores, combined with their longevity,
have made M. roreri a formidable invader once it escaped the relatively nar-
row confines of its origin, on the western slopes of the Andes. If there are no
extensive geographic barriers then there is little that can be done to prevent
the entry of this pathogen into new territories. Evans (1986) considered that
natural spread to the Bahian region from a hypothetical Amazonian source
is a remote possibility, given that there are over 1500 km of land without
cacao and most of this is semi-arid, thorn forest (Fig. 7.4).
The chances of long-distance dissemination by man, however, are con-
siderable because of the cryptic, latent period within the pod, which can fool
even professional collectors, as evidenced by interceptions at Kew Gardens
(Evans, 1986). Once these ripening pods are opened and found to be ‘useless’,
sporulation on the cut surface is both rapid and abundant. The powdery spores
could also adhere to budwood or similar planting material and remain viable
for many months. However, since all such material is routinely passed through
intermediate quarantine stations, which follow the Food and Agricultural
Organization guidelines (Frison and Feliu, 1989), then this route to the Old
World plantations has effectively been blocked. Could it cross the Atlantic, as
purportedly happened with coffee leaf rust, Hemileia vastatrix Berk. & Broome
(Bawden et al., 1971)? This is open to debate and speculation.
Cultural
Fulton (1989) pondered on the fact that, since the infective stage of M. roreri
is limited to those spores produced on the pod surface, then field management
of the disease through good cultural practices, particularly crop sanitation,
104 H.C. Evans
should be highly effective. He then outlined the reasons why this has not
proved to be the case. For example, the cryptic nature of the disease deceives
the farmer as to its potential impact on crop loss. If he is not vigilant, then
the extremely rapid switch from apparently healthy to necrotic pod, with
massive sporulation (estimated at 44 million spores cm–2; Evans, 1981b), cre-
ates infection foci which are then difficult to eradicate and, in fact, removal
at this stage may only exacerbate the problem as the disturbed pods release
clouds of spores. Evans (1981b, 1986) had advocated removal of all
mummified pods in the canopy during the intercrop or dry season in order to
reduce and delay the build-up of infection during the following season.
However, erratic cropping cycles, poor follow-up sanitation (diseased pods
must be removed or buried) and contamination from badly managed, neigh-
bouring farms can all prejudice cultural control.
Chemical
As with witches’ broom disease, the maintenance of a continuous fungicide
cover on rapidly expanding, susceptible pods is a daunting task, illustrated
by Rorer’s experiences over 8 years in Ecuador as he battled to control
the ‘new’ disease with sulphur and copper products: ‘. . . These [fungicide]
experiments showed that many applications at frequent intervals were neces-
sary to control the disease at all successfully and that the cost of the work was
absolutely prohibitive’ (Rorer, 1926). Nevertheless, copper-based protectants
have proved to be economic in Ecuador when cropping is heavy but have failed
to give acceptable returns in Costa Rica (Evans, 1986). Fulton (1989) has
advocated the addition of oil to copper formulations in order to delay sporu-
lation, enhance droplet spread and increase residual persistence.
Biological
It is only relatively recently that this control strategy has been explored and
two different approaches are being evaluated, both based on the classical
biocontrol premise that suitable agents will only be found in the natural range
of the target pest. In the search for specific mycoparasites, wild populations of
Theobroma gileri have been surveyed in remnant primary forest on the western
slopes of the Ecuadorian Andes (700–800 m a.s.l.). M. roreri-infected pods
have been located but the pathogen never appears to be a critical constraint.
A range of mycoparasites colonizes the pseudostroma of M. roreri on the pod
surface, including species of Nectria and their Clonostachys anamorphs, and
appears to impact on and significantly reduce sporulation. The other, ongoing
approach is to assess the potential of benign endophytes which have been
found within the stem tissues of wild T. gileri, and which may confer induced
resistance to malign endophytes, such as M. roreri, through passive exclusion
or antagonism.
Resistance
All Theobroma and Herrania species would appear to be highly susceptible to
M. roreri, although some resistance, in the form of reduced lesion size and low
sporulation, has been detected in the ‘Nacional’ cacao of coastal Ecuador
(Rorer, 1926), and the hybrids derived from it (Evans, 1981b). Resistance
reported in other ‘refrectario’ trees has been ascribed to disease escape due to
a cropping pattern in which most pods develop during the dry season when
conditions are unfavourable for the pathogen. Based on this principle, crop
manipulation techniques, using artificial stimulation of flowering combined
with hand-pollination during the drier months of the year, have given prom-
ising results in Ecuador (Evans et al., 1977).
Conclusions
The fungal pathogens comprising this trinity of neotropical plant diseases
share many historical and biological traits. All rose to prominence at approx-
imately the same time when countries in northern South America began to
exploit their natural resources and cultivate indigenous forest tree species on
a large scale, in particular rubber and cacao, mainly for export to Europe.
Previously unknown natural enemies, especially fungal pathogens, emerged
from the obscurity of their forest habitats to wreak havoc in these new mono-
cultures. Over the intervening 80–90 years, they have been on an invasive
front, catching up with their hosts in most if not all the neotropical regions
where the crops have been introduced. The socio-economic and ecological
ramifications have been and continue to be severe.
Perhaps not surprisingly, the same pathologists and mycologists figure
prominently in the early history of these diseases and some, such as G. Stahel
and J. Rorer, made outstanding contributions, which, in general, have stood
the test of time. Given the uniqueness and complexity of the pathogens
involved, and of their disease cycles, it was inevitable that other fungi would
be implicated initially as the causal agents. In particular, the cryptic nature
of the cacao diseases, with a prolonged incubation period from infection to
symptom expression, compounded in the case of witches’ broom by an even
longer time lag to sporulation, created additional problems. The latter
pathogen is now undoubtedly correctly placed in the genus Crinipellis, despite
the prolonged and illogical resistance to the name change from Marasmius,
where it had been accommodated for over 25 years. Crinipellis species are
well represented in the Neotropics (Singer, 1942), and there is increasing
evidence that some species are obligate, and benign endophytes. For example,
basidiomata of C. siparunae Singer have been encountered regularly by the
author in Amazonia on the trunks of living healthy trees of the genus
Siparuna. This taxon was originally described from a Siparuna tree growing in
the Leningrad Botanical Garden (Singer, 1942) and, undoubtedly, the fungus
106 H.C. Evans
had been imported with its host from Brazil. Whereas most endophytes colo-
nize and grow within their hosts without causing abnormal reactions, the
swollen, intercellular mycelium of C. perniciosa and also that of M. roreri
appear to have a ‘haustorial’ or absorption function, sequestering nutrients
which diffuse out of host cells. It is postulated that this ‘leakiness’ is due to
the release of fungal metabolites which alter the permeability of the host cell
walls, a side-effect of which is to provoke a hormonal imbalance, resulting in
gross hyperplasia and hypertrophy (Evans, 1980). The hemibiotrophic nutri-
tion of these two cacao pathogens is also shared by Microcyclus ulei and, dur-
ing the biotrophic phase, tissue malformations are also induced in the rubber
host (Fig. 7.1). Indeed, Stahel (1919) illustrated amorphous, haustorial-like
structures within the cortical cells colonized by the pathogen. The
necrotrophic phase is characterized by the rapid invasion and death of the
host tissues by a saprotrophic mycelium. It has been hypothesized, for C. per-
niciosa at least, that these mycelial types are genetically and physiologically
distinct (Evans, 1980). If this holds true for M. roreri, then meiosis must occur
during sporogenesis and, controversially, the conidiogenous cell is, in fact, a
modified basidium.
It took nearly 20 years for the frosty pod pathogen to be formally assigned
to a genus, and a further 45 years for it to be recognized as an anamorphic
basidiomycete and accommodated in a new genus. Hopefully, the story will be
completed in the near future, using modern molecular techniques, when its
basidiomycete status is confirmed in the genus Crinipellis, close to C. perniciosa
(Evans, 1981b).
The correct taxonomic placement of the rubber leaf blight pathogen has
been less problematic, although it took over 10 years to link the teleomorph
and anamorph stages and nearly 50 years to determine its relationship to
the genus Microcyclus. Nevertheless, von Arx (1983) has since expressed
reservations about both the teleomorph and anamorph names. A more
critical evaluation and comparison of both its morphological and molecular
characters may reveal that this pathogen has close affinities with the genera
Mycosphaerella and Passalora.
All three pathogens have destabilized economies wherever they have
invaded in the Neotropics and, potentially, their impact in the Palaeotropics
could be even more dramatic, none more so than South America leaf blight,
which has been recognized for many years as the number one threat to nat-
ural rubber production. As Davis (1997) pointed out, the short-sightedness of
bureaucrats has meant that the considerable investment made in collecting
and selecting sources of disease-resistant material from Amazonia has been
wasted, and there is no existing germplasm collection in the Neotropics
on which to base a long-term breeding programme. Thus, South American
leaf blight of rubber ‘continues to hang like a Damoclean sword over the neck
of the industrial world’ (Davis, 1997).
The knock-on effects resulting from invasion by the cacao pathogens have
proved to be even more diverse and complex. The arrival of M. roreri in Central
America, for example, has threatened the livelihood of the small-scale cacao
farmers in the region. If they should decide to abandon the crop in the face
of decreasing yields and low prices, then this could result in widespread
deforestation as farms turn to annual cash or subsistence crops. With the loss
of these forest corridors, bird migration would be seriously disrupted, perhaps
permanently. At the other extreme of its current range in Peru, M. roreri is
now posing a threat to the alternative crops programme, in which cacao
occupies a key position. In the worst-case scenario, the farmers may become
disillusioned and return to cocao cultivation as cacao-growing becomes
uneconomic. The Peru experience has demonstrated that, in the ‘pecking
order’ of cacao diseases, M. roreri occupies the top position, probably by virtue
of its massive sporulation capacity, having rapidly ousted C. perniciosa as a
major constraint to production (Evans et al., 1998). This bodes ill, of course,
for Bahia, where witches’ broom disease on its own has seriously affected
the economic viability of the entire region. Moreover, abandonment of the
traditional old cacao farms, which are based on cultivation under remnants
of the highly biodiverse Atlantic forest, could result in the loss of these forest
refuges, home to many unique plants and animals.
Unlike rubber, however, invaluable cacao germplasm collections still exist,
thanks largely to the efforts of the chocolate industry. However, their future
is uncertain and, if the UK apple germplasm collection can be threatened
with extinction, as it was some years ago, the risk to cacao collections must
be that much greater. The long-term strategy of breeding for resistance to both
diseases should eventually benefit Latin American, as well as African
cacao-growing countries, if either of these pathogens should ever cross the
Atlantic.
Acknowledgements
I should like to thank Pauline Oldfield and Robert Reeder for invaluable assis-
tance with the preparation of this chapter.
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Lichens of Tropical Forests 8
B.J. COPPINS1 AND P. WOLSELEY2
1
Royal Botanic Garden Edinburgh, Edinburgh EH3 5LR, UK;
2
Department of Botany, The Natural History Museum, Cromwell
Road, London SW7 5BD, UK
Introduction
The tropical zone occupies about one-fifth of the earth’s surface between the
Tropic of Cancer and the Tropic of Capricorn. In this zone, tropical forests
have been the dominant vegetation over large areas of the landmass since
the Tertiary period. The forests extend in altitude from coastal mangroves, to
lowland areas supporting tropical rainforest, to montane cloud forest, and in
latitude from the tropical rainforest with c. 2000–3000 mm year–1 of rain
northwards to deciduous savannah forests with extended dry periods in a
monsoon climate. Of the 5 million ha of land area in this zone, c. 37% is still
forested, despite an annual deforestation rate of 15.4 million ha or 0.8% (Food
and Agriculture Organization of the United Nations (FAO),1997).
Total Numbers of Species in the Tropics
The diversity of tropical forests is well established for groups of larger organ-
isms but for fungi and their lichenized counterparts there are still enormous
gaps in our taxonomic and ecological knowledge. Aptroot and Sipman (1997)
estimated that between one-third and one-half of the world’s lichen diversity
occurs in the tropics, but more precise figures or estimates are currently
impossible to give because of the lack of suitable checklists. Even for extra-
tropical regions the figures are difficult to give. Ainsworth and Bisby’s Dictionary
of the Fungi (Kirk et al., 2001) accounts for 17,000–20,000 species of
lichenized fungi (most of which are ascomycetes), but other estimates range

114 B.J. Coppins and P. Wolseley
from 18,000 (Nash and Egan, 1988) and 17,000–20,000 (Galloway, 1992),
to 20,000–30,000 (Aptroot, 1997b).
The latest North American checklist has c. 3400 species (Egan, 1987),
and the equivalent figure for Europe is probably slightly higher, given the more
extensive coverage. Although there is no European checklist, most European
countries have recent lichen checklists available in print or from web sites.
Approximate species totals are: Austria (2100), British Isles (1760), Finland
(1500), Germany (1700), Italy (2100) and Norway and Sweden combined
(2300). These totals are remarkably similar to the estimated totals of
1500–2000 and 2000–2500 for Papua New Guinea (PNG) and Colombia,
respectively (Aptroot and Sipman, 1997).
How reliable are these estimates for tropical regions? Aptroot (1997a)
stated that 1079 species were reliably reported for New Guinea, but that a fur-
ther 300 species were as yet unidentified among the collections made by him-
self and his colleagues. A brief and probably not totally comprehensive search
of recent lichen literature for 1997–1999 revealed the addition of 105 species
for New Guinea, including 55 species new to science; these figures exclude
the report of many new additions by Aptroot et al. (1997), which were pre-
sumably included in the ‘1079’. A major contribution to these additional
figures concerns Pertusaria, a large but hitherto little studied genus in tropical
regions. A revision of Australasian species added 48 species to the lichen flora
of PNG, 31 being new to science (Elix et al., 1997; Archer and Elix, 1998a,b).
Other important papers concerning the numbers of lichens in New Guinea
are studies on Parmotrema (18 added species, six new to science) by Louwhoff
and Elix (1999) and of miscellaneous genera (20 added species, three new to
science) by Aptroot (1998). Given that the net additions (taking account of
synonyms) to the ‘well-known’ British lichen flora have continued to average
about ten species year–1 since the mid-1960s, the average of 35 species year–1
for 1997–1999 should easily result in an accepted total for New Guinea of
exceeding 1500 by the year 2013.
Analyses of tropical lichen floras in most countries are difficult to do, given
the dearth of modern checklists or ‘floras’. Since 1997, the only checklists to
appear for anywhere in the tropics are for Hong Kong (Aptroot and Seaward,
1999) and a catalogue of the lichens of the smaller Pacific Islands (Elix and
McCarthy, 1998). However, checklists are in preparation for Colombia,
Ecuador and Thailand, and the latest checklist for Australia (Filson, 1996)
includes tropical species but does not specifically identify them. The Hong
Kong list included only 261 species, but 176 were new to Hong Kong, 132
were new to China, and 43 were new to East Asia. Aptroot and Sipman’s
(1997) estimate that 50% of the tropical lichen flora is still unknown is prob-
ably a fair estimate overall, but for most tropical countries the percentage is
far higher.
Site Diversity
‘Within-site’ diversity of lichens is surprisingly similar in both tropical and

temperate conditions. Aptroot (1997a) reported 400–450 species (from all
habitats) for each lowland, intermediate and high-altitude area in PNG. These
species’ totals equate well with recent site surveys carried out in areas little
affected by atmospheric pollution in Scotland: 402 from Craig Clunie and
Lion’s Face Site of Special Scientific Interest in the North East Highlands
(Coppins and Coppins, 1999), 467 from Glen Shira in the West Highlands
(Coppins and Coppins, 1996), and 466 from the high ground of Ben Lawers
National Nature Reserve in the Central Highlands (Gilbert et al., 1988; Fryday,
1989). In all situations lichen diversity drops rapidly where forest destruction
and/or atmospheric pollution is occurring.
Substrata
Although in temperate and boreal zones a large proportion of the lichen

diversity is associated with terrestrial and saxicolous substrata, in tropical
climates the highest lichen diversity is found among epiphytes. Where temper-
atures and rainfall are relatively stable for much of the year, foliicolous lichen
diversity may be very high, contributing to c. 30% of the total lichen flora on
trees and shrubs in lowland tropical rainforest (Aptroot, 1997a). In seasonal
deciduous forests, diversity is highest on corticolous substrata, and foliicolous
species are found only along watercourses (Wolseley and Aguirre-Hudson,
1997a). Within a tropical rainforest, much of the corticolous diversity is not
readily accessible as the diversity increases markedly with distance from the
ground. Sipman (in Gradstein et al., 1996) pointed out that a reliable canopy
inventory cannot be carried out by sampling fallen branches, because most of
these branches are from the lower canopy and soon become covered in sapro-
trophic moulds. The best results are obtained from the study of recently fallen,
or felled, trees, or by cutting and carefully lowering down selected branches (ter
Steege and Cornelissen, 1988). Aptroot (1997a) recorded 173 lichen taxa on a
recently fallen Elaeocarpus tree. Of these only 41 (24%) occurred on the lower
trunk below 5 m. A recent, somewhat more sophisticated, technique has been
employed in Project Surumoni in a tropical lowland rainforest in southern
Venezuela, using a 40 m high tower crane mounted on a 120 m long railway,
with a 40 m jib-boom (Komposch and Hafellner, 2000a). From their study of
nine trees in a single 1.5 ha plot, Komposch and Hafellner (1999, 2000a)
recorded 268 species. Of these only 9% occurred on the lower trunk, with the
most diverse zone being the middle canopy. Accordingly, they believed that
Aptroot and Sipman’s (1997) estimation that the ‘total number of corticolous
and foliicolous lichens per km2 of tropical rainforest may well be over 300’ was
cautious, and that the figure should be changed to 300 species ha–1.
Foliicolous lichens
Evergreen leaves form a constantly renewable surface for colonization by both

lichens and bryophytes, and, as the specimens have been more accessible than
many epiphytes of tropical forests, advances in this area were made relatively
early on. Santesson’s (1952) monumental treatise on the taxonomy and
identification of foliicolous lichens was a landmark, in which he reduced
the accepted c. 1000 species to 250 and provided workable keys. With this
foundation, other lichenologists, especially Antonin Vezda, Emmanuël
Sérusiaux and Robert Lücking, have increased the number of foliicolous
lichens to beyond 600 species, and have also provided good illustrations and
updated keys to genera and species where necessary. A comprehensive
reference list to the taxonomic works on foliicolous lichens is provided by
Farkas and Sipman (1997) and the more important subsequent works are by
Aptroot et al. (1997); Lücking (1997a,b, 1998a, 1999a); Lücking and Vezda
(1998); Lücking and Cáceras (1999). An additional advantage for studying
foliicolous lichens is that samples are easily collected – on the ‘micro’ scale, a
whole substratum and its community of colonizers can be quickly plucked
into a collecting packet for comfortable examination in the laboratory.
However, this is countered by the need for good microscopical equipment and
great patience and skill in sectioning and examining the individual compo-
nents of the community! For ecological studies, experience is needed, in order
to reliably identify juvenile, poorly developed, parasitized or sterile thalli.
Taxonomists embarked on purely taxonomic studies have the luxury of
discarding poor specimens, but this possibility is denied them if carrying out
ecological investigations.
Saxicolous lichens
Although rich saxicolous lichen communities, and associated terricolous

lichens, can be found on some high mountains within the tropics, rocky
habitats within the rainforest zones are usually dismissed as ‘poor’. The rocks
are usually either too shaded for lichens or, if exposed, too eroded or sun-
baked. Also, where rock surfaces are in an appropriate condition for lichen
colonization, the flora is often composed mostly of normally corticolous
species (e.g. species of Graphidaceae, Thelotremataceae, Parmeliaceae,
Heterodermia or Pertusaria). However, careful scrutiny for suitable niches can
be rewarding. The specialist lichens of rocks in and alongside streams
and rivers in temperate regions are reasonably well known and appreciated,
but in tropical rainforests have scarcely been mentioned. During a short stay
in Khao Yai National Park, Thailand in 1997, such lichens were found within
the evergreen rainforest zone. These included the widely distributed
Endocarpon adscendens (Anzi) Müll. Arg., the recently described Ionaspis tropica
Aptroot from New Guinea, undescribed species of Staurothele and Verrucaria,

and three new species of Porina (Boonpragob et al., 1998; McCarthy, 1999).
Although, admittedly not likely to be as ‘rich’ as their temperate counterparts,
the rocky habitats within rainforests have potential and should not be ignored.
Their previous neglect could be a combination of such simple factors as the
explorers being overwhelmed and diverted by the richness and newness of the
corticolous and foliicolous habitats, and by not having a hammer and chisel
in their collecting bag!
Taxonomic Literature and Identification Keys
In many areas the literature is still poor. There is an urgent need for taxonomic
revisions of families of tropical lichens, particularly large families of crustose
taxa, such as Graphidaceae and Thelotremataceae, both of which contribute
significantly to biodiversity and may also be indicators of environmental
conditions. These two families contain c. 1000 and 800 species, respectively.
Some advances are being made in this direction, with the recent revision of
Acanthothecis (Graphidaceae) by Staiger and Kalb (1999), although several
studies are not yet published. A study of 767 collections of Thelotremataceae
from Thailand and Malaysia by Homchantara (1999) recognized 124 species,
35 of which were unnamed and perhaps undescribed. Klaus Kalb and his
students are carrying out further studies on these two families, including one
on African thelotremes by Andreas Frisch.
A survey of the taxonomic literature on lichens from 1997 to 1999
revealed from 40 publications that 193 new species had been described from
the tropics, including new species of macrolichens (e.g. Pooprang et al., 1999).
Excellent though most of these papers are, only 14 of them provide keys to
the genera or groups concerned. Global keys are given for Acanthothecis
(Staiger and Kalb, 1999), foliicolous Arthoniaceae (Ferraro and Lücking,
1997), foliicolous Chroodiscus (Santesson and Lücking, 1999), Fellhanera p.p.
(Lücking, 1997c), Porina epiphylla group (Lücking and Vezda, 1998) and the
Trichotheliaceae (Lücking, 1998a). Regional keys have been provided for:
foliicolous Coenogonium and Dimerella (Lücking, 1997a), Lobaria (Yoshimura,
1998) and Peltigera (Vitikainen, 1998) in the neotropics, the Gomphillaceae in
Costa Rica (Lücking, 1999a), and Anisomeridium and Pyrenula (Aptroot et al.,
1997), Parmotrema (Louwhoff and Elix, 1999), Pertusaria (Archer and Elix,
1998a) and Stereocaulon (Sipman, 1998) for New Guinea.
These keys are much welcomed but were written for the specialist. There
are no widely available, illustrated keys to genera for the benefit of the
generalist and the potential specialists of the future, especially those residing
in tropical countries. The latter may well have been able to acquire the
necessary microscopical and chemical equipment, and have full access
to the Internet, but progress with identification is hampered by the lack of
literature. The production of a user-friendly, illustrated identification manual

for the genera of tropical lichens should be considered a priority by the licheno-
logical community. Such a manual would perhaps be best available from a web
site, so as to be easily updated, and to avoid currency problems. An Internet
location (www.mycology.net/lias/index.cfm) for specialist keys already exists in
the form of LIAS (The Global Information System for Lichenized and Non-lich-
enized Ascomycetes), coordinated by Gerhard Rambold at Munich, and mono-
graphers are urged to contribute their data for the benefit of all.
Taxonomic studies, often undertaken in a rather piecemeal fashion, with
short-term grants, would be much enhanced by long-term, well-coordinated,
reliably funded international programmes. Lichenologists should consider set-
ting up international specialist groups, as exist for many families of vascular
plants, to improve communications and provide a better focus for funding.
Recent Ecological Studies on Lichens in Tropical Forests
The structural diversity of habitats in tropical forests was outlined by Richards

(1984), related to cryptogams by Gradstein (1992) and described for canopy
specialists by Rhoades (1995). The contrast between the dense shade of the
ground and lower trunks and the exposed canopy branches is extreme. In the
intermediate zones, there may be a great number of ecological niches with
characteristic lichen communities. In the outer canopy, lichens may have
to withstand high UV levels and also be subject to extremes of wetting and
drying. In contrast, the trunk receives very low light levels, but humidity
is ± 100% most of the time. Lichens, such as Trypetheliaceae, that dominate
the outer canopy are often highly coloured by anthraquinones and other sec-
ondary products, which act as a sun and radiation screen for the photobiont,
whereas lichens on the bole are pale or dark coloured and often have
hydrophobic secondary products, which protect their surface from excess
water (Kantvilas et al., 1985; Wolseley, 1997). The high diversity along the
vertical gradient is reflected in the species turnover (beta diversity) of rather
distinct communities adapted to the micro-conditions. Cornelissen and ter
Steege (1989) distinguished 13 cryptogamic communities along a vertical
gradient in evergreen forest in Guyana. At high altitudes the forests have less
vertical stratification and macrolichens are a conspicuous feature of the forest,
whereas in lowland rainforest the diversity is dominated by crustose species,
many of which present problems of identification. For this reason, much
lichen research concentrated on high-altitude zones (Wolf, 1993a,b;
Pentecost, 1998). The importance of crustose communities in the lowland
rainforests of Guyana has been documented by Montfoort and Ek (1990), and
changes in crustose communities from trunk to canopy in a lowland forest in
Venezuela have been investigated by Komposch and Hafellner (2000a), who
characterized six lichen zones, all of which were dominated by crustose
species, with foliose species occurring only on the lowest trunk (zone 1) and
in the well-lit middle canopy of zone 5. There was no species overlap between
zones 1 and 6, whereas between zones 1 and 2 there was a 23% species
similarity. The pattern throughout lowland rainforests is remarkably similar,
with minor variations according to local conditions (Sipman and Harris,
1989; Wolseley et al., 1998; Komposch and Hafellner, 1999), with
Thelotremataceae dominant at lower levels together with Crocynia, Eschatagonia
and species of Porina (Trichotheliaceae). Species of Graphidaceae and
Arthoniaceae increase towards the upper zones, and in the outer canopy
Pyrenulaceae and Trypetheliaceae become the dominant families. Crustose
species increase in diameter with age and in undisturbed forests a single
thallus may be a metre or more in diameter and encircle the trunk, suggest-
ing that a lichen may colonize a young tree and grow with the tree (Fig. 8.1).
On slow-growing trees the circumference is round, but on fast-growing trees
the lichen thallus is stretched into an ellipse (Wolseley and Aguirre-Hudson,
1997a).
Fig. 8.1. Large smooth-barked emergent tree in the 50 ha Forest Dynamic Plot at
Pasoh Forest Reserve at Negri Sembilan, Malaysia, showing large rounded thalli
of Thelotremataceae and Graphidaceae.
Reproduction
Research on foliicolous lichens has led the way in ecological studies of lichens
in tropical forests, due to accessibility of the habitat and the sound taxonomic
basis available. In a study of the community ecology of foliicolous lichens,
Lücking (1999b) demonstrated two major groups governed by microclimatic
factors: one characteristic of the shady understorey and the other confined to
light gaps. The shady understorey community is dominated by species of the
Arthoniaceae, Opegraphaceae, Trichotheliaceae and Pilocarpaceae, which pre-
dominantly have photobionts of the Trentepohliaceae, thin thalli, abundant sex-
ual reproduction, small ascospores produced in high numbers and pycnidial
conidiomata. The light-gap community is dominated by species of
Gomphillaceae and Ectolechiaceae, with Trebouxia as photobiont, thickly
crystalline or whitish, dispersed thalli, frequent asexual reproduction, large
ascospores produced in low numbers and specialized conidiomata, such as
campylidia and hyphophores. The complexities involved in interpreting the
ecological significance of these reproductive mechanisms are discussed in
detail by Lücking (1999b), and are clearly a fascinating area for further
research. It is well known that the proportion of species producing large,
multi-celled ascospores is much higher in the tropical forests than in temperate
or boreal regions (Sipman and Harris, 1989), but the ecological interpreta-
tion of this is far from clear. It is further complicated by observations that
large, muriform ascospores are often secondarily divided into minute simple
conidia within the asci (e.g. Hafellner and Bellemère, 1983; Sipman and
Harris, 1989), or following release (as in Agonimia pacifica (Harada) Diederich).
Of the 268 species collected from nine trees in lowland rainforest at
Surumoni, 94% were crustose species and 90% reproduced sexually, including
12% that also produced vegetative propagules (Komposch and Hafellner,
2000b). Komposch and Hafellner (2000b) further found that asexual propaga-
tion was highest at ground level (60% of observations), and continuously
decreased upwards, being absent altogether in the outer canopy. In plots in the
more open seasonal deciduous forests of northern Thailand, Wolseley (1997)
found that asexual propagules were more frequent in fire-damaged areas, and
that in undisturbed forest sexual reproduction was more frequent. On 20 trees
in two 100 m plots in fire-damaged deciduous dipterocarp forest, 62% and 85%
of lichens on trunks (up to 3 m), respectively, produced propagules, whereas in
a dipterocarp forest protected from fire for 23 years, 53% were reproducing
sexually, and in unburned seasonal forest, 58% of lichens were reproducing sex-
ually (Wolseley, 1997).
Lichens as Indicators of Ecological Continuity
Detailed studies in the British Isles and other parts of western Europe revealed
that many species were confined to woodlands with a long ecological

continuity and presence of mature trees. From these observations, lists of ‘old
forest indicator’ species were drawn up, and indices of ecological continuity
devised (Rose, 1976, 1992). The most important species in the fagaceous
woodlands and forests of temperate western Europe are found in epiphytic
communities belonging to the Lobarion pulmonariae alliance. Wolseley (1991)
demonstrated the same pattern of association with ancient, little-disturbed
forest for similar ‘Lobarion’ lichens in montane fagaceous (hill evergreen)
forest zones in South-East Asia. These ‘old forest’ lichens are tolerant of some
forms of management, e.g. conversion to wood pasture in Europe, and
traditional slash and burn in Asia. However, they are intolerant of more
drastic management, such as clear-felling or extensive burning. Very few of
these ‘old forest’ lichens are to be found in secondary forest or in formerly
cleared forest that has been re-established with native trees, even after a
century or two. This is perhaps not surprising, as many of these lichens require
the bark substratum provided by mature or ancient trees, and they are
intolerant of dense shade as would occur following a coppice cycle or
secondary forest development.
What is more surprising is that some foliicolous lichens are equally good
indicators of ecological continuity, given that the lifespan of their substratum
is limited to rarely more than 2 years (Lücking, 1992, 1995). From 99 sites
within the rainforests of Costa Rica, Lücking (1995) compared species and
‘form’ diversity with environmental conditions, including leaf age, phorophyte
(host) identity, seasonality, light intensity, relative humidity and altitude. The
highest species number was found at low-altitude sites with a slight dry sea-
son, and these were in primary forest. The species total for primary forests was
283, with secondary forest supporting little more than half that number, and
anthropogenic vegetation (plantations and fruit trees) 78 species. The results
of further ecological studies have followed (Lücking, 1998b,c) and, with the
information gained, Lücking (1997d) analysed the use of foliicolous lichens
as bioindicators. He proposed that their use as indicators of altitudinal zona-
tion and seasonality was rather restricted but that they had great potential as
indicators of anthropogenic disturbance and microclimatic conditions.
Lichens as Indicators of Changes Induced by Fire
In the seasonal climate of Thailand, deciduous dipterocarp forests are wide-

spread, often occurring in a mosaic with evergreen forests. Fire occurs natu-
rally in the dry season, but its incidence has been increased by man, causing
a shift in the balance of forest types in favour of the fire-tolerant deciduous
dipterocarp forests. The lichen communities of these forest types were found
to be markedly different in composition at the family and generic levels, the
evergreen forests being dominated by lichen families with trentepohlioid algae,
while the deciduous dipterocarp forests were dominated by families with

trebouxioid algae. Species diversity was high in both types of forest where
ecological continuity was maintained, but, where there had been an envi-
ronmental shift from evergreen to deciduous forest, lichen diversity was very
low, with a marked absence of species characteristic of ‘natural’ deciduous
dipterocarp forest, and the remnants of evergreen forest had species-poor
communities. In sites where the deciduous forest was native and of long-
standing continuity, lichen communities were highly diverse, with many
species adapted to conditions of high light intensity, temperature and UV
radiation, some having characteristic, highly coloured anthraquinone and
xanthone pigments (Figs 8.2, 8.3). Conversely, in the evergreen forests
the lichen communities were more characteristic of those found in rainforests
throughout South-East Asia, and dominated by the Thelotremataceae pyreno-
carps, and species of Crocynia, Eschatagonia and Phyllopsora. The disjunct
nature of these communities, with adaptations to very different environ-
mental conditions, suggests that the species-rich communities of both forest
Fig. 8.2. Fire-tolerant Dipterocarpus obtusifolius Teysm. ex Miq. in dry diptero-

carp forest (DDF) in Doi Suthep National Park, Chiang Mai,Thailand, showing
conspicuously coloured mainly foliose lichens Pyxine coccifera (Fée) Nyl. (red)
and Relicinopsis rahengensis (Vain.) Elix & Verdon (yellow-green) above the fire
zone.
Fig. 8.3. Quadrat on Dipterocarpus tuberculatus Roxb. in DDF in a protected

area at Wat Phalad in Doi Suthep NP, showing high lichen diversity with a mix-
ture of highly coloured fire-tolerant and pale-coloured fire-sensitive species, and
presence of fertile crustose species.
types evolved under different climatic conditions. Those of the deciduous

dipterocarp forests probably developed in the drier glacial periods of the
Holocene when fires were frequent, and those of the evergreen forest are an
extension of evergreen forest that was widespread in the Tertiary (Wolseley
and Aguirre-Hudson, 1997b).
Lichens and the Ecosystem
The dominance of lichens in the montane forest contributes considerably

to the biomass of the forest (Pentecost, 1998). The role of cyanobacteria in
nitrogen fixation may also be important in these montane areas of high rain-
fall where soil nitrogen is rapidly leached (Forman, 1975). However, the
importance of lichens to other microorganisms is still little understood.
Recent research in Borneo demonstrated a preference of the lichen-
feeding termite Hospitalitermes for large emergent dipterocarps (Fig. 8.4). A
Fig. 8.4. Termites, Hospitalitermes hospitalis Haviland, feeding on lichens on the

trunk of a tree in a recently logged rainforest site, Tabalong district, South
Kalimatan, Indonesia. The termites leave their nest in the afternoon and travel in
columns across the forest floor until they find a suitable tree on which to forage.
The termites climb the tree and forage throughout the night, with workers return-
ing to the nest carrying balls of food held in their mandibles.
colony will feed on lichens and bryophytes in the canopy of one tree for c. 4
days, chopping the epiphytes into food balls and carrying these back to the
nest, before moving on to another tree (Jones and Gathorne-Hardy, 1995).
Investigation of the food balls has identified lichen spores (Collins, 1979), and
bryophyte, lichen and algal tissue. Loss of dipterocarps from logged forest may
cause Hospitalitermes to feed on lichens and on other bryophyte and algal tis-
sue on other trees. In recently logged forest in Kalimantan, Hospitalitermes was
feeding on a range of crustose lichens on many different species and ages of
tree, leaving highly grazed patches of crustose lichens with only fungal
tissue remaining (Wolseley et al., 2001; Fig. 8.4). Other canopy feeders on
lichens are oribatid mites, which are recorded in very high densities of 1000
to 10,000 mites m–2 of leaf in rainforest canopies in Queensland (Walter and
O’Dowd, 1995). Apart from the food source, some species are known to
camouflage themselves with lichen propagules (Stubbs, 1995). The role
of both organisms in the lateral and vertical transport of lichen propagules
is important in tropical rainforests, where there is often very little wind
below the outer canopy and only rain contributes to the downward transport
of propagules.
Forest Management and Factors Affecting Lichens
Although the loss of tropical forests is now occurring in all forest zones from
lowland to montane, there is still little research on the effects of this on diver-
sity of cryptogamic or invertebrate populations. Loss of lowland rainforest is
still mainly due to logging, and between 1981 and 1990 averaged 4.6 million
ha annum–1; in moist deciduous forests the rate of loss was 6.1 million ha
annum–1, and in the dry forests, where there are few valuable timber trees,
the loss was 2.2 million ha annum–1. Montane and hill forest do
not contain many useful timber trees, but a loss of 2.5 million ha annum–1
has been recorded (FAO, 1997), mainly due to the increasing demand for
agricultural land.
Logging has become an increasingly technical process, dependent on large
machinery and road access to all sites. In order to obtain selected timber trees,
many other trees are lost or damaged. What effect does this have on
cryptogamic diversity? In an area of lowland forest in Kalimantan, where the
forest had been clear-felled, then slashed and burned for agricultural land for
1–2 years, and converted to plantation forest of exotic species, there was
almost complete absence of forest species of lichens, bryophytes and termites.
This was in marked contrast to a site logged in 1999, where lichen and termite
diversity was not much lower than that of a plot logged 17 years before. In
the 1999 plot, the topography was complex, so that pockets of forest
remained, contributing to the availability of propagules, and, with increased
light on the trunks, many of the canopy species were able to invade sites lower
down the trunks. In the 1950s, selective logging of a site in Malaysia adja-
cent to a 50 ha plot of old-growth forest had encouraged the retention of trees
as seed trees. In 1996, these trees were surrounded by a rapidly regenerating
stand of young dipterocarps with a considerable diversity of lichen species,
albeit few specialist species (Wolseley et al., 1998). Although there is a need
to describe primary site diversity in tropical forests and to define and protect
the areas of outstanding species richness, there is also an urgent need to assess
management practices in tropical forests so that best practices can be
established.
Fire is also an increasing hazard in tropical forests that are not fire-
adapted, especially in montane evergreen forests or in lowland forest
established on peat. The increased use of fire by man has brought about the
spread of deciduous forest at the expense of the evergreen forest in areas of
monsoon climate with an extended dry period. Lichen species of the original
forest are lost and, as lichens of the new forest type colonize slowly, the species
composition of epiphytic lichens can be used to chart the time period over
which the change has been occurring (Wolseley and Aguirre-Hudson, 1997b:
Fig 8.5). Lichens are well established as indicators of the ecological continu-
ity associated with old-growth forests in temperate Europe and America, and
recent work suggests that this will be similar in the tropics, but there is still a
Fig. 8.5. Quadrat in fire-damaged seasonal evergreen forest in Huay Kha Khaeng
Wildlife Sanctuary showing invasion by Pyxine consocians Vain. following death
of the pyrenocarp.
considerable amount of taxonomic and survey work to be done to establish

which species can be used to detect ecological continuity in tropical forests
and to define indicators of environmental change.
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The Importance of Invertebrate- 9
pathogenic Fungi from the
Tropics
N.L. HYWEL-JONES
BIOTEC–Mycology, Yothi Laboratories, 73/1 Rama 6 Road,

Introduction
Fungal pathogens of invertebrates (mostly Insecta and Araneae are consid-

ered here)1 have assumed increasing importance as potential biological con-
trol agents and as a source of novel secondary metabolites for industry in the
last 20 years (Nisbet and Porter, 1989; Nisbet and Fox, 1991; Rossman,
1996). Despite this, little is known about the basic biology of these fungi.
Samson et al. (1988) provided an overall picture of ‘entomopathogenic fungi’,
with chapters on topics such as taxonomy, pathogenesis, ecology and biology,
biotechnology and biological control. Consequently, these authors provided
the most modern synthesis of the broad subject, and yet this work pre-dated
the revolution provided by the development of molecular phylogenetic analy-
sis as a tool for clarifying evolutionary relationships.
Importantly, in the last 10 years the significance of fungi in assessments
of global biodiversity has also increased, largely due to Hawksworth (1991),
who produced estimates of fungal diversity based on an analysis of a wide
range of literature. At that date c. 68,000 species of fungi were considered to
have been described for the world. Compared with plants and animals, where
it is acknowledged that more than 85% of species diversity have been named,
the fungi come poorly down the list with < 5% having been reliably named.
The Hawksworth estimate of 1.5 million has often been considered conser-
vative, although it has been challenged and reduced to one-third of that figure
1
In this chapter, I refer to invertebrate-pathogenic fungi in recognition of the many species
that are pathogenic to spiders and mites.

134 N.L. Hywel-Jones
(A. Aptroot, Hong Kong, China, 2000, personal communication). Even at one-
third, there is still > 85% of global fungal diversity hidden from current
view. For invertebrate-pathogenic fungi there is every indication that our
knowledge of such fungi is even more poorly realized, and that these may con-
tribute a still further hidden and large element of the overall fungal diversity
on our planet. The aim of this chapter is to examine this component from the
standpoint of the tropics, where fungi are a major component of biodiversity
(Hawksworth, Chapter 1, this volume).
What are the Invertebrate-pathogenic Fungi?
Apart from work on a few species with broad host ranges, there are few stud-
ies that have fully examined details of pathogenesis in invertebrate-pathogenic
fungi. Where studies have been done, these have been limited to a few ‘model’
species such as Metarhizium anisopliae (Metschn.) Sorokin, Beauveria bassiana
(Bals.) Vuill. and Nomuraea rileyi (Farlow) Samson. For the rest, it is assumed
they have a truly pathogenic life cycle, based on their restricted host ranges
and relationships to those which have adopted a wider host range and have
lent themselves to laboratory study. Invertebrate-pathogenic fungi include
seemingly highly coevolved pathogens (e.g. Cordyceps), broad-range oppor-
tunistic pathogens (M. anisopliae) and opportunistic necrotrophs (Conidiobolus
coronatus (Costantin) Batko).
Within the fungal kingdom at least five ‘hot spots’ have evolved associa-
tions with invertebrates. These are:
• Zygomycota – Entomophthorales
• Zygomycota – Eccrinales
• Ascomycota – Laboulbeniales
• Ascomycota – Hypocreales
• Basidiomycota – Septobasidiales
In keeping with modern phylogenetic studies, invertebrate-pathogenic
Chytridiomycota and Oomycota are excluded, but see Samson et al. (1988). It
seems that, when the jump is made from a presumptive plant host to an
invertebrate host, there is great opportunity for rapid speciation of the fungi
as they seek out new invertebrate hosts. This, not surprisingly, can be
accounted for by the fact that invertebrates are the largest component of
diversity on the planet.
Within the five ‘hot spots’, the ascomycete order Laboulbeniales is
completely invertebrate-associated and currently numbers c. 2000 species.
Few systematic studies have been made in the tropics and undoubtedly many
new species of Laboulbeniales are waiting to be found here (Tavares, 1985; Weir
and Hammond, 1997). A significant limit to further taxonomic work on this
order is the present lack of culturability and opportunities for in vitro study.
Invertebrate-pathogenic Fungi from the Tropics 135
With increasing emphasis on the use of molecular phylogenetics in many

areas of systematics, this is a hindrance. However, this need not be limiting
in the future, as techniques are available for securing DNA from minute
samples, albeit at a cost. Further, it must surely be only a matter of time before
efficient and routine methods are devised by which Laboulbeniales can be
cultured in vitro, providing DNA in larger amounts.
A second, similarly species-rich ‘hot spot’ is the Septobasidiales
(Basidiomycota). These are wholly restricted to one insect order, Homoptera,
and are furthermore restricted to the immobile larval stages of scale insects.
However, the large number of species recorded for the Septobasidiales is an
indication of their restricted host ranges. As with the Laboulbeniales, absence
of routine methods of isolation is currently a hindrance to further work on
this order. The monograph of Couch (1938) still stands as a defining text
on the subject. Although tropical to subtropical in distribution, there has been
no modern systematic study of the order. This is long overdue.
A third ‘hot spot’ is the Entomophthorales (Zygomycota), which stands out
as an order with many invertebrate pathogens. The name of the order means
‘insect destroyer’. Most of the work on these has been from temperate (specif-
ically North American and European) regions and, again, little systematic
study has been done in the tropics. Evans (1982) implied diversity of
Entomophthorales was low in the tropics ‘in contrast to temperate habitats
where Entomophthora species (Entomophthorales; Zygomycota) predominate’.
There is, however, increasing evidence that the Entomophthorales are more
numerous in the tropics than previously considered. Continuous study in
Thailand shows that members of this order are found in the early morning,
and there is every indication that death of an insect and sporulation are con-
fined to the night and early morning, when temperatures are lowered and
humidity high. This contrasts with temperate regions, where sporulation can
occur over a period of days under favourable circumstances, making it more
apparent. A full survey of the Entomophthorales in natural forests in the tropics
is needed. Also within the Zygomycota, the Trichomycetes is a fourth ‘hot spot’
that has received comparatively little attention in the tropics. Like the
Laboulbeniales, these are external parasites albeit in the gut. They do not cross
the cuticle barrier and invade the haemolymph.
The ‘hot spot’ that has received by far the most attention is that within
the Hypocreales, especially the Clavicipitaceae. It is this group that will be
covered in further detail. Although most work has been done on the
Clavicipitaceae (especially in the tropics), this chapter must still highlight what
little is known. By inference, knowledge of the other four ‘hot spots’ in the
tropics will be seen to lag further behind the Clavicipitaceae.
These five ‘hot spots’ account for c. 3000 of the currently accepted 75,000
fungal species known, i.e. c. 4% of total fungal species diversity described
for the planet. However, there has been a disproportionately higher level of
study on ‘plant’-associated fungi compared with ‘invertebrate’-associated
fungi. If the Hawksworth (1991) figure is correct then ‘invertebrate’ fungi

may, in the future, account for c. 60,000 species, assuming they continue to
represent 4% of total fungal biodiversity.
It is significant that all estimates of fungal biodiversity are currently based
on plant–fungus ratios. In the future, fungus–invertebrate ratios may provide
a more reliable estimate of overall fungal biodiversity. However, with both
groups, fungi and invertebrates, the current problem is that there is no ade-
quate approximation of either total. By contrast, there are well-established
estimates of plant biodiversity, which are likely to prove accurate to within
5–10%. The conclusion is that invertebrate-pathogenic fungi probably
account for more than 4% of fungal biodiversity and may account for a sig-
nificant proportion of the hidden fungal diversity.
The Clavicipitaceae: a Significant ‘Hot Spot’ for Invertebrate

Pathogens
Mycologists rely upon Ainsworth and Bisby’s Dictionary of the Fungi (Kirk et al.,
2001) as a rough guide to numbers. However, there is every indication that
the estimates in the Dictionary are grossly below the actual figures for many
major groups. The Dictionary records 100 species of Cordyceps and ten of
Torrubiella. However, Kobayasi (1982) listed 282 Cordyceps and 59 Torrubiella.
Similarly, the Dictionary records 30 Hypocrella, whereas Evans and Hywel-
Jones (1990) recorded 38. Significantly, there are many instances where an
anamorph is known but no association has yet been made with a teleomorph
(Hywel-Jones, 1996, 1997b; Hywel-Jones et al., 1998). In Thailand to date,
286 taxa of invertebrate-pathogenic fungi have been recorded. Of these, 257
taxa are Clavicipitaceae with the remainder being either Entomophthorales or
non-clavicipitaceous Hypocreales. Of the Clavicipitaceae, 114 taxa (44%) are
teleomorphs. Significantly, of the 143 clavicipitaceous anamorphs recorded,
only 55 (38%) have been linked with teleomorphs.
In the field there are instances where teleomorphs and anamorphs are
regularly seen together. The genus Hypocrella often has an Aschersonia
state present either within the same stroma or within the same population of
stromata on a leaf: e.g. Hypocrella raciborski Zimm. – Aschersonia placenta B. &
Br.; Hypocrella discoidea (B. & Br.) Sacc. – Aschersonia samoensis P. Henn; and
Hypocrella javanica (Penz. & Sacc.) Petch – Aschersonia coffeae P. Henn. Of 12
species of Hypocrella from Thailand, ten spontaneously produced an
Aschersonia state in culture. However, Hypocrella schizostachyi P. Henn. and
Hypocrella scutata (Cooke) Sacc. did not produce an Aschersonia state in cul-
ture or in the field, calling into question whether these are best placed in this
genus. Hypocrella schizostachyi, in particular, is of questionable placement, as
it produced anamorphs in culture with affinities to Nectria (Hywel-Jones and
Samuels, 1998). Although members of the genus Hypocrella sensu stricto
appear to be the most recently evolved of invertebrate-pathogenic

Clavicipitaceae (Artjariyasripong et al., 2002), our observation that H.
schizostachyi produces a ‘nectriaceous’ anamorph suggests this could be more
ancestral within the Clavicipitaceae.
Although there does not seem to be a significant temporal component to
the presence of teleomorph and anamorph states in Hypocrella, this is not
the case for Cordyceps. Many species of Cordyceps recorded from Thailand are
found first as the anamorph, while the teleomorph develops over 4–8 weeks.
Notable examples of this development include: Hymenostilbe nutans Samson &
Evans and Cordyceps nutans Pat. (Hywel-Jones, 1995b); Hirsutella brunnea-
punctata Hywel-Jones and Cordyceps brunneapunctata Hywel-Jones (Hywel-
Jones, 1995c).
Are the Origins of the Clavicipitaceae Tropical?

Undoubtedly, the diversity of the invertebrate-pathogenic Clavicipitaceae is
greater in the tropics than in temperate regions (Evans, 1982). Six man-hours
of searching in tropical forest in Thailand usually produces 25–30 species.
By comparison, the same collecting in temperate forest in Japan produced
10–15 species. Rogerson (1970) reviewed the Hypocrealean fungi and con-
cluded, based on morphology, that the clavicipitaceous genera could stand as
a separate order – the Clavicipitales. Later molecular phylogenetic studies,
however, did not support the clavicipitaceous fungi as a separate order but
placed them within the Hypocreales (Spatafora and Blackwell, 1993; Rehner
and Samuels, 1995). These and further molecular studies (Nikoh and Fukatsu,
2000; Artjariyasripong et al., 2002) supported the view that the Clavicipitaceae
are monophyletic within the Hypocreales, with other hypocrealean families
apparently basal to the Clavicipitaceae. By examining all the genera considered
by Rogerson (1970) and by Kirk et al. (2001), the conclusion can be drawn
that the majority are wholly tropical in distribution. Within the invertebrate-
pathogenic genera the two largest genera, Cordyceps and Torrubiella, have
many tropical species, while smaller genera such as Atricordyceps,
Cordycepioideus, Hyperdermium and Hypocrella are either wholly tropical or
tropical/sub-tropical in their distribution (Petch, 1921b; Blackwell and
Gilbertson, 1981; Samuels, 1983; Sullivan et al., 2000).
These considerations beg the question: what is the ancestor of the
Clavicipitaceae? There are invertebrate pathogens within the Hypocreaceae
and these also are tropical. Notably, in Thailand, Cosmospora diploa (Berk. &
M.A. Curtis) Rossman & Samuels has been reported from scale insects
(Homoptera). Petch (1921a) described many species of hypocrealean
fungi from ‘scale insects’ and commented on the extent of the bambusicolous
nature of many of these associations. A hypothesis (Hywel-Jones’ basis for
Artjariyasripong, 1999) was that Hypocrella (wholly tropical in distribution)
was ancestral for the Clavicipitaceae, with Torrubiella and Cordyceps evolving
after radiation from homopteran scale insects into other insect orders and,
ultimately, other invertebrates, such as spiders, as well as into other fungi.
Recent studies involving species from all three genera suggest, however, that
Cordyceps is basal to the clade, with Hypocrella being the most recently derived
(Artjariyasripong et al., 2002). The problem is clearly more complex than hith-
erto expected. There is natural interest in inter-kingdom host jumping (Nikoh
and Fukatsu, 2000) and it is here posited that the scale insects (largely trop-
ical in distribution) hold the key to understanding the evolutionary develop-
ments within the Clavicipitaceae. Furthermore, given the observations of Petch
(1921b) and of work in Thailand (Hywel-Jones, 1997a), a significant role for
bambusicolous scale insects and other invertebrates can be considered, espe-
cially as there are many plant-pathogenic genera of Clavicipitaceae that are
found only on tropical bamboo (Rogerson, 1970).
As the DNA of more of these fungi is sequenced, it is expected that many
of these questions will be answered in the next few years and many new ones
posed. The tropical invertebrate-pathogenic Clavicipitaceae offer interesting
problems for study. At the morphological level Rogerson (1970) was able to
demonstrate that there were enough features of clavicipitalean morphology
to separate them from the Hypocreales as an order in their own right. Molecular
evidence does not support this view. It is suggested here that the switch
to invertebrate hosts has forced the Clavicipitaceae to evolve morphological
characteristics that aid their new ‘lifestyle’ and make them stand out from
their relatives – the Hypocreales. Is a new order developing?
The Clavicipitaceae as Biocontrol Agents
Most of the tropical invertebrate-pathogenic Clavicipitaceae have highly

evolved associations with their hosts. Consequently, it appears that the teleo-
morph has been largely retained in most species. A few species, however, have
made the transition to being ‘broad-band’ pathogens. Consequently, most of
the literature on clavicipitaceous invertebrate pathogens is concerned with a
few species that are commonly encountered in agricultural ecosystems. Most
important among these are:
• Beauveria bassiana – pan-global, with a wide host range in the Insecta
• Metarhizium anisopliae – pan-global, limited to Insecta and with a wide host
range
• Verticillium lecanii (Zimm.) Viégas – pan-global, a wide host range within
and without the Insecta (also infects Araneae and Fungi)
• Nomuraea rileyi (Farlow) Samson – pan-global, restricted to Lepidoptera
within Insecta
• Hirsutella citriformis Speare – pan-tropical, restricted to a single order,
Homoptera, and most common on Delphacidae within Insecta
All five of the above species have been considered for use as biocontrol agents.
Four of the five genera are encountered as the anamorph with teleomorphs
either only anecdotally known or very rare. The exception is Hirsutella,
where strong relationships with Cordyceps are known. Although H. citriformis
has no known teleomorph described, recent cultural and morphological
studies with tropical isolates of H. citriformis and H. saussurei (Cooke) Speare
suggest a close relationship between the two. The teleomorph of
H. saussurei is Cordyceps humberti Robin, which has close morphological
affinities with C. unilateralis (Tull.) Sacc. from ants. Future molecular studies
on these should clarify their relationships.
For Nomuraea, there is a teleomorph recorded for N. atypicola (Yasuda)
Samson – Cordyceps cylindrica Petch (Evans, 1982; Hywel-Jones and Sivichai,
1995). However, the relationship of N. atypicola to N. rileyi is in doubt and no
teleomorph has yet been reported for N. rileyi (O. Boucias, Bangkok, Thailand,
2001, personal communication). More than 2500 man-hours of survey work
in natural forest in Thailand has failed to find N. rileyi and yet it can be com-
monly collected in agricultural ecosystems in Thailand. Although N. rileyi is
now pan-global, it is possible that its centre of origin is not South-East Asia
but that it has been imported to Thailand as a pathogen of crop pests over
time. The alternative is that it is present in natural forest, but is rare and has
yet to be found.
Within Beauveria, a teleomorph, Cordyceps brongniartii Shimazu, has been
described for B. brongniartii (Sacc.) Petch (Shimazu et al., 1988) and has
recently been reported from Thailand (Hywel-Jones, 2002). Metarhizium
anisopliae has received much attention as a potential biocontrol agent.
Significantly, this also has not had a teleomorph reliably reported. Recently, a
Metarhizium state was described as the anamorph of Cordyceps taii Liang et
Liu (Zongqi et al., 1994). M. anisopliae has a worldwide distribution and has
been recorded from a wide range of hosts in many insect orders. However, the
continued reporting of Metarhizium species associated with Cordyceps from
sub-tropical southern China hints at this area being the centre of origin for
Cordyceps spp. producing a Metarhizium state. A Cordyceps–Metarhizium com-
bination is occasionally found in Thai forest. Further work is needed to
determine if China/South-East Asia is the centre of origin for Metarhizium-
producing Cordyceps.
In anamorph genera, where there has been a move out of natural forest
and into agricultural ecosystems, it is notable that these contain few species.
Nomuraea has three species, Metarhizium c. five species and Beauveria also c.
five species. Variation is apparently intraspecific. An exception is the genus
Hirsutella which is linked with many Cordyceps species and is an important
part of the life cycle. As originally conceived by Patouillard (1892) and
recognized by Speare (1920), the genus was a sound one. The genus Hirsutella
is now in need of revision. Evans (in Hawksworth, 1991) speculated that each
species of thrips may have its own unique Hirsutella species. Other anamorph
genera linked with Cordyceps are equally rich in species despite a poor record
of research on them. For example, Hymenostilbe (mostly tropical in distribu-
tion) is only dealt with in < 20 papers and yet it contains c. ten published
species with many more awaiting description (Samson and Evans, 1975;
Hywel-Jones, 1995a,b,d).
It must be presumed that the few (compared to those that have been
recorded from natural forest) agricultural invertebrate pathogens have
migrated either from natural forest to the agricultural ecosystem or have
been imported from elsewhere by man to Thailand. More than ten species
of Aschersonia have been recorded from natural forest in Thailand while only
one species, A. placenta, has been found in agricultural ecosystems.
Significantly, the Hypocrella state of A. placenta has apparently not made
the transition from forest to agricultural ecosystem. In conclusion, few
invertebrate-pathogenic Clavicipitaceae have moved from the forest to agricul-
tural ecosystems. Most fungi being assessed for biocontrol originate from
isolates taken from agricultural ecosystems. The potential for new biocontrol
agents from natural forest is vast.
Cordyceps from an Ethnomycological Point of View
Although several species of Cordyceps are used as herbal medicines, the most
famous is the Chinese Cordyceps, C. sinensis Berk. (Sacc.). This made the
popular press in the mid-1990s with news that it was one of the ingredients
in tonics used by Chinese long-distance runners (Pegler et al., 1994;
Steinkraus and Whitfield, 1994). A casual search of the Internet reveals thou-
sands of ‘hits’ for the genus Cordyceps. The majority of these refer to the medic-
inal properties of C. sinensis. Apart from C. sinensis, which is popular
throughout East Asia, other species that have been developed into herbal med-
icines include: Paecilomyces tenuipes (Peck) Samson in Korea (R.A. Samson,
Bangkok, Thailand, 1999, personal communication), Paecilomyces cicadae
(Miquel) Samson in Korea, Japan and China and Cordyceps militaris (L.:Fr.)
Link in Japan.
Despite the many species of Cordyceps (and related anamorphs) recorded
from Thailand, there is no example of a Thai Cordyceps being used as a herbal
medicine. The probable reason is that, while species diversity is high in the
tropics, there are few examples where large numbers of stromata can be har-
vested on a regular and predictable basis, but the increasing interest in the
west in ‘alternative herbal medicines’ from the east is driving research into
the therapeutic values of these eastern herbals. This has also resulted in
increased interest in invertebrate-pathogenic Clavicipitaceae as sources of novel
secondary metabolites.
Tropical Invertebrate-pathogenic Clavicipitaceae as Sources

of Novel Secondary Metabolites
The invertebrate-pathogenic Clavicipitaceae are not easy to find. In Thailand,
the author could locate c. 70–200 specimens (representing 15–30 species)
in a 4-day survey and secure c. 40–80 isolates representing 10–15 species. A
similar survey in temperate Japan produced c. 70 isolations (21 species) from
226 specimens (40 species) collected in 7 days.
However, the Hypocreales (especially the Clavicipitaceae) and Eurotiales
are two closely related orders that have proved to be important sources of
secondary metabolites used in the pharmaceutical industry (Rossman, 1996).
Significantly, few species have had their novel secondary metabolites studied.
However, the extensive programme of isolating insect fungi from Thai forest
has now resulted in a large number of species being available for culture. Seifert
et al. (2000) concluded that ‘the importance of fungal cultures is increasing’.
The pharmaceutical industry still has a mentality of preferring to work
with easily acquired, easily handled and easily maintained organisms. In the
early days, this was achieved by working with fungi isolated from soil samples.
Increasingly, industry goes through trends of interest in different organisms.
Recent examples have included marine fungi, endophytes and xylariaceous
fungi. It is as if a seam is mined before moving on to a new one. Similarly,
there are organism trends, e.g. fungi versus actinomycetes. Recently, novel
secondary metabolite discovery has also had to compete with combinatorial
chemistry, which has been made possible with the advent of powerful
computing systems.
The random discoveries of combinatorial chemists cannot possibly
compete in the long run with the organized natural selection of which
mankind is also an integral part. The author fully expects that the important
‘drug leads’ of the future will continue to be found from nature’s great diver-
sity. Four billion years of genetic evolution will continue to supply the drugs
of the future. Combinatorial chemistry and 40 years of ‘genetic modification’
will merely help to fine-tune the process. For this to occur, the greatest diver-
sity of genes must occur where there is greatest species diversity, assuming
that general genetic diversity equates to metabolic diversity. The tropics pro-
vide that diversity. Within the animal kingdom, Insecta also provide high
genetic diversity, and within the fungal kingdom it is likely that the tropical
invertebrate-pathogenic fungi will provide a great level of diversity (Bills et al.,
Chapter 11, this volume).
Conclusions
In the future, if we are to obtain a reasonable estimate of global fungal bio-

diversity, more attention must be paid to the tropical invertebrate-pathogenic
fungi. With their rich diversity, the tropics are expected to provide a large pool
of material for future research. The clavicipitaceous fungi have long been
considered as a source of biocontrol agents. More recently, they have been
considered to be a source of novel metabolites and, if industry is prepared to
invest some time, energy and money into a detailed investigation that goes
beyond the routine, then it is anybody’s guess what metabolites of benefit to
mankind might come from tropical invertebrate Clavicipitaceae.
Acknowledgements
The considerable support of BIOTEC over the years is thanked for presenting
the opportunity to conduct this work. Recent grants from the Biodiversity
Research Training Programme have helped in this regard. Especially, it is a
pleasure to thank Drs Sakarindr Bhumiratana and Morakot Tanticharoen.
Significant discussions have been had over the last few years with colleagues
who share an interest in the Clavicipitaceae; Harry Evans, Julian Mitchell, Rob
Samson, Gary Samuels, Keith Seifert, Joey Spatafora and Jim White have been
especially, significant ‘sounding boards’. I would particularly wish to record
my deepest gratitude for the support over the years from the late Stephen Moss.
The Clavicipitaceae are not easy to find and it is a pleasure to thank Somsak
Sivichai and, especially, Rungtip Nasit for locating many of the species which
my tired eyes now pass over. JISTEC provided the opportunity to compare and
contrast Japanese fungi with those from Thailand. Lastly, it is a pleasure to
thank Roy Watling for the support he provided to get this work completed.
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Tropical Mycoses: Hazards to 10
Travellers
E.G.V. EVANS* AND H.R. ASHBEE
Mycology Reference Centre, Division of Microbiology, University of

Leeds, Leeds LS2 9JT, UK and General Infirmary, Leeds LS1 3EX, UK
Introduction
Fungi are now recognized as an important cause of disease in humans. Some

of these fungi lie in wait for tropical hitch-hikers.
The incidence of fungal disease has increased markedly over the last
decade. Infections of skin, hair, nail and mucous membranes affect up to 20%
of the population. Potentially fatal deep-seated infections have increased,
mainly because of the larger numbers of people with a defective immune
system (immunocompromised), for example, transplant patients and those
with acquired immune defiency syndrome (AIDS). Some fungal infections
occur mainly in tropical or sub-tropical parts of the world and, in general,
people from temperate zones, particularly Europe, have little immunity to
them. Increased leisure and business travel to these exotic tropical locations
have resulted in a higher prevalence of unusual fungal diseases.
In general, skin infections are caused mainly by ringworm fungi (der-
matophytes) and several types of ringworm (tinea) are specific to tropical areas,
often producing different lesions from those seen in temperate zones. Some of
these are animal ringworms, for example, from monkeys. In addition to these
superficial diseases, there are large numbers of fungi in soils and on plant debris
in tropical countries that are capable of infecting tissues beneath the skin,
including bone, causing disfiguring and debilitating conditions. These infec-
tions include mycetoma, chromoblastomycosis and sporotrichosis, all of which
*Present address: Department of Medical Microbiology, University of Wales College of

Medicine, Heath Park, Cardiff CF14 4XN, UK.

146 E.G.V. Evans and H.R. Ashbee
tend to affect the limbs. All are caused by the introduction of fungi through
unprotected skin into the underlying tissues by thorns or splinters. The last and
most serious group of diseases is the deep-seated infections, which are fre-
quently fatal. As the name suggests, these can affect any or many organs of
the body, although they usually begin by inhalation of fungal spores into the
lungs. A lung infection then develops, which in some cases spreads to other
organs. Diseases such as histoplasmosis and coccidioidomycosis are included
in this group. Both are restricted to hot climates where the causal fungi are
present in the soil, but a surprising number of cases of infection are seen in
Europe in people who have visited the disease areas. Immunocompromised peo-
ple, such as those with AIDS, are especially vulnerable. Histoplasmosis, for
example, is acquired from the droppings of birds and bats and so presents a
particular threat to those tropical hitch-hikers who also enjoy hanging around
in dark caves! The types of fungal infections are reviewed below.
Fungal Diseases of Humans
About 180 species of fungi are recognized as able to cause disease (mycosis) in
humans and animals. Most of these are moulds, but there are a number of path-
ogenic yeasts and many are dimorphic. The dimorphic fungi usually assume the
mould form when growing as saprotrophs in nature and the yeast form when
causing infection; both morphological forms can be induced in the laboratory by
varying the culture conditions. Some fungi, for example, the systemic pathogens
Histoplasma capsulatum Darling and Coccidioides immitis Rixford & Gilchrist, are
highly pathogenic and are capable of establishing an infection in all exposed indi-
viduals. Others, such as Candida and Aspergillus species, are opportunist
pathogens, which in general cause disease only in immunocompromised hosts.
The form and severity of infections often depend on the degree of exposure to the
fungus and the extent to which the person is immunocompromised.
Most fungal infections are caused by fungi which grow as saprotrophs
in the environment. Some yeasts are commensals of humans and cause
endogenous infections when there is some imbalance in the host. Only ring-
worm infections are truly contagious. Many fungal diseases have a worldwide
distribution, but some are endemic to specific geographical regions, usually
because the causal agents are saprotrophs that are restricted in their distri-
bution by soil and climatic conditions.
Superficial mycoses
Ringworm
Diseases of the skin, hair and nail are the most common of all fungal infec-
tions and have a worldwide distribution. They are caused by a group of closely
Tropical Mycoses 147
related mould fungi called dermatophytes that have the ability to colonize and
digest keratin. Ringworm infections occur in both humans and animals.
Ringworm infections are also referred to as dermatophytoses.
There are about 20 species of dermatophyte fungi that cause ringworm
and they are grouped into three genera, Trichophyton, Microsporum and
Epidermophyton (Table 10.1). A number of dermatophytes are primarily ani-
mal pathogens that may also infect humans (Table 10.2), and some species
are restricted to or are more common in certain parts of the world. Ringworm
infections are spread by direct or indirect contact with an infected individual
or animal. The infective particle is usually a fragment of keratin containing
viable fungus. Indirect transfer may occur via the floors of swimming pools
and showers or on brushes, combs, towels and animal-grooming implements.
In addition to exposure to the fungus, some abnormality of the epidermis,
such as slight peeling or minor trauma, is probably necessary for the estab-
lishment of infection.
In the industrialized countries, ringworm of the scalp accounts for only
a small proportion of infections, although it is on the increase in Europe.
However, the use of communal bathing facilities has resulted in a consider-
able increase in the incidence of foot ringworm (Fig. 10.1) and associated nail
(Fig. 10.2) and groin infections. The predominant cause of these infections is
Trichophyton rubrum (Castell.) Sabour., which is responsible for c. 75% of all
ringworm infections diagnosed in temperate zones. In developing countries,
Table 10.1. Common dermatophyte pathogens of humans.
Common site(s) Main area of

Species of infections distribution
Epidermophyton floccosum
(Harz)
Langeron & Milchevitch Groin, feet (nail) Worldwide
Microsporum audouinii
Gruby Scalp (body) Africa, America and
Europe
M. ferrugineum Ota Scalp (body) Africa, Balkans and Asia
Trichophyton mentagrophytes
var. interdigitale (Robin)
Blanchard Feet (nail, groin) Worldwide
T. concentricum Blanchard Body South Pacific
T. rubrum (Castell.) Sabour. Feet, nail, groin, body Worldwide
T. schoenleinii (Lebert)
Langeron & Milochevitch Scalp (body, nail) Eurasia and North Africa
T. soudanense Joyeux Scalp (body) Africa
T. tonsurans Malmsten Scalp, body (nail) Europe and America
T. violaceum Sabour. Scalp, body (nail) Africa and Eurasia
Parentheses in second column indicate secondary sites of infection.
Table 10.2. Common dermatophyte species of animals.
Animals commonly Main area of

Species affected distribution
Microsporum canis Bodin Cat, dog Worldwide

M. distortum Di Menna
& Marples Cat, dog Australasia, USA
M. nanum Fuentes Pig Worldwide
M. persicolor (Sabour.)
Guiart & Grigor. Bank field voles Europe
Trichophyton mentagrophytes
var. mentagrophytes (Robin)
Blanchard Rodents (horse, cat, dog) Worldwide
T. equinum (Matr. &
Dassonville) Gedoelst Horse Worldwide
T. erinacei (Smith et
Marples) Padhye & J.Carm. Hedgehog UK, New Zealand
T. quinckeanum (Zopf)
MacLeod & Muende Mice Europe, North America
T. simii (Pinoy) Stockdale,
Mackenzie et Austwick Monkey, chicken India
T. verrucosum Bodin Cattle Worldwide
Fig. 10.1. Athlete’s foot caused by Trichophyton rubrum.

Fig. 10.2. Destruction of nail due to Trichophyton rubrum infection.
Fig. 10.3. Extensive body lesions due to cattle ringworm (Trichophyton

verrucosum Bodin).
Fig. 10.4. Ringworm infection of the scalp caused by Trichophyton violaceum.
particularly in warm climates, body (Fig. 10.3), scalp (Fig. 10.4) and groin
infections predominate, with T. rubrum and T. violaceum Sabour. apud Bodin
among the most common causes, and visitors to these regions are exposed to
these fungi. There is no evidence of natural immunity to ringworm, but scalp
ringworm is predominantly a disease of children and foot ringworm a disease
mainly of adult males.
In culture, many dermatophyte species produce two types of asexual
spore, macroconidia and microconidia. Classification into the three genera
Trichophyton, Microsporum and Epidermophyton is based on the morphology of
the macroconidia, although the identification of species is also based on the
shape and disposition of the microconidia and the macroscopic appearance
of the colony.
Ringworm lesions vary considerably according to the site of the infection
and the species of fungus involved. Sometimes there is only dry scaling, but
more commonly there is irritation, reddening, swelling and sometimes
vesicles. More inflammatory lesions with weeping vesicles, pustules and ulcer-
ation are usually caused by animal species of dermatophyte. In skin infections
of the body, face and scalp, spreading circular lesions with a raised, inflam-
matory border are produced. It is the appearance of these lesions that led to
the name ringworm. In scalp infections there is scaling and hair loss, the
extent of which depends on the causal fungus. Some species from animals
cause a highly inflammatory, raised pustular lesion called a kerion. It is impor-
tant that scalp ringworm is recognized and treated promptly since it can lead
to scarring and permanent hair loss.
Ringworm infections may be reliably diagnosed in the laboratory by direct
microscopical examination and culture of skin, crusts, hair and nail.
Microscopy of wet-mounts of keratinous material in potassium hydroxide is
simple and reliable. Dermatophytes are seen in skin and nail as branching,
septate hyphae that often fragment into vegetative spores (arthroconidia) (Fig.
10.5). For culture, small fragments of infected human material are scattered
on Sabouraud glucose agar and incubated at 27–30°C for up to 3 weeks.
Topical therapy, with creams and ointments, is satisfactory for treating
most dermatophyte infections of skin, but oral antifungals are required to treat
infections of the nail and scalp. Topical agents include terbinafine and azole
compounds. Oral griseofulvin is useful for scalp, skin and fingernail infections,
but it is poor for toenail infections. The newer oral antifungals such as
terbinafine and itraconazole are gradually replacing griseofulvin for the treat-
ment of all forms of ringworm, since they give much better cure rates with
much shorter periods of treatment.
Yeast infections
Candida: superficial Candida infections are very common throughout the world
although they are not especially prevalent in warm climates. These mycoses
involve the skin, nails and mucous membranes of the mouth and vagina;
infection of the mucous membranes is commonly referred to as thrush (Fig.
10.6). One species, Candida albicans (Robin) Berkhout, accounts for 80–90%
of cases. C. albicans and occasionally other Candida species are found in small
Fig. 10.5. Dermatophyte mycelium and arthroconidia seen in skin (KOH mount).
Fig. 10.6. Florid Candida infection of the mouth showing typical white plaques.
numbers in the commensal flora (mouth, gastrointestinal tract, vagina and

skin) of about 20% of the normal population. The carriage rate tends to increase
with age and is higher in the vagina during pregnancy. Chronic, intractable
oropharyngeal candidosis is common in AIDS patients (< 100%) and may
extend to give infection of the oesophagus. The appearance of this infection is
often the indicator of the transition from HIV-positive to full-blown AIDS.
However, in developed countries, where the newer, so called ‘triple-therapy’ is
available for treatment of HIV infection, the prevalence is much lower (10%).
Pityriasis versicolor: this is a mild, chronic infection of the stratum
corneum that produces a patchy discoloration of the skin and is caused by
yeasts of the genus Malassezia, species of which are common members of the
normal skin flora, and most infections are thought to be endogenous.
Development of the disease is probably related to host or environmental fac-
tors. It is very common in the tropics and it is most prevalent in young adults.
There is no evidence that it is contagious. The organisms are dimorphic and
both yeasts and short hyphae are associated with pityriasis versicolor. The
lesions of pityriasis versicolor are non-inflammatory; scaling is usually present
on the upper trunk or neck and in white-skinned people are brownish in
colour, whereas in dark-skinned individuals they appear as light-coloured
patches (Fig. 10.7a,b).
Malassezia species require lipids for growth, and media containing Tween
and lipid supplements have been developed specifically for growing the
organism. Malassezia has also been associated with conditions such as
dandruff and seborrhoeic dermatitis, where only yeasts that characteristically
produce buds on a broad base are seen.
Diagnosis of yeast infections can be confirmed reliably by demonstration
(a)
(b)
Fig. 10.7. Pityriasis versicolor showing (a) hyper-pigmented and (b) hypo-pig-
mented lesions. Photographs courtesy of Dr D.T. Roberts.
of the organism in skin scales or mouth/vaginal swab smears by microscopy

and culture. For pityriasis versicolor, culture is unnecessary. In the tissues,
Candida appears as budding yeast cells, often with mycelium, and Malassezia
as numerous clusters of round yeast cells along with short, stout hyphae.
Candida infections and pityriasis versicolor respond well to treatment with

antifungal creams. Oral therapy, with either fluconazole or itraconazole, is
sometimes needed for widespread infections. Relapse of pityriasis versicolor is
common, particularly in hot climates.
Other superficial infections

Skin and nail: certain moulds (non-dermatophyte) may cause infection of skin
and nail. It is important that these are recognized since they are often resist-
ant to the agents used to treat ringworm and superficial candidosis.
In the UK, non-dermatophyte moulds cause about 5% of fungal nail infec-
tions. Scopulariopsis brevicaulis Bainier, a ubiquitous saprotroph of soil, is the
most common, although other saprotrophic moulds, such as Fusarium,
Aspergillus and Penicillium species, are also occasionally implicated. Infection
usually follows trauma.
Scytalidium dimidiatum (Penz.) B. Sutton & Dyko, a pathogen of fruit trees
and a soil fungus, can cause nail and skin infection among those in tropical
areas, and visitors to these areas will be exposed to this fungus. Scytalidium
infections are diagnosed by microscopy and culture, but they are not amenable
to treatment with any of the currently available antifungals.
Tinea nigra: this is a superficial, asymptomatic skin disease characterized
by pigmented brown areas, usually on the palms and soles. It is caused by a
black mould, Exophiala werneckii (Horta) v. Arx, and occurs mainly in the
tropics. Tinea nigra is not contagious but is contracted by contact with
the fungus in soil. Again, those in the endemic areas will be exposed to this
fungus. However, this infection responds well to treatment with antifungal
creams.
Mycotic keratitis: fungal infections of the cornea of the eye are usually
secondary to injury. They are caused by moulds that occur commonly as
saprotrophs in soil or on plants, in particular species of Aspergillus and
Fusarium. These infections are serious and any splinter, thorn or soil in the
eye that develops into a lesion requires medical attention immediately.
Treatment is with topical antifungal agents, in particular natamycin.
Subcutaneous mycoses
Mycoses of the skin, subcutaneous tissues and bone occur mainly in the tropics
and subtropics. They result from the inoculation of saprotrophic fungi from soil
or decaying vegetation into the subcutaneous tissue. The principal subcutaneous
mycoses are mycetoma, chromoblastomycosis and sporotrichosis. Anyone visit-
ing the endemic areas is exposed to the fungi that cause these diseases.
Mycetoma
This is a chronic infection of the skin, subcutaneous tissues and bone that is
characterized by deep abscesses, with pus draining to the surface through

channels in the tissue. It most often affects the foot or the hand. One of a
number of different actinomycetes (actinomycetoma) or moulds (eumyce-
toma) can cause it. The disease is most prevalent in tropical and subtropical
regions of Africa, Asia and Central America. Infection results from inocula-
tion of the organism into the subcutaneous tissue from soil or plant sources,
usually on thorns or splinters. Consequently, infection occurs most frequently
in those who have close contact with soil. A number of organisms have been
implicated in this disease, including the moulds Madurella, Exophiala,
Acremonium, Pseudallescheria, and the actinomycetes Actinomadura, Nocardia
and Streptomyces. The organisms form compacted colonies (grains) < 2 mm
across in the tissues, the colour of which depends on the organism responsi-
ble; for example, Madurella grains are black and Actinomadura pelletieri
(Laverau) Lechevalier & Lechevalier grains are red.
Mycetoma usually takes the form of localized swollen lesions which are
usually found on the limbs. The foot is often involved (Fig. 10.8) and the infec-
tion can spread up the long bones of the leg. There is often a long period
between the initial infection and formation of the characteristic lesions.
Diagnosis is made by examination of pus or biopsy material. The grains are
usually visible to the naked eye and their colour may help to identify the
organism responsible. Microscopical examination enables differentiation
between actinomycetoma and eumycetoma (Fig. 10.9). This is important since
actinomycetoma responds well to antibiotics (e.g. rifampicin in combination
with sulphonamides or co-trimoxazole), but eumycetoma does not respond to
antifungal treatment, and amputation of the foot or limb is usually necessary.
However, some of the newer antifungals have yet to be properly evaluated in
treating this condition.
Chromoblastomycosis
This disease is a chronic disease of the skin and subcutaneous tissues,
characterized by crusted, warty lesions, usually of the limbs (Fig. 10.10). It
is mainly encountered in the tropics and can be very disfiguring. The principal
causes are the dematiaceous moulds Fonsecaea pedrosoi (Brumpt) Negroni, F.
compacta Carrion, Phialophora verrucosa Medlar and Cladosporium carrionii
Trejos. Like mycetoma, the disease is seen most often in rural areas. Diagnosis
is straightforward, as the dark-coloured fungal elements are easy to see on
microscopical examination of skin scrapings, crusts and pus, but the fungi
are difficult to identify. The disease responds well to antifungal therapy with
the newer drugs, such as itraconazole and terbinafine.
Sporotrichosis
This is a chronic, nodular infection of the skin and subcutaneous tissues in
which there are abscesses and pus. Unlike the other subcutaneous mycoses,
Fig. 10.8. Mycetoma of the foot caused by Madurella grisea Mackinnon, Ferrada
& Montemayer.
Fig. 10.9. Grain of Madurella grisea in tissues of the foot.
it can spread through the lymphatic system (Fig. 10.11). Typically, the pri-
mary lesion is on the hand with secondary lesions extending up the arm,
although in immunocompromised individuals it can spread to involve the
bones, joints, lungs and, in rare cases, the central nervous system. The dis-
ease occurs mainly in Central and South America, parts of the USA and
Africa, and Australia. The localized form responds to treatment with a num-
ber of systemic antifungals but disseminated disease is more difficult to treat.
Fig. 10.10. Chromoblastomycosis of the leg due to Fonsecaea pedrosoi with

raised, warty lesions.
Systemic mycoses
Deep-seated fungal infections generally result from the inhalation of airborne

spores produced by the causal moulds, present as saprotrophs on plant
material and in soil. Many are caused by dimorphic fungi, which assume a
yeast phase in tissues. The principal diseases are coccidioidomycosis (caused
by Coccidioides immitis), blastomycosis (Blastomyces dermatitidis Gilchrist &
Stokes), histoplasmosis (Histoplasma capsulatum) and paracoccidioidomycosis
(Paracoccidioides brasiliensis (Splendore) Almeida).
Systemic mycoses caused by opportunistic pathogens, such as Aspergillus,
Candida and Cryptococcus species, have a more widespread distribution, and
are being seen with increasing frequency in patients compromised by disease
or drug treatment. In transplant patients, for example, these fungi are among
the most frequent causes of mortality due to infection.
Systemic mycoses occur most frequently in workers in agriculture or the
construction industry, especially following disturbance of soils containing
the causal fungi. The incidence of systemic infections caused by opportunis-
tic pathogens has also increased with developments in medical and surgical
Fig. 10.11. Sporotrichosis of the arm with a chain of ulcerated lesions due to
lymphatic spread of the organism (Sporothrix schenckii Hektoen & Perkins).
practice and with the increased number of immunocompromised individuals,

e.g. neutropenic and AIDS patients.
Cryptococcosis
This is caused by the encapsulated yeast Cryptococcus neoformans (Sanfelice)
Vuill., and is most frequently manifested as a disease of the central nervous
system, although the primary site of infection is the lung. It occurs sporadi-
cally throughout the world, but it is most frequently associated with patients
suffering from AIDS.
Four serotypes of C. neoformans can be differentiated (A, B, C, D) which rep-
resent two varieties of the organism, namely, C. neoformans var. neoformans (A,
D) and C. neoformans var. gattii Vanbr. & Takashio apud DeVroey & Gattii (B, C).
The majority of infections are caused by var. neoformans, which is found in large
quantities in the excreta of wild and domesticated birds throughout the world.
C. neoformans var. gattii causes infection in warmer climates. It is associated with
the flowers of the Red River gum tree (Eucalyptus camaldulensis Dehnh.) and so
infections caused by this variety coincide with the distribution of this tree.
Inhalation of C. neoformans may lead to a mild, self-limiting pulmonary

infection, which is the most common form. Symptomatic pulmonary infec-
tion causes the formation of small nodules that heal leaving scar tissue, or
enlarge to form a chronic cryptococcoma. The meningeal form of the disease
may also occur in healthy individuals, but is most commonly seen in people
with cell-mediated immune defects. It develops as a headache, low-grade fever
followed by changes in mental state, weight loss, visual disturbances and ulti-
mately coma. Unless cryptococcal meningitis is treated, it is uniformly fatal.
Lesions may occur at other sites, including the skin, mucosa, major internal
organs and bone.
Diagnosis of cryptococcosis is relatively straightforward. The yeast can
often be seen in direct microscopy of cerebrospinal fluid (CSF), sputum or pus
as cells of 4–10 µm diameter. In unstained wet preparations of CSF, the
capsule surrounding the yeast may be visualized by the addition of a drop of
India ink, which is excluded by the capsule, with the resultant appearance
of a halo around the cell (Fig. 10.12).
Although cultures can be ‘performed’, a much quicker method for the
diagnosis of cryptococcal meningitis is the use of a latex agglutination test to
determine the presence of capsular polysaccharide material in the CSF. The
test is highly sensitive and specific and gives better results than microscopy
and culture. Over 90% of infected patients will give a positive reaction with
this test, with the highest levels seen in patients with AIDS.
Patients with the mild, self-limiting form of cryptococcosis need no treat-
ment. However, patients with meningitis require intravenous therapy with
amphotericin B and flucytosine, and AIDS patients will require lifelong main-
tenance therapy to prevent relapses.
Fig. 10.12. Yeast cells of Cryptococcus neoformans in cerebrospinal fluid. Note

the large transparent capsule.
Histoplasmosis
The causal organism is Histoplasma capsulatum. Histoplasmosis is usually an
asymptomatic or mild self-limiting pulmonary infection but there are also
chronic and acute disseminated forms of the disease. The fungus occurs in
nature in soil enriched with bird or bat droppings in specific endemic areas.
However, histoplasmosis has been reported in most countries of the world in
those who have travelled to the endemic areas, which include central and
eastern North America, particularly the Mississippi and Ohio river valleys,
Kentucky, Arkansas and Missouri. Caves in these areas are particularly likely
to be a source of infection. In the environment, H. capsulatum grows as a
mould, but in human tissue it grows as an intracellular yeast.
Manifestations of histoplasmosis vary considerably, depending on the level
of exposure and the health of the person exposed. In most cases, infections
are asymptomatic, but an acute influenza-like illness may occur if large
numbers of the organism are inhaled. Chronic pulmonary histoplasmosis may
occur in middle-aged men with pre-existing lung disease. Symptoms include
pneumonia, weight loss and night sweats, resulting in fibrosis, tissue destruc-
tion and lung cavitation, which may lead to disseminated disease or death due
to lung failure in extreme cases. Disseminated histoplasmosis is usually seen
at the extremes of age or in those with some impairment of the immune
system. Dissemination in immunocompetent people is usually indolent over
months or years, with liver, adrenal and mucosal lesions common. In
immunocompromised patients disease progression may be much more
aggressive and rapid.
Diagnosis of histoplasmosis can be carried out using several methods, but,
as none is totally reliable, several are usually undertaken simultaneously.
Culture of the organism is the ‘gold standard’, but as the organism can take
several weeks to grow, it does not give quick results. Serological detection of
either antibodies or antigen may also be useful. Antibody detection can be
carried out using complement fixation and immunodiffusion, but cross-
reaction with antibodies to other organisms may occur. A radioimmunoassay
to detect antigen has been developed but is not widely available. The treat-
ment of choice for disseminated histoplasmosis in immunocompromised
patients is intravenous amphotericin B. Itraconazole may be used in immuno-
competent patients and as maintenance therapy in AIDS patients.
A second form of histoplasmosis, called African histoplasmosis, is caused
by H. duboisii Vanbr. and is restricted to the continent of Africa. It mainly
affects the skin and underlying structures, sparing the lungs. The organism is
a variety of H. capsulatum and it is identical to it in culture but has larger yeast
cells (12–15 µm diameter) in the tissues (Fig. 10.13). Treatment is the same
as for infections with H. capsulatum.
Fig. 10.13. Histology of a lymph node showing large yeast cells of Histoplasma
duboisii.
Fig. 10.14. Cutaneous lesions caused by Penicillium marneffei in an AIDS

patient. Photograph courtesy of Dr A.J. Hamilton.
Penicillium marneffei infections

Penicillium marneffei Segretain has only recently emerged as a significant cause
of infection. It infects both immunocompetent and immunocompromised
patients in South-East Asia and people who have travelled to the region. It is
now the fourth most common cause of death in AIDS patients in this region.
P. marneffei was first identified and cultured from a bamboo rat in South
Vietnam. Initially, it appeared to be a mycological curiosity, but the rising
number of cases of infection due to this organism in patients with AIDS and
also in some healthy individuals changed that. The exact relationship between
the fungus, the bamboo rat and humans remains unknown. P. marneffei can
be isolated from some bamboo rats and their burrows, but is only rarely
isolated from soil and so its environmental reservoir is still unknown.
Infection with P. marneffei results in fever, chronic cough, anaemia, weight
loss and skin lesions which are considered to be characteristic of the disease
(Fig. 10.14). In AIDS patients, the onset of symptoms is often more intense,
particularly in children. Diagnosis of this disease is often difficult. P. marneffei
can resemble H. capsulatum in vivo as both are intracellular parasites. Culture
of the organism is the definitive diagnostic test, but confirmation of the
identity of the organism may be problematic. Serology is not yet useful for
diagnosis, a combination of diagnostic tests is therefore required. Mild cases
may respond to itraconazole, but more severe forms will require amphotericin
B. As with cryptococcosis and histoplasmosis, long-term maintenance ther-
apy is required in patients with AIDS to prevent relapse.
Other tropical mycoses
There are several other fungal infections that occur in tropical countries and
may pose a threat to travellers to endemic areas. All are caused by dimorphic
fungi. Coccidioidomycosis, due to Coccidioides immitis, is endemic to the
western states of North America, through Central America into Venezuela,
Colombia and Argentina. Blastomycosis, caused by Blastomyces dermatitidis,
is endemic to the Mid-West and south-eastern regions of North America and
many countries in Africa. Paracoccidioidomycosis, caused by Paracoccidioides
brasiliensis, is endemic to all Latin American countries, except Chile and
Nicaragua. In common with the previous infections, these diseases have a
range of presentations from asymptomatic, acute or chronic to disseminated
disease. Diagnosis in most cases is relatively straightforward, but treatment
with amphotericin B is necessary for the more serious forms of these
diseases.
Summary
In summary, potentially pathogenic fungi capable of causing a whole range

of fungal infections, from the trivial to the life-threatening, are found in soil
and vegetation in tropical climes. People who visit these areas, particularly
those whose travels bring them into close contact with nature, run the risk of
developing exotic fungal diseases. It is advisable to be aware of the risks so
that the infections can be prevented or dealt with promptly should they occur.
General reading
Ashbee, H.R. and Evans, E.G.V. (2000) Fungi and skin. Microbiology Today 27,
132–134.
Clayton, Y. and Midgley, G. (1985) Medical Mycology. Pocket Picture Guide Series.
Gower, London, UK.
Evans, E.G.V. (1997) Fungi. In: Greenwood, D., Slack, R.C.B. and Peutherer, J.E. (eds)
Medical Microbiology, 15th edn. Churchill Livingstone, Edinburgh, UK, pp.
556–576.
Evans, E.G.V. and Richardson, M.D. (eds) (1989) Medical Mycology. A Practical Approach.
Oxford University Press, Oxford, UK.
Kwon-Chung, K.J. and Bennett, J.E. (1992) Medical Mycology. Lea and Febiger,
Philadelphia, USA.
Richardson, M.D. and Warnock, D.W. (1997) Fungal Infection: Diagnosis and
Management, 2nd edn. Blackwell Scientific, Oxford, UK.
Recent and Future Discoveries 11
of Pharmacologically Active
Metabolites from Tropical Fungi
G. BILLS,1 A. DOMBROWSKI,2 F. PELÁEZ,1 J. POLISHOOK2
AND ZHIQIANG AN2
1
Centro de Investigación Básica, Merck, Sharp & Dohme de España
S.A., Josefa Valcárcel 38, 28027 Madrid, Spain; 2Merck Research
Laboratories, Rahway, NJ 07065, USA
Introduction
All major commercial medicines and agrochemicals based on fermentation

products of fungi are derived from metabolites of strains that originated from
temperate regions (Table 11.1). These natural products (NP), semisynthetic
derivatives and synthetic analogues include the b-lactams, cyclosporin,
mycophenolic acid, ergonovine, ergotamine, lovastatin, pneumocandins and
echinocandins, and the strobilurins. The vast numbers of untested taxa, the
few but highly successful fungal-derived products, ease of laboratory manip-
ulations and, to a lesser degree, hypotheses about chemically mediated rela-
tionships between fungi and other organisms are frequently cited reasons for
advocating fungi as a resource for low-molecular-weight organic compounds
for drug discovery (Dreyfuss and Chapela, 1994; Gloer, 1997; Pearce, 1997).
Since the late 1980s, the unrealized potential of metabolites of tropical fungi
as sources of starting molecules for drug development has been emphasized
(Nisbet and Porter, 1989; Petrini et al., 1992; Fox, 1993).
Despite an increasing awareness of and discussion regarding the poten-
tial for metabolites from tropical fungal species, arguably the numbers of
metabolites discovered from tropical fungi, as judged from the patent and NP
literature, at best have only equalled those from temperate fungi (Wildman,
1997). Some of the most significant NP discoveries of the 1990s have resulted
from temperate fungi, a few of which are mentioned in this chapter.
Nevertheless, some important lead compounds have been discovered from
tropical fungi, and a selection resulting from projects carried out at Merck
Research Laboratories (MRL) are included here. Some misconceptions about

166
Table 11.1. Major medicinal and agrochemical products developed, or in development, that are based on fungal metabolites. The
common name of the fungal metabolite is given followed by its biosynthetic origin, the fungal taxon that produces the metabolite,
and generic and trade names of the commercial products or derivatives.
Metabolite Biosynthetic family Fungi Commercial productsa

Penicillins Peptide Penicillium, Aspergillus spp. Penicillin G, ampicillin, carbenicillin, methi-
cillin, amoxicillin
Cephalosporins Peptide Acremonium, Emericellopsis Cephalosporin N, Cefoxitin, Rocephin, Cefzil,
spp. Monocid
Lovastatin, pravastatin Polyketide Aspergillus terreus, Penicillium Mevacor, Zocor, Pravachol
spp., Monascus ruber,
Pleurotus spp.
Cyclosporin A Peptide Tolypocladium spp., other Sandimmune
Hypocreales
Ergotamine Tryptophan-isoprenoid Claviceps spp. Ergostat, Cafergot, Bellergal-S, Maxalt
Mycophenolic acid Polyketide-isoprenoid Penicillium spp. CellCept (mycophenolate mofetil)
Pneumocandin B0 Peptide-polyketide Glarea lozoyensis Cancidasb (caspofungin acetate)
Strobilurins Polyketide-amino acid Strobilurus tenacellus and Allegro, Brio (kresoxim-methyl), Amistar,
other basidiomycetes Heritage (azoxystrobin)
a
Registered trade names are capitalized. Some of the products are semi-synthetic derivatives, or wholly synthetic derivatives based
on a fungal-derived natural product.
G. Bills et al.
b
In phase III clinical trials.
Pharmacologically Active Metabolites from Tropical Fungi 167
the priorities and goals of fungal metabolite screening programmes and ques-
tions of the metabolic distinctiveness of tropical fungi are also addressed.
Recent sweeping changes in pharmaceutical research, molecular biology of
biosynthetic pathways, high-throughput screening (HTS), and the Convention
on Biological Diversity (CBD) have redirected MRL’s approaches to fungal
screening. In the light of this experience, recommendations are made on how
to ensure that tropical fungi contribute to future discoveries.
An Evolving Role for Natural Product Research in

Drug Discovery
The landscape for NP screening within drug discovery has changed dramati-
cally during the last 10 years (MacIlwain, 1998; Archer, 1999; Service,
1999a). Most pharmaceutical companies have established large collections of
structurally distinct, small molecules that are used as the starting point for
drug lead identification. A corporate compound collection may consist of
100,000–500,000 discrete chemical entities. The initial task of screening
hundreds of thousands of compounds is usually performed by HTS laborato-
ries, which are operated by scientists in applied biology and chemistry. They
utilize specialized assay technologies, dedicated robotic systems and data-
handling software to screen the chemical samples and evaluate the primary
screening data within a time frame of weeks to a few months.
Access to chemical diversity may no longer be the rate-limiting step in
drug discovery. As a result, NPs have shifted from being a primary chemical
source to a complementary source of chemical structural diversity.
Combinatorial synthesis, the automated synthesis of structurally related small
molecules, can generate millions of new structures in the search for an active
drug or probe molecule for research or diagnosis (Ellman et al., 1997; Karet,
2000). Most compounds do not bind or interact with a target, and therefore
many researchers in industrial and academic laboratories are mixing the
power of combinatorial chemistry with the specificity of targeted, or rational,
drug design to create focused libraries of compounds more likely to ‘hit’ their
targets. Still, much structural diversity found in NPs cannot be achieved by
synthetic means, and many experts argue that NPs offer a source of often
unpredictable chemical structures that can lead to unanticipated and alter-
native medicinal chemistry programmes (Shu, 1998; Henckel et al., 1999;
Service, 1999b; Harvey, 2000; Strohl, 2000).
HTS technology obliges researchers to use natural extract collections dis-
pensed in ‘screen-ready’ formats in microwell plates. Sample-testing formats
have continuously moved towards higher-density and smaller-volume micro-
well plates (96-, 384- and 1536-well plates), and the pace at which synthetic
chemical collections, combinatorial chemistry libraries and NPs are tested in
parallel has increased exponentially. To accrue and deliver large numbers of
168 G. Bills et al.
testable NP extracts for HTS stations, most laboratories have abandoned past
approaches to microbial NP testing, in which a dedicated team continually
screens fermentations of freshly isolated strains. Instead, large extract collec-
tions, often called NP libraries, are assembled either in-house or obtained from
a number of speciality companies or research institutes that offer NP extracts
or purified NPs. Collections of organism extracts are carefully assembled, with
each microbial extract backed by a preserved microbial strain. If an extract
needs extensive retesting, the microbe is revived to reproduce the fermentation
extract. Plant and animal extracts are backed by well-documented voucher
specimens that allow rapid recollection of the organism when more of its tis-
sue is sought for isolation chemistry and biological evaluation.
The extract collection/screening model has several advantages over the
traditional continuous isolation/fermentation model and allows for system-
atic exploration of biological diversity. Organism extracts are accumulated and
predispensed over time. They are immediately available for screening and facil-
itate integration of NPs into the HTS laboratory. Representative organisms can
be selected to ensure thorough coverage of chemically important species. In
very metabolite-rich taxa, e.g. Eurotiales, sampling the entire range of phylo-
genetic diversity may be desirable. The screening strategy shifts efforts from
continuous collection and screening of easy-to-collect speciose organisms
(Bills, 1995), towards a focus on difficult-to-obtain organisms, which are
stored and retested instead of being grown once and discarded. Fungi are usu-
ally grown in a variety of conditions to obtain a full array of metabolites. Since
the collections are used repeatedly for many tests, efforts previously devoted
to regularly obtaining new sets of organisms for screening are redirected to
ensure that each organism is distinct, kept in a stable form, grown to produce
a metabolite-rich fermentation, and that its extract uniquely contributes to
the overall chemical diversity in the collection (Julian et al., 1998).
Extract collections, however, have their own liabilities, most of which cen-
tre on maintenance of the biological and chemical integrity and adequate
inventory of extracts. The need to return to collections after years of storage
requires meticulous and stable preservation of strains. Chemical stability of
stored natural product extracts, how to reconstitute them so that they give
results representative of a fresh extract and inventory management are criti-
cal considerations. However, the success of the extract library versus the tra-
ditional continuous isolation/fermentation model as a discovery tool still
awaits validation.
What are Tropical Fungi? Are They Metabolically Distinct

from Other Fungi?
Using a geographic definition of tropical, the experience in the modern HTS
environment at MRL has been that an extract of a tropical fungus is equally
likely to produce a new lead compound as one of boreal or austral origin. It

is generally accepted that in the tropics, especially the humid tropics, certain
taxonomic and ecological groups of fungi are more speciose than in temper-
ate habitats. Less sampling effort yields more species than in temperate or
boreal regions, and certain taxa appear to be uniquely tropical. At the same
time, temperate habitats, e.g. high-elevation forests, are also present in tropical
latitudes. The rich diversity of tropical fungi and varied habitats within the
tropics are the primary consideration that leads to the incorporation of a
tropical component into a metabolite screening programme.
Although fungal NPs display an extraordinary degree of structural diver-
sity, most of the major structural classes share their biosynthetic origins. The
genes required for biosynthesis of polyketides, non-ribosomal peptides and ter-
penoids are now known to be clustered and to occupy a significant propor-
tion of chromosome loci. Other fungal NPs are derived from shikimic acid,
carbohydrates and fatty acids. Tropical fungi can produce the same com-
pounds as their temperate counterparts, and undiscovered metabolites are fre-
quently found among both. Examples of similar or identical secondary
metabolites produced by taxonomically disparate fungi abound in the litera-
ture (Turner and Aldridge, 1983; Anon., 1999).
The redundancy of NP biosynthetic pathways among fungi may be
explained by several hypotheses on the evolution of secondary metabolites.
One proposal was that NPs evolved from ancestral low-molecular-weight sub-
stances that facilitated primordial biochemical reactions (Davies, 1990). If this
is true, fungi would share various NP pathways because they share a com-
mon ancestry. Another hypothesis for NP evolution is that some secondary
metabolic pathways are modified primary pathways. This is strongly supported
by the similarities between pathways involved in polyketide and fatty acid
biosynthesis. The main differences are that polyketide synthases (PKSs) can
incorporate a large variety of acyl starting units and lack the partial or com-
plete reductions of the b-keto groups formed after each chain extension.
Furthermore, the primary nucleotide sequences and functional domain organ-
ization of known fungal PKS genes are highly conserved (Fig. 11.1).
Horizontal gene transfer hypotheses of evolution may explain why some sec-
ondary metabolic pathways are redundant among fungi (Walton, 2000). The
b-lactam biosynthetic pathways in fungi and bacteria share significant
homologies, suggesting that fungi may have acquired the genes from bacteria.
The incongruencies between metabolite redundancy among unrelated
species and projections of infinite chemical diversity from taxonomic diversity
should be considered when interpreting the articles of the CBD that pertain
to sovereign rights over genetic resources and benefit sharing. Metabolite
redundancy also affects assignment of intangible intellectual property rights.
Breakthrough products like cyclosporin and lovastatin are examples of redun-
dant metabolites. Retrospective data indicate that valuable fungal metabolites
are likely to be widespread. It is predicted that cyclosporin-, strobilurin-, and
170 G. Bills et al.
Fig. 11.1. Organization of fungal type I iterative polyketide synthase gene clus-
ters. KS (ketoacyl synthase), AT (acyltransferase), DH (dehydratase), MeT (methyl-
transferase), ER (enoyl reductase), KR (ketoreductase), ACP (acyl-carrier protein,
possibly more than one per gene cluster), TE (thioesterase-cyclase).
lovastatin-producing fungi could be found in every country of the world

(Gunde-Cimerman et al., 1993; Möller et al., 1996; Traber and Dreyfuss, 1996;
Anke and Steglich, 1999). Despite the confusing redundancy, the distributions
of many metabolite families occur frequently and predictably among certain
taxa, and often with striking taxonomic distributions (Turner and Aldridge,
1983; Frisvad et al., 1998). Some fungal metabolites are claimed to be rare or
strain-specific because they have been reported once from a single strain. In
some instances this may be true, perhaps because of recent recombinations
of biosynthetic genes in rapidly evolving populations (Schardl and Wilkinson,
2000), or because a species is rare and geographically isolated. However, in
most instances, it is suspected that populations of producing organisms were
not systematically compared using the same detection methods. In other
cases, incorrect identifications have prompted misguided comparative

searches for metabolites (Frisvad, 1989; Bills et al., 1999). Therefore, lack of
data or erroneous data should be considered with respect to apparent discov-
eries of rare and strain-specific metabolites.
The widespread distribution of metabolites raises questions concerning
assignment of economic value to any particular fungal strain or any partic-
ular fungal habitat as a source for drug discovery. The first discovery of a
compound may be the most valuable, while subsequent discoveries of that
compound will probably be of insignificant worth (Simpson and Sedjo,
1996a,b). New uses for known biological products further complicate the
question of the value of the producing organisms, since the chemical
structures and methods for production are already publicly known and not
generally patentable. Nevertheless, the advent of new targets and screening
tools makes re-exploration of familiar fungal species as exciting as exploration
of new and exotic species. Undoubtedly, the novel application of known
metabolites can constitute as important a breakthrough as that of novel
chemicals. When expediency in discovery can determine economic value, does
the over-regulation of biological resources which probably transcend political
boundaries make sense, or would it be more advantageous to test resources
as quickly and as often as possible to achieve discovery first, and capture the
potential benefit?
Metabolic redundancy can be a problem in screening NPs. Many
compounds recur even among seemingly unrelated species from different
geographical regions and habitats. Fermentations produce mixtures of known
and unknown metabolites. Primary and constitutive metabolites produced
during active growth, as well as secondary metabolites, are extracted
simultaneously. Although redundancy can be alleviated to some extent by
careful strain selection, isolation chemists are still burdened with the task of
sorting out known and ‘nuisance’ compounds from unknown components
(Corely and Durley, 1994). Fungi, like bacteria and plants, produce their own
sets of ‘nuisance’ compounds (Fig. 11.2). Certain metabolites can be trouble-
some and highly reactive in sensitive biochemical assays because of highly
oxidized or reactive chemical groups, such as multiple hydroxyls, quinones,
epoxides, or pigmented chromophores. Other characteristics include produc-
tion in high titres, often causing extracts to behave like potent active samples.
Such compounds are frequently produced under varied growth conditions,
thus increasing the probability that they may be detected from different
fermentations of the same strain and found interfering with assays.
During the period from 1995 to 1997, more than 13,000 fungal isolates
were examined for production of bioactive secondary metabolites at MRL’s
Centro de Investigación de España (CIBE). An approximate distribution of the
geographical origins and types of substrata are listed in Table 11.2. Certain
ubiquitous taxa, e.g. Bionectria ochroleuca (Schwein.) Schroers & Samuels
and Lasiodiplodia theobromae (Pat.) Griffiths & Maubl., may have been screened
172 G. Bills et al.
OH
O
OH
O
HO
NH
O O
H OH O
Lambertellin
Deacety-cytochalasin C from Encoelia heteromera
from Beltraniella portoricensis
OH O OH OH OH O
OH HO O
O O HO O
HO
OH OH O
OH O OH
Skyrin
from Hyperdermium bertonii Cephalochromin
from Nectria vilior
O N
O
COOH
O O
O O
N N OCCH3
O
O O
O OCCH3
O
Beauvericin Helvolic acid

from Paecilomyces tenuipes from Metarrhizium anisopliae
Fig. 11.2. Examples of ‘nuisance’ metabolites from tropical fungi commonly

encountered during natural products screening.
multiple times from among the different substrata. The percentages of active
strains refer to those of strains scored as active in a broad array of biological
assays targeting therapeutic areas such as antifungal and antibacterial
antibiotics, antivirals, insecticides, antihelminthic agents, cancer, diabetes
mellitus, inflammation, and endocrinology. Each strain was usually fermented
by two to four methods, and at least one and sometimes two solvents were
used to extract each fermentation. The data are based on the unrefined
primary active extracts, before applying secondary assays for prioritization. If
one or multiple extracts from a strain were scored as active in at least one
assay, then the strain was considered to be active. Only a small fraction of the
active components were purified and fully elucidated to give chemical
structures.
Fungi from temperate and tropical regions performed similarly. A c2
goodness-of-fit test revealed significant differences (P < 0.01) only between
the ‘hit rates’ of endophytes (fungi isolated from living plants) from tropical
versus temperate areas. Tropical endophytes appear to provide a higher ‘hit
rate’ than their temperate counterparts. Likewise, the ‘hit rate’ obtained
with the tropical endophytes was significantly higher than with fungi from
other tropical substrata according to a c2 test. Ascribing meaning to differ-
ences in rates of detection of actives in HTS needs cautious interpretation.
The perception of higher bioactivity may or may not translate into increased
discovery opportunities. Endophyte isolates from humid-tropical plants are
taxonomically biased towards the Xylariales and Hypocreales, two especially
metabolite-rich taxa. However, high rates of active extracts are not always
Table 11.2. Performancea of strains from tropical and temperate areas in a

screening programme for bioactive natural products. Results were obtained at
CIBE during the years 1995–1997. Only those types of substrata giving statisti-
cally relevant numbers of isolates are considered as separate categories.
Tropicalb Temperate and boreal Total

Substratum Isolates Per cent Isolates Per cent Isolates Per cent
class tested active tested active tested active
Living plants 3,005 12.2 3,217 14.6 6,222 13.4
Leaf litter 3,463 11.4 511 12.0 3,974 11.9
Dung 396 11.6 387 10.9 783 11.2
Othersc 209 12.4 1,827 21.5 2,036 13.3
Total 7,073 12.1 5,942 13.3 13,015 12.8
a
A strain is scored as active if one or more of its extracts are active in at least
one biological assay.
b
Tropical is defined geographically, i.e. fungi isolated from substrata collected
between the Tropics of Cancer and Capricorn.
c
Soils, marine, freshwater, other substrata and macroscopic fungi.
174 G. Bills et al.
related to pharmaceutically desirable products. A high proportion of the

strains could produce reactive and toxic classes of compounds, e.g.
trichothecenes and cytochalasins, or high concentrations of primary metabo-
lites, e.g. unsaturated fatty acids, which interfere non-specifically with bio-
chemical assay mixtures to generate false positives. Either way, differences in
rates of activity from tropical endophytes did not result in a higher number
of novel structures with respect to other fungi. Twenty different compounds
(or new biological activities for known compounds) were discovered from all
the fungal isolates tested during the period. The ratio of the compounds’
geographical distributions was the same as that of the tested isolates, i.e. 11
compounds were from tropical areas and nine from temperate areas. Data
reported by Glaxo’s NP laboratory during the early 1990s (Wildman, 1997)
resulted in a similar ratio of activities and new compounds between tropical
and temperate fungi.
Important Metabolites from Tropical Fungi
Examples of discoveries from tropical fungi
Six case studies from MRL illustrate a range of discovery scenarios involving
tropical fungal metabolites. Most of the discoveries were potentially important
because new modes of action for drug targets were uncovered or because the
fungal metabolite provided the first proof that the target was valid and could
be affected by a small organic molecule. For some of the leads, extensive medic-
inal chemistry investigations were carried out to modify and improve the
potency and spectrum of the lead molecule’s activity. To sustain delivery
of the metabolite to a preclinical investigation, gram or kilogram quantities
of purified compound must be produced. The case studies include some of
MRL’s experiences with metabolite titre improvement and the difficulties in
predicting the physiological responses of wild fungal species.
Flutimide, an inhibitor of flu transcriptase

Flutimide (Fig. 11.3) was discovered in the MRL screening programme seek-
ing inhibitors of cap-dependent transcription of influenza viruses (Hensens et
al., 1995). Flutimide is a substituted 2,6-diketopiperazine, structurally related
to the aspergillic acid family (Turner and Aldridge, 1983). Its mode of action
is that of an inhibitor of cap-dependent transcriptase of influenza A and B
viruses that specifically targets the endonuclease activity of the transcriptase.
Other viral polymerases were not affected by flutimide (Tomassini et al., 1996).
Flutimide was the only NP structure isolated after screening several thousand
microbial extracts. The compound is produced by Delitschia confertospora
Peláez, Polishook, Valldosera and Guarro, a new fungal species isolated from
dung of a dassie (Procavia sp.) collected in Namibia (Peláez et al., 1994).
O
N O
N
H
N NH N
H
O N N OCH3
O O
OH
O
Flutimide O
from Delitschia confertaspora
Apicidin
fom Fusarium spp.
O NH
OH CO2H
CHO
OH
OH
O
O CH3O O
N
H OH
Sordarin
Demethyl-asterriquinone B-1 from Sordaria araneosa and unidentified
from Pseudomassaria sp. endophyte
O O
COOH COOH
N N
O
HO OH HO
O O
Nodulisporic acid A Nodulisporic acid A1

from Nodulisporium sp. from Nodulisporium sp.
Fig. 11.3. Examples of pharmacologically active metabolites from tropical fungi.
Flutimide was isolated only from a single strain, but an extensive search for
additional strains was not carried out after the lead was identified. Shortly
thereafter, the molecule was made synthetically to verify the structure and
produce sufficient material for preclinical testing (Singh, 1995).
Apicidin, an antiprotozoal agent

A number of human and animal parasitic diseases, including malaria,
cryptosporidiosis, toxoplasmosis and coccidiosis, are caused by protozoa of the
subphylum Apicomplexa. The cyclic tetrapeptide apicidin (Fig. 11.3) was
identified from MRL’s NP screening programme and has been described as a
broad-spectrum agent effective against a range of apicomplexan parasites
(Darkin-Rattray et al., 1996; Singh et al., 1996). It acts as a potent inhibitor
(IC50 1–2 nM) of the parasite’s histone deacetylase. Apicidin exhibits a broad
176 G. Bills et al.
range of activity in vitro (minimum inhibitory concentrations of 4–70 ng ml–1)

against the Apicomplexa, as well as in vivo activity against Plasmodium berghei
Vinke & Lips. malaria in mice.
Fusarium pallidoroseum (Cooke) Sacc. and an unidentified Fusarium
species, both isolated from Costa Rican plants, were found to produce apicidin
(Cannova et al., 1997). The first strain, F. pallidoroseum, produced 50–80 µg
g–1 in solid substratum fermentation but less in liquid media. Discovery of the
second Fusarium species accelerated delivery of apicidin for evaluation. In ver-
miculite-based screening medium, the second strain produced 10-fold higher
levels than those by F. pallidoroseum. Empirical fermentation studies with the
second strain eventually improved titres to over 1 mg ml–1 in liquid media in
shake flasks and 300–400 µg ml–1 in 600-l fermenters (A. Dombrowski et al.,
unpublished).
It is predicted that apicidin and other tetrapeptides with histone-deacety-
lase activity, e.g. HC toxin, are likely to be geographically widespread among
Fusarium species. Apicidin has recently been implicated in a haemorrhagic
disease syndrome of livestock in Korea (Park et al., 1999). Fusarium isolates
recovered from soybeans produced titres of apicidin in the range of 340–680
µg g–1 in wheat grain cultures, which is of the same order of magnitude as
the titres MPL obtained in our titre improvement project (A. Dombrowski et
al., unpublished).
A potent insecticide from a pantropical Hypoxylon

Nodulisporic acids (Fig. 11.3) are novel indole diterpenes, the first compounds
of this class reported from the Xylariales that exhibit potent insecticidal
properties against the larvae of the blowfly, Lucilia sericata Meigen (Ondeyka
et al., 1997; Ostlind et al., 1997; Hensens et al., 1999). Levels of toxicity are
in ranges intermediate between those of two of the most potent NP insecti-
cides, the avermectins and paraherquamide (Ostlind et al., 1997). The mode
of action of nodulisporic acid is that of an activator of insect glutamate-gated
chloride channels (Smith et al., 2000). Nodulisporic acids are structurally
similar to janthitrems, shearinines, paspalines, and lolitrems. However, they
differ in having a five-membered substituted ring on the indole moiety, an
inversion of the tetramethyl pyran ring system and a five-membered hemi-
ketal ring instead of a six-membered ring.
The compounds were first isolated from a fermentation broth of an endo-
phytic Nodulisporium species. This endophyte was cultured from a surface-
sterilized dried voucher specimen (woody stem) of Bontia daphnoides L.
collected from Kuaui Island, Hawaii, during a collaborative project between
MRL and the New York Botanical Garden (Hensens et al., 1999). An intensive
search was made for other Nodulisporium strains and other xylariaceous
ascomycetes to discover more potent nodulisporic acid analogues or strains
with improved fermentation titres. Twelve more isolates of Nodulisporium
were found to produce nodulisporic acids (J. Polishook et al., 2001). The strains
were isolated from diverse substrata, not only plants, from six different tropi-
cal countries on four continents (Table 11.3).
Alignment and parsimony analysis of the rDNA intertranscribed spacer
regions and morphological comparisons indicated that the 13 nodulisporic
acid-producing Nodulisporium strains were conspecific. The xylariaceous
anamorphs were characterized by faster radial growth at 37°C than at 23ºC,
a dark brown soluble pigment in agar and liquid cultures, a sweet, medicinal
odour, and deposits of a melanin-like pigment on the conidiophores. No
particular isolate exhibited a striking advantage in nodulisporic-acid titre;
titres of nodulisporic acid A ranged from 2 to 10 µg ml–1. Comparisons of ITS
sequences with a sequence database from other xylariaceous fungi (Sánchez
et al., 2000) failed to link the nodulisporic acid-producing Nodulisporium
species to a teleomorph species but demonstrated that it is a Hypoxylon species,
phylogenetically close to H. fendleri Berk. ex Cooke. To date, nodulisporic acids
may be the only examples of exclusively tropical fungal metabolites that have
been observed at MRL.
A non-peptidyl insulin mimetic

Bis-demethyl-asterriquinone B-1 (Fig. 11.3) was discovered as an orally
available insulin mimetic agent through a screening assay designed to detect
non-peptidyl small molecules that activate the human insulin receptor
tyrosine kinase (Zhang et al., 1999). The compound was shown to be selec-
tive for the insulin receptor versus other receptor tyrosine kinases, such as the
insulin-like growth factor receptor or the epidermal growth factor (EGF) recep-
tor. It mimicked insulin in several biochemical and cellular assays, including
stimulation of glucose uptake in whole cells. Moreover, the oral administra-
tion of the compound in two mouse models of diabetes resulted in significant
lowering of glucose levels and suppression of the elevated plasma insulin lev-
els associated with one of these mouse models. Although quinones are noto-
riously reactive and generally considered an undesirable molecular platform
for medicinal chemistry, the discovery was celebrated as a breakthrough in
the field for the development of orally active antidiabetic agents. For the first
time, a small, non-peptidyl molecule was demonstrated to mimic the in vitro
and in vivo function of a protein hormone by interacting with and activating
its receptor.
The above compound belongs to the family of the asterriquinones, widely
known fungal bis-indolyl-quinones, which have been found in diverse
ascomycetes from tropical and temperate regions, including Aspergillus species
(and the teleomorph Petromyces muricatus Udagawa, Uchiyama & Kamiya),
Chaetomium spp., Chrysosporium merdarium (Ehrenb.) J.W. Carmichael,
Botryotrichum species and in Humicola species (Sekita, 1983; De Guzman et
al., 1994; Mocek et al., 1996; Fredenhagen et al., 1997; Ooike et al., 1997).
The asterriquinones have been studied as potential anti-tumour agents
because of their DNA-intercalating properties (Yamamoto et al., 1976; Kaji et
178
Table 11.3. Distribution and origin of nodulisporic acid-producing Nodulisporium speciesa.
Isolate Source Location Continent

MF5954 (ATCC 74245) Stems, Bontia daphnoides Kuaui Island, Hawaii, USA Oceania
MF6230 Horse dung Hiva Oa, Marquesas Islands, French Polynesia Oceania
MF6263 Bush twig, unknown host Capurgana, Colombia South America
MF6262 Bark disc, unknown host Capurgana, Colombia South America
MF6324 Leaf litter, unknown host Lago Sandoval, Peru South America
MF6245 Fruticose lichen, Usnea sp. Playa La Parguera, Puerto Rico North America
MF6321 Leaf litter, unknown host Curepipe, Mauritius Africa
MF6315 Leaf litter, Coffea sp. La Region de Sept Couleurs, Mauritius Africa
MF6380 Twig, Coula edulis Acan-Bot Esaveng, Equatorial Guinea Africa
MF6246 Bark disc, Griffonia tessmannii Bolondo, Bata, Equatorial Guinea Africa
MF6379 Twig, Dorstenia elliptica Acan-Bot Esaveng, Equatorial Guinea Africa
MF6378 Twig, Anacardium occidentale Bata, Equatorial Guinea Africa
MF6377 Twig, Scaevola plumieri Bome, Bata, Equatorial Guinea Africa
a
Polishook et al., unpublished data.
G. Bills et al.
al., 1998). They are also HIV-1 protease inhibitors (Fredenhagen et al., 1997)
and toxic to insects (De Guzman et al., 1994). The compound found in MRL’s
screening had been previously obtained by chemical treatment of aster-
riquinone B-1(Arai et al., 1981), but its insulin-mimetic properties were
unknown. Other asterriquinones are ineffective as insulin-mimetic agents. For
example, the similar asterriquinone, hinnuliquinone, is about 100 times less
active in the assay (Zhang et al., 1999). However, other asterriquinones have
been shown to be inhibitors of the EGF receptor tyrosine kinase, which is
closely related to the insulin receptor (Fredenhagen et al., 1997).
Bis-demethylasterriquinone was detected in a liquid fermentation of an
endophytic fungus isolated from unidentified living leaves collected near
Kinshasa, Democratic Republic of the Congo. Sporulation was unachievable
in agar culture, but cultivation of the fungus on sterilized wood strips induced
formation of a mature ascoma that permitted tentative classification in the
ascomycete genus Pseudomassaria. The compound was detected only once
among more than 5000 fungal isolates tested from tropical and temperate
regions.
A new class of antifungal agents: inhibitors of the protein synthesis

elongation
The rediscovery of sordarins and related compounds has given rise to a
promising new class of agents for treatment of human and plant fungal
infections. Analyses of the discovery of sordarin and development of its
analogues as antifungal agents for human mycoses illustrate the difficulties
in interpretation of the CBD. Sordarin (Fig. 11.3) was first discovered in a soil
isolate of Sordaria araneosa Cain from Sri Lanka by researchers at Sandoz
searching for antifungal agents (Sigg and Stoll, 1969; Hauser and Sigg, 1971).
Sandoz investigators patented and published their discovery, but sordarin was
overlooked for more than two decades. During the mid-1990s, several labo-
ratories independently reported the discovery of sordarin, zofimarin (Ogita et
al., 1987), and other antifungal diterpene glycosides with the sordaricin agly-
cone (Okada et al., 1994; Coval et al., 1995; Schneider et al., 1995; Daferner
et al., 1999). GlaxoWellcome’s HTS programme for inhibitors of Candida
albicans (C.P. Robin) Berkhout protein synthesis resulted in the discovery of
GB135402, a new sordarin analogue from Graphium putredinis (Corda) S.
Hughes from the UK, possessing promising potency and selectivity for fungal
versus mammalian protein synthesis (Kinsman et al., 1998). At about the
same time, sordarins were rediscovered at MRL through non-mode-of-action
screening for antifungal agents (Bills et al., 1998).
The exceptional potency and biological spectrum of sordarin derivatives
stimulated investigations to solve the molecular basis for their antifungal
activity. Sordarin and its analogues are tetracyclic diterpene glycosides that
selectively inhibit fungal protein synthesis by impairing the function of
eukaryotic elongation factor 2 (eEF2) (Domínguez et al., 1998; Justice et al.,
180 G. Bills et al.
1998, 1999). Sordarin and its derivatives bind to the eEF2–ribosome–

nucleotide complex in sensitive fungi, stabilizing the post-translocational GDP
form.
Sordarin and similar compounds occur widely among ascomycetous
fungi, especially in the Sordariales and Xylariales (Daferner et al., 1999). MRL’s
investigations of sordarin relied on the compound produced by a non-
sporulating endophyte from living roots of a mangrove shrub, Conocarpus
erectus L. from Costa Rica (MF6232) and from Rosellinia subiculata (Schwein.)
Sacc. collected in New Jersey (Bills et al., 1998). Although studies at Glaxo-
Wellcome initially focused on GB135402 from Graphium putredinis, further
scale-up of sordarin analogues relied on superior titres provided by the original
sordarin-producing strain, Sordaria araneosa (ATCC 36386) (Hayes et al.,
1996). MRL obtained the same species of Sordaria (ATCC 36386) and
compared it with strains isolated in their laboratory. Fermentations on solid
substrata were converted to liquid fermentation media and manipulated to
improve the titres. The titres of the sterile strain, MF6232, were more readily
improved, obtaining production levels up to 700 µg ml–1 in shake flasks.
Eventually, MF6232 was scaled up successfully in 800-l and 19,000-l stirred
tanks. Rosellinia subiculata produced only 80–100 µg ml–1 in shake flasks.
Sordaria araneosa, in shake flasks, initially produced 250 µg ml–1 sordarin,
but the production was increased nearly threefold by media manipulation
(A. Dombrowski et al., unpublished).
Antifungal sphingolipid inhibitors

Sphingolipid synthesis is a vital pathway for cell membrane components.
Fungal sphingolipid biosynthesis (Fig. 11.4) has been a target for discovery of
new human health antifungal agents for candidiasis, aspergillosis, and other
mycoses because certain enzymes in the pathway are unique to fungi
(Dickson, 1998). Identification of specific and potent inhibitors of fungal
enzymes could be potential starting points for lead development. Over the
course of several years, an assay for detection of fungal pathway inhibitors
was run at CIBE. Natural product inhibitors for three different enzymes, serine
palmitoyltransferase, ceramide synthase, inositol phosphoceramide synthase,
and for the fatty acid elongation pathway were discovered from temperate and
tropical fungi and actinomycetes (Mandala and Harris, 1999).
Inhibitors derived from tropical fungi and the specific enzymes they affect
are shown in Fig. 11.4. Viridiofungin A (Fig. 11.4), B, and C form a novel
family of amino alkyl citrates and are potent broad-spectrum antifungals
(Mandala et al., 1997b; Onishi et al., 1997). They were isolated from a strain
identified as Trichoderma viride Pers. recovered from soil collected in Micronesia
(Harris et al., 1993). Although viridiofungins are moderate inhibitors of yeast
squalene synthase, they do not specifically inhibit fungal ergosterol bio-
synthesis in vivo. Their antifungal mode of action is based on their nanomolar-
level inhibition of serine palmitolytransferase (Mandala et al., 1997b). An
O CO2
–
SCoA + +H
3N OH
H CO2H O
palmitoyl CoA serine HO
HO2C
OH
O NH
serine palmitoyltransferase Viridiofungin A
HO2C
OH
ketodihydrosphingosine +
NH3 OH MAMMALIAN MEMBRANES
ceramide (DHS)
OH
OH
OH NH
+
dihydrosphingosine NH3
O
ceramide
synthase HO2C O
HO2C Fumonisin B-1
OH O OH OH
OH
+
OH NH3 OH NH2
phytosphingosine O
ceramide HO2C
synthase HO2C O
O OH
OC O CH3
N
H
OH
Colletotrichum metabolite
O O
+
SCoA SCoA
long chain fatty n
acid synthesis
O
OH O
N
OH
OH HN N
OO HO
HO NH OH
HO
N
O H
ceramide (PHS)
Minimoidin
inositol
O OHOH phosphoceramide OH
O O PO OH O O OH
O O– HO synthase
O
OH
OH HO
O O
O OH
phosphatidylinositol
diacylglycerol
OH
O OHOH Khafrefungin
O P O
HO NH H O– HO
OH
O
OH
inositol-P-ceramide
FUNGAL MEMBRANES
Fig. 11.4. Examples of fungal natural product inhibitors of sphingolipid biosyn-

thesis. Viridiofungin A, the Colletotrichum metabolite, minimoidin and khafrefun-
gin originated from tropical fungi. White arrows indicate enzymatic steps
inhibited. Adapted from Mandala and Harris (1999).
unnamed sphingosine-like metabolite isolated from a Costa Rican isolate of

Colletotrichum acutatum J.H. Simmonds (Fig. 11.4) inhibits fungal ceramide
synthase (Bolessa et al., 1996). Australifungin is another fungal-specific
inhibitor of this step of the pathway, while the mycotoxin fumonisin is a potent
inhibitor of mammalian ceramide synthase (Mandala and Harris, 1999).
Khafrefungin (Fig. 11.4), a linear polyketide isolated from a non-sporulating
endophyte of Tetragastris panamensis Kuntze collected in Costa Rica, is a
182 G. Bills et al.
fungal-specific subnanomolar inhibitor of inositol phosphoceramide synthase

(Bills et al., 1997; Mandala et al., 1997a; Mandala and Harris, 1999). Inositol
phosphoceramide synthase is essential for fungal growth and is the target of
the antifungal fungal metabolite aureobasidin (Zhong et al., 2000). Finally,
minimoidin (Fig. 11.4) is a potent inhibitor of sphingolipid synthesis that acts
by inhibiting the fatty acid elongation pathway. Minimoidin contains a novel
heterocyclic ring system acylated with a b-keto fatty acid (Clapp-Shapiro et
al., 1998; Mandala and Harris, 1999). It was discovered from a strain of
Sporormiella minimoides S.I. Ahmed & Cain isolated from giraffe dung collected
in Namibia. Minimoidin has minimum inhibitory concentrations against
Candida spp., Cryptococcus neoformans (San Felice) Vuill. and Aspergillus
fumigatus Fresen. in the range of 0.125–2 µg ml–1 (Clapp-Shapiro et al., 1998),
and therefore is the most potent metabolite found among the antifungal
sphingolipid pathway inhibitors.
Although numerous potent and specific NP inhibitors of the sphingolipid
target were discovered, many of the structures were disadvantaged by poor
solubility or stability and limited whole-cell activity, while others were lytic to
red blood cells, and were therefore eliminated as candidates for further
development.
Fungal Natural Product Discovery in the Genomic Era
Discoveries of new NPs from easily culturable fungi will continue, but expan-
sion of sources of fungal natural products to unculturable and slow-growing
fungal species might lead to even more new structural classes. Improved
cultivation techniques could take advantage of less-manageable fungi, but the
task is likely to become increasingly intractable, and some fungi may never be
cultivated. Advances in gene sequencing, molecular biology and genetic
engineering make it possible to express genes from unculturable organisms in
laboratory fungal strains, therefore providing an alternative route for realizing
the genetic potential.
A molecular genetic approach is being tested (Fig. 11.5). This approach
is based on the fact that genes involved in fungal and bacterial secondary
metabolite biosynthesis are often clustered within 30–60 kb of DNA (Martin,
1992; Kimura and Tsuge, 1993; Keller and Adams, 1995; Brown et al., 1996;
Kennedy et al., 1999). Therefore, it is feasible to transfer large pieces of
DNA that include most, if not all, of the gene cluster for a particular second-
ary metabolite from unculturable fungi, as exemplified by cosmid clones
(35–45 kb) and bacterial and yeast artificial chromosome clones (> 100 kb).
An assumption underlying this approach is that genes or clusters of genes
from fungi can be expressed in laboratory strains, because transcription and
translation control sequences from one organism often function among closely
related organisms, and sometimes even in distantly related organisms (Punt
Fig. 11.5. Capture and engineering of secondary metabolite biosynthetic genes

from nature and their heterologous expression in a surrogate fungus for discovery
of new natural products. BAC, bacterial artificial chromosome; YAC, yeast artifi-
cial chromosome.
et al., 1987; Barrett et al., 1990; Mooibroek et al., 1990; Smith et al., 1990;
Hondel et al., 1991; Romanos et al., 1992; Kennedy et al., 1999). Although
the studies suggest that foreign genes may be expressed in laboratory
strains, donor–recipient combinations must be carefully matched; not all tran-
scription control sequences from one fungus function in other fungal
hosts. Several strategies, such as mRNA analysis, reporter gene analysis, and
184 G. Bills et al.
complementation of auxotrophic markers, can be applied to test gene expres-

sion. The recipient strains for expressing genes from donor fungi should be
easily manipulated genetic model organisms. These host fungi often grow
quickly and are easily fermented on a large scale. The availability of multiple-
recipient laboratory strains increases the chance for heterologous expression
of foreign genes.
Undirected transfer of genetic material from unculturable and slow-
growing organisms to a surrogate host will generate large numbers of trans-
genic strains. Screening huge numbers of extracts of random transformants
would be costly and impractical. One of the key obstacles to realizing the
approach is how to eliminate non-productive transformants before they
are sent for NP screening. Some type of sensitive and high-throughput pre-
screen(s) is needed to discern novel extracts (Fig. 11.5). At the same time,
prescreens should not discard potentially productive candidates. If the pre-
screen employs biological readouts, they should reflect broad biological
signals, such as general stress responses and signal transduction. A possible
strategy would be to fuse transcription regulatory sequences of broad inter-
est to sensitive reporters in cell-based prescreens.
Many microbial secondary metabolite-encoding genes have been cloned,
sequenced, and characterized, and more will become available as more micro-
bial genomes are determined. Conserved biosynthetic genes can be used as
probes to preselect clones that are most likely to contain secondary metabo-
lite-encoding gene(s). This approach will work only if the prescreen is in a
high-throughput mode. Significant progress has been made in DNA array
technology during the last 5 years, some of which can be practically applied
to search for secondary metabolite-encoding genes from a larger collection of
cloned DNA. Briefly, a secondary metabolite-encoding gene library can be
created by including consensus DNA fragments applied from genes encoding
various secondary metabolites, such as polyketides, non-ribosomal peptides
and alkaloids. The DNA fragments can be used individually as probes to screen
libraries for secondary metabolite gene clones. The DNA fragments can also
be placed on membranes or glass to create a secondary metabolite-encoding
DNA array.
Another new concept of NP drug discovery, known as the biocom-
binatorial production of synthetic NPs, is evolving. Several biotechnology
companies have been formed based on the promise of this genetic engineer-
ing/chemistry hybrid approach for developing novel NPs. Understanding of
genetic programming of bacterial PKSs has progressed tremendously, leading
to the rational design of novel polyketides (McDaniel et al., 1993, 1994, 1995;
Fu et al., 1994; Kim et al., 1995; Tang et al., 2000). As mentioned above,
fungal polyketides represent a structurally diverse chemical class, but the
genes that encode these compounds are conserved in primary nucleotide
sequences and the organization of functional motifs (Fig. 11.1). Most current
efforts focus on polyketides of bacterial origin, because the genetic systems for
bacteria are well developed, but the same principles are applicable to fungal
NP systems.
In summary, exploitation of the genetic diversity of unculturable fungi by
manipulation of ‘biocombinatorial’ diversity immeasurably expands opportu-
nities for discovering novel or ‘unnatural’ secondary metabolites. Since recip-
ient strains are fast-growing, ‘industrial’ organisms, when drug-producing
transformants are identified, scale-up fermentation for commercial production
can be quickly implemented.
Obstacles to Commercialization of Tropical Fungal

Metabolites
Considering the increased awareness of fungal diversity, one might ask
why tropical fungi have not made a greater impact in NP discovery.
Pharmaceutical companies have established few projects to screen tropical
fungi, because demands for large numbers of test species have been tempered
by alternative sources of chemical diversity. The number of industrial labora-
tories engaged in NP research has declined and, of those that remain active,
many have filled their extract collections to capacity in order to adapt to the
HTS environment of industrialized drug discovery. Mature NP collections may
continue to expand, but the need for strains should not increase substantially.
The issues faced by those who collect tropical fungi parallel those of
researchers exploring tropical plants and animals. Geographic separation
of the major discovery and fermentation facilities in industrialized temperate
countries in North America, Europe and Asia from forests in the tropics has
complicated utilization of tropical fungi as a drug discovery resource. At least
two NP laboratories of multinational pharmaceutical companies have been
established in tropical countries: the Centre for Natural Product Research rep-
resents a major initiative in drug discovery, jointly funded by GlaxoWellcome,
the Singapore Economic Development Board and the Institute of Molecular
and Cell Biology; similarly Hoechst Marion Roussel has a long-established
research laboratory for natural products chemistry in India. However, a lack
of adequate research facilities and microbiological/mycological expertise in
many biologically rich regions remains a limiting factor. Until biocombinato-
rial approaches are realized on an industrial scale, the search for secondary
metabolites from fungi will continue to require that the organisms be cultured
successfully to produce sufficient quantities of compounds to fuel medicinal
chemistry and preclinical investigation programmes. Many fungi are not
amenable to long-distance transport or commonly used culturing techniques
(Bills, 1995). To culture perishable species, transport time between the field
and laboratory should be minimal; therefore, a laboratory near the habitats
is needed. Facilities need to be extended; skilled personnel are required to
work in often suboptimal conditions, knowing how to select organisms,
186 G. Bills et al.
document their collections, culture them cleanly, and avoid repetitive

collection of common species.
Many tropical fungi will never be used for pharmaceutical exploration
because of the lack of adequate facilities and personnel in their source
countries. Even more crucial is the need for a regulatory environment that
encourages collaboration with industry, or at least allows independent con-
tractual agreements. Exploration can be integrated into ongoing research at
government or academic microbiology, plant pathology, or other biological
research laboratories. Simple agreements in accordance with the CBD can be
set rapidly into place if the regulatory environment permits and financial
expectations for success are realistic. Such complications increase the costs
and delays in acquiring tropical species. Given that temperate and boreal
habitats may be equally productive, and, to an unknown degree, metabolically
redundant, the difficulties in establishing a tropical collaborative project can
favour establishment of explorations of fungi in the temperate zones.
MRL supports the spirit and recommendations of the CBD, but the authors
have witnessed some unintended consequences brought about by unrealistic
expectations raised by publicity and discussions surrounding the treaty. The
time, perseverance and expense required to negotiate contracts to transfer
microorganisms severely limits the number of exploration projects undertaken
(Gollin, 1999). Fears about hidden past claims to unrealized products favour
agreements in which a clean title to microorganisms is acquired directly from
the original source. Simple two-party agreements avoid the unneeded
expenses of intermediaries and reduce uncertainties about ownership. The
authors are concerned that the CBD environment will stifle use of strains
accumulated in academic research collections and public culture collections
because of complex requirements to track the history of strains and wariness
of unexpected claims to potential products dictated in material transfer
agreements.
Ten years ago, collecting easy-to-transport-and-culture organisms world-
wide, e.g. soil actinomycetes, was a common strategy to feed screening
programmes. Today that strategy is impossible. NP laboratories have shifted
from worldwide collections to geographically focused collections and to
cultivating long-term collaborations with partners at the sources of microbial
diversity. An unfortunate consequence of geographically restricted sampling
is that valuable revelations about the taxonomic and biogeographical patterns
of metabolites, e.g. in the case of the cyclosporins (Traber and Dreyfuss, 1996),
zaragozic acids (Bergstrom et al., 1995), sordarins, and nodulisporic acids, will
be obscured. Maximizing microbial diversity based on collections from one or
a few locations means shifting from dependence on speciose organisms from
one substratum, e.g. endophytic fungi or litter fungi, to methodically mining
out organisms from a few forests, deserts or estuaries. On the other hand, long-
term collaborations and stratified collection strategies meld well with semi-
permanent extract collections needed for the HTS environment, because
expensive and difficult-to-obtain fungi are held and reused. It is predicted that
the gold rush for tropical microorganisms, if it ever appeared, will dissipate,
and that means fewer viable opportunities for tropical fungi to contribute to
the drug discovery process.
Conclusions and Outlook
A major drug or agrochemical from a tropical fungus has yet to reach the
market. Evidence from academic laboratories that are focusing on isolating
unusual organic structures from fungal fermentations has shown that fungi
produce large numbers of novel compounds (Gloer, 1997), but the goal of NP
research in the pharmaceutical industry is to find new health care products,
not novel compounds. Therefore, new uses for known metabolites can be as
interesting as novel metabolites. During the 1990s, a more profound under-
standing of how fungal metabolites are biosynthesized and the commonality
among biosynthetic genes has been gained, leading to new ways to recom-
bine and express biosynthetic genes.
Several models have been proposed to utilize organisms from threatened
habitats as sources for drug discovery or other types of biotechnological
innovations (Baker et al., 1995; Porzecanski et al., 1999). These models
assume that, to obtain the benefits of revenue sharing from the discovery of
biologically derived, commercial products, biologists in tropical countries must
actively participate in the discovery process. However, participation also
means risk-sharing. Discovery at the leading edge of NP research is not unlike
playing the lottery. Luck and persistence are essential components for
winning. The financial risks and capital investment in basic research and
development are extremely high for drug discovery, while the investment
and risk in selecting a single fungal strain or a substratum from which to
isolate fungi is minimal in proportion to the potential rewards. Investigators
who believe they have potentially valuable organisms, and who are willing
to assume part of the risk, should not wait to be contacted by a commercial
group interested in product development because that invitation may never
arrive. Scientists who are eager to promote sustainable use of microbial
resources must be proactive in formulating strategies for microbial collection
and cultivation, identifying and contacting serious collaborators, and finding
ways to streamline negotiations for legal exchange of germplasm and intel-
lectual property. Instead of asking how to gain short-term profit from selling
biological resources, perhaps the better question is how to make tropical
resources competitive with other sources of chemical diversity and maximize
exposure to opportunities over the long term, so that those much-needed and
successful examples of the value of tropical fungi will be realized.
188 G. Bills et al.
Acknowledgements
We thank Jan Tkacz, Guy Harris and Michael Dreyfuss for helpful comments
and suggestions on earlier versions of this manuscript.
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Index
Page numbers in bold refer to figures and tables
accidental introduction 89, 95–96, 101 teleomorph connections 41–42, 44,

see also anthropogenic factors; 45, 46, 47
migration see also reproduction
Acicomplexa 175 Andes barrier 101
acquired immune deficiency syndrome animal disease caused by fungus
(AIDS) 152 145–163
aeroquatic hypomycetes xii–xiii, 41–47 animal/fungi interrelationship xiii, xiv,
agricultural ecosystems 138–140 133–142, 145–163
agrochemical products 166 anthropogenic factors
Ainsworth and Bisby’s Dictionary of the fire 121–123, 125–126
Fungi 113, 136 forest management 125–126
Aithaloderma 34, 35, 36, 37–39 lichens xiii, 125–126
alien fungal plant pathogens 83 Microcyclus ulei introduction 89
amphigyny 76 Moniliophthora roreri introduction
see also anamorph; reproduction 101
anabolic sterol metabolic pathways 69 origin graminicolous downy mildew
anamorph 74, 76
asexual propagules 68–69 Trans-Amazonian highway 95–96
Cordyceps 137 trans-Andean oil pipeline 101
Fusicladium 85 witches’ broom introduction 97
genera 139 see also human
Hypocrella 136 antifungal agents 179–182
Ingoldian hyphomycetes 45 antiprotozoal agent 175–176
new species 42 apicidin 175–176
stage, lignicolous freshwater higher Araneae 133–142
fungi 41–47 arm sporotrichosis 158
195
196 Index
Aschersonia 140 Lantana camara L. 5

ascomatic centra, sooty moulds 36 Moniliophthora roreri 104
ascomycete organisms xiv, 133–142
cacao xiii, 83–107 programme 7
on Freycinetia 54, 57 South American leaf blight 90
fungi from the Pandanaceae 57, 58 witches’ broom disease 97–98
genera, new 57 biological invasion 83–107
on Pandanus 53, 57 biotechnology 184–185
rubber xiii, 83–91, 106 bis-demethyl-asterriquinone 175, 177,
on Saranga 57 179
see also mildew; moulds; yeasts bis-indolyl-quinones 177
Ascomycota 134 blastomycosis 157, 162
asexual propagules 68–69 body infections 149
see also anamorph; reproduction Bolivian cacao 101
Asian plantation industry 84 Borneo 123–124
Aspects of Tropical Mycology, proceedings Botrytis 90
British Mycological Society Brazilian cacao organization (CEPLAC)
meeting 1992 xi, 3 95–96
Aspergillus 146, 154, 157, 183 breeding for resistance 107
asterriquinones 175, 177, 179 British Isles studies 120–121
athletes foot 148 British Mycological Society xi
Australifungin 181
cacao xiii, 83, 91–107

Bahia 96, 97, 103, 107 Caldariomyces 33, 34, 35, 36, 37–39
Bambusoideae 70 Candida 146, 151–154, 157, 182
barrier, Andes 101 Candida albicans, inhibitors 179
basidiomycetes xiii, 58, 83–107 canopy feeders 124
basidiospores 93 canopy zone species 118–120
Beauveria 138, 139 Capnodium citri Pers 33
Belgrave, W.N.C. 89 carbon cycle 4
Bellis perennis L. 5 cattle ringworm 149
Berkeley, M.J. (1803–1889) 33 causal agents
Bignoniaceae 94 Colletotrichum 92
bioactive compounds, novel 4–5 Frosty (Monilia) pod rot of cacao
bioactive natural products screening 99–100
programme 173 Microcyclus ulei 85–89
biocombinatorial production of Moniliophthora roreri 99–100
synthetic NPs 184–185 South American leaf blight 85–87
biocontrol see biological control witches’ broom disease 92–94
biodiversity component 2–3 CEPLAC 95–96
Biodiversity of Tropical Microfungi 2 Chaetopsina 46, 47
biofungicide 90 checklists 114
biological control chemical control 90, 97, 104
agents see also biological control
Clavicipitaceae 138–140 chestnut blight 83
source 5 Chinese Cordyceps 140
Trichoderma stromaticum 98 chirimoyas 95
Index 197
Chloridoideae 72, 73 see also biological control

chromoblastomycosis 145–146, 155, cupuaçu 96
157 cutaneous lesions, Penicillium marneffei
Ciferri, R. 99 161
Ciferrioxyphium 34, 35–36, 37, 38 Cyperaceae 64
Cladobotryum amazonense 97–98
Clavicipitaceae 135, 136–140, 141–142
Coccidiodes immitis 146, 157 deep-seated infections, human 146
Coccidioidomycosis 157, 162 Delitschia confertospora 174–175
cocoa see cacao Dennis, Reginald W.G. vii
coelomycete on Pandanus 54 dermatophyte
coevolution animal species 148
host, grasses, downy mildews xiii, arthroconidia, skin 151
63–77 fungi genera 147
Sclerosporales and Poaceae 71–76 mycelium, skin 151
Colletotrichum 92 pathogens of humans 147
combinatorial chemistry 141 ringworm 145, 146–151
commercialization, tropical fungal Desmaziéres, J.B.H.J. 33
metabolites, obstacles to destabilized economies 106
185–187 dimorphic fungi 146, 162
conidia 100 Dipterocarpus 122, 123
Conidiocarpus 36, 37, 38 discomycete connections 43–44, 45
Conidiocarpus Woron 34, 36 Discula destructiva 83
conidiophores 46, 47 disease
Conidioxyphium 38 fungal 145–163
connections, Ingoldian fungi 41–47 human 145–163
see also anamorph; teleomorph resistance 90–91, 98, 105, 107
control tree crop 84–107
Moniliophthora roreri 103–105 see also infections; pathogen
South American leaf blight 89–91 Diseases of Cultivated Plants and Trees 92
witches’ broom disease 97–98 disjunct communities 122–123
see also biological control distribution and impact
Convention on Biological Diversity 3, 5, Crinipellis perniciosa 94
167, 169, 179, 186 Frosty (Monilia) Pod rot 100–103
cordon sanitaire 96 Microcyclus ulei 88
Cordyceps 137, 138, 139, 140 Moniliophthora roreri 102
corticolous species 116 Nodulisporium sp. 178
Costa Rica 121 Peronosclerospora sp. 73
Couch, J.N. (1896–1986) 135 Sclerospora and Sclerophthora 75
Crinipellis perniciosa 92–94, 96, 106 South American leaf blight 87–89
crustose communities 118–119 witches’ broom disease 94–96
crustose taxa 117 see also migration; mycogeography
Cryphonectria parasitica 83 dogwood anthracnose 83
cryptococcal meningitis 159 Dothidella ulei 84
cryptococcosis 158–159 downy mildew of grasses xiii, 63–77
Cryptococcus 157, 159, 182 drug discovery, natural product research
cultural control 97, 103–104 167–168
198 Index
ecology 4, 68–70, 118–119 human interrelationship xiv,

economies, destabilized 106 145–163
ecosystem, lichens 123–124 plant interrelationships xii, 51–60,
Ecuador 95, 98, 100–103, 105 63–77, 83–107
endophyte isolates 173 fungicidal isoxazoles 69
Entomophthorales (Zygomycota) 134, fungicides 69, 70, 90, 97, 104
135 Fungus Flora of Venezuela and Adjacent
epidemiology 68–70 Countries vii
Epidermophyton 147, 150 Fusarium xii, 6, 154, 176
ethnomycology 140 Fusicladium 85, 90
European studies 120–121
Eurotiales 141
evolved associations with invertebrates, Ganoderma-based preparations 7
hot-spots 134–136 genetic modification 141
expeditions xi genomic era 182–185
geographical area of origin, gramini-
colous downy mildew xiii,
fire 121–123, 125–126 71–76
floras 114 germplasm collection 107
flu transcriptase inhibitor 174–175 Global Biodiversity Assessment 2–3
flutimide 174–175 global ecological processes 4
foliicolous lichens 116, 120 Global Information System for
Fonesecaea pedrosoi 157 Lichenized and Non-lichenized
foot mycetoma 155, 156 Ascomycetes (LIAS) 118
foot ringworm 148 global keys 117
Ford, Henry (1863–1947) 88–89 graminicolous downy mildew xiii,
forest management, factors affecting 71–77
lichens 125–126 graminicolous Peronosporomycetidae
forests, tropical 113–126 species 65–66
fosetyl-A1 70 Graphidiaceae, rounded thalli 119
freshwater fungi 41–47 Graphium putredinis 179, 180
Freycinetia 51–52, 54, 55–58 grasses 70–74
frost 98–99 groin infections 147
Frosty (Monilia) Pod rot, cacao 98–105, Guyana 95
106 Guzmán, G. 9
Fumago Pers 33, 34
Fumonisin B-1 181
fungal diseases 145–163 hallucinogenic effect, mushrooms 7
Fungal Diversity 2 Hawksworth, D.L. xiii, 133, 136
fungal species occurring on, Pandanaceae hazards to travellers 145–163
58 helada 98–99
fungal sphingolipid biosynthesis Helicomyces roseus 45–47, 46
180–182, 181 Hennings, P. 84
fungi herbal medicines 140
animal interrelationship xiv, see also natural products; novel sec-
145–163 ondary metabolites;
described from the Pandanaceae pharmaceuticals
52–55 Herrania spp. 100, 101, 105
Index 199
Hevea 84, 86, 87 Hypocreales 135, 141, 173

hidden inoculum 97 Hypocrella 136–138
high-throughput screening (HTS) hypoxylon 176–177
technology 167–168, 173 Hywel-Jones, N. 9
Hirsutella 138, 139–140
Histoplasma 146, 160, 161
histoplasmosis 157, 160–161 identification keys
HIV-1 protease inhibitors 179 lichens 117–118
Homoptera 135, 137 Nectria-like fungi, tropical species key
Hospitalitermes 123–124 13–30
host indicators of changes induced by fire,
co-evolution, grasses/downy mildews lichens 121–123
xiii, 63–77 indicators of ecological continuity,
genera, graminicolous downy mildew lichens 120–121
67, 71 industrial mycology xiv, 165–187
Microcyclus ulei 89 infections
–pathogen interaction human 145–163
Peronosclerospora/Andropogoneae pod 100
67, 72, 73 Ingoldian fungi 41–47
Peronosclerospora/Zea 72 inhibitors
preference, graminicolous downy antifungal sphingolipid 180–182
mildew 64–67 Candida albicans 179
range flu transcriptase inhibitor 174–175
graminicolous downy mildew natural products, sphingolipid
64–67 biosynthesis 181
invertebrate–pathogenic fungi protein synthesis elongation
134–136 179–180
Phytophthora 71–72 inoculum, hidden 97
Pythium 71–72 inositol phosphoceramide synthase 182
rubber tissue malformations 86, Insecta 133–142
106 insecticide 176–177
hot-spots, evolved associations with insects 135, 137
invertebrates 134–136 insulin mimic 177–179
HTS (high-throughput screening) Inter-African Phytosanitary Commission
technology 167–168, 173 90
Hughes, Stan xii International Biological Programme 8
human International Mycological Association 9
culture 7 invasive Neotropical pathogens, tree
fungal diseases 145–163 crops 83–107
fungal/human interrelationship xiv invertebrate-pathogenic fungi xiii-xiv,
health 4–5, 6 133–142
infections 145–163
mushroom consumption 6–7
pathogens 6 Kew Bulletin vii
see also anthropogenic factors keys
Hyde, K.D. 9 lichen identification 117–118
Hymexazol 69, 70 Nectria-like fungi, tropical species key
hyphomycetes 45, 53–54, 56–58 13–30
200 Index
Khafrefungin 181–182 Melanochaeta 43, 45, 46, 47

Khao Yai National Park, Thailand 116 Melanopsammopsis 84
kranz anatomy 72 Merck Research Laboratories (MRL)
165
metabolic redundancy 169–171
Laboulbeniales 134–135
metabolites
Lantana camara L., control 5
commercialization, obstacles to
leaf blight 84–91, 106
185–187
Lecanicillium acerosum 97
pharmacologically active xii,
leg chromoblastomycosis 157
165–187
Leptolegniaceae 77
see also secondary metabolites
Leptoxyphium 34, 35, 36, 37–39
Metalaxyl resistance 69
LIAS 118
Metarhizium 138, 139
lichenologists 116
Mibey, R.K. 9
lichens
Microcyclus 84, 85–88, 89, 106
ecological studies 118–119
Microcyclus ulei 85–89
ecosystem 123–124
Microsporum 147, 150
forest management affecting
Migration xiii, 6, 74, 101
125–126
see also accidental introduction;
identification keys 117–118
distribution and impact;
indicators of changes induced by fire
mycogeography
121–123
mildew xiii, 63–77
indicators of ecological continuity
see also ascomycete; moulds; yeasts
120–121
Millennium Symposium, British
reproduction 120
Mycological Society, Liverpool
role in plant assemblages xiii
John Moores University, April
saxicolous 116–117
2000 xi
site diversity 115
Minimoidin 181, 182
species 113–114
misidentified species 37–39, 74, 76,
substrata 115
92
taxonomic literature 117–118
mitosporic centra, sooty moulds 35,
lignicolous fungi xiii, 41–47
36
Lobarion pulmonariae alliance 121
mitosporic fungi 57, 58
logging 125
mitosporic structure, sooty moulds 34,
Lücking, Robert 116
35
models 187
Madurella grisea 156 Monilia
Malassezia 152, 153 Cryptococcus species 157
mancha 98–99 Frosty (Monilia) Pod rot, cacao
Marasmius 92, 93 98–105
Massee, G. 92 Microsporum 147, 150
medicinal products 166 Penicillium 154, 161, 162
medicine ringworm 145, 146–151
herbal 140 Moniliophthora roreri 99–107
traditional 7 morphology 68
see also natural products; novel moulds
secondary metabolites; saptrophic 154
pharmaceuticals sooty, xii, 33–39
Index 201
taxonomy reassessment 33–39 obligate biotrophic parasitism 68

water 77 oomycetes 58, 63–77
see also ascomycete; mildew; yeasts organisms survival 3–4
mushroom 6–7
mutualisms 3–4
mycetoma 145–146, 154–155, 156 Pandanaceae xiii, 51–60
mycochemical fungicide 97 Panicoideae 72, 74
mycogeography xii, 71–76 pantropical hypoxylon 176–177
see also distribution and impact; paracoccidioidomycosis 157, 162
migration parasites 64, 72, 135
mycoparasites 97–98, 104 Parodi, E. 99
mycoses 145–163 parthenocarpic pods 95
see also disease; infections; pathogen pathogen
Mycotic keratitis 154 dermatophyte 147
mycotoxins 6 frosty pod 106
myxomycetes 58 host interaction 67, 72, 73
human 6, 147
invasive Neotropical xiii, 83–107
‘Nacional’ variety 95, 105 plant 5–6
nail infections 147, 149, 154 rubber leaf blight, taxonomic
natural products placement 106
biocombinatorial production of see also disease; infections; mycoses
synthetic NPs 184–185 pathway inhibitors 180
discovery, genomic era 182–185 penicillin 4
drug discovery 167–168 Penicillium 154, 161, 162
fungal-derived 165 Peronophythora 76
inhibitors, sphingolipid biosynthesis Peronosclerospora sp., distribution 73
181 Peronosporomycetes 63–77
laboratories 185 Persoon, C.H. (1762–1836) 34
screening 172, 173 Peru 102, 103, 107
see also medicine; pharmaceuticals; Petch, T. 84, 137, 138
secondary metabolites Phaeoisaria clematidis 45, 46, 47
Nectria xii, 13–30, 43, 46–47 pharmaceuticals 4, 141
Neotestudina 6 pharmacological compounds xiv, 4
Neotropical pathogens xiii, 83–107 pharmacologically active metabolites xii,
new species 41, 42 165–187
New Zealand 51–60 phenylamides 69
nodulisporic acids 175, 176–177 phosphonate fungicides 70
Nodulisporium sp. origin and distribution physiology 68–70
178 Phytophthora 70, 76, 83
Nomuraea 138, 139 Pityriasis versicolor 152, 153
Non-Green Revolution 7 plant diseases 84–107
non-peptidyl insulin mimic 177–179 plant pathogens 5–6
novel secondary metabolites 133, 140, plant pathology perspectives 63–77
141–142 Plant Protection Committee for the
see also metabolites; secondary Southeast Asia and the Pacific
metabolites Region 90
nuisance metabolites 172 Poaceae 64–65, 70–71
202 Index
pod disease types 98 Sclerophthora 69, 75

pod infections 100 Sclerospora 75
pod rot 98–105, 106 Sclerosporales 64–77
poisonous fungi 6 Scolecoxyphium 34, 36
Polychaeton 34, 36, 37 Scopulariopsis brevicaulis 154
polyketides 169, 170, 184–185 Scorias Fr. 35–36
Pound, F.J. 98 Scotland, surveys 115
prevention Scytalidium dimidiatum 154
Frosty (Monilia) Pod rot, cacao 103 secondary metabolites
South American leaf blight 89–90 biosynthetic genes 183
witches’ broom disease 97 pharmaceutical compounds xiv
Project Surumoni 115 sources 133, 140, 141–142
propagules transport 124 see also metabolites; natural products
protein synthesis elongation inhibitors Seibert, R. 91
179–180 Septobasidiales (Basidiomycota) 134,
Pythium 4 135
Pyxine conscians Vain 126 Sérusiaux, Emmanuël 116
sexual reproductive organs
Peronosporomycetes 68
Quevedo disease 99 see also anamorph; reproduction
Singer, Rolf (1906–1994) 92
site diversity, lichens 115
Sixth General Meeting, British Mycology
rainforest loss 125–126
Society, Birmingham 1988 xi
regional keys 117
skin infections 145–146, 151,
re-identified herbarium specimens 38
154–155, 156
reproduction 68–69, 120
Smith, R.E. 99
see also anamorph
sooty moulds xii, 33–39
research laboratories 185
see also ascomycete; mildew; moulds;
resistance, Moniliophthora roreri 104
yeasts
resistance breeding 90–91, 98, 105,
sordarins 179–180
107
South American leaf blight of rubber
ringworm 145, 146–151
84–91, 106
Rondonia 96, 101
South-East Asia xiii, xiv, 41–47
Rorer, J.B. 87, 92, 99, 100–101, 105
species
rubber xiii, 83–91, 106
canopy zones 118–120
rust 5
keys 13–30, 117–118
misidentified 37–39, 74, 76, 92
new 41, 42
saprobic phylloplane fungi 33–39 taxonomically misplaced 136–137
Saprolegniomycetidae 69, 77 teleomorph, new 42
Sapromyces 70 timber 42
saprotrophic moulds 154 total numbers, lichens, tropics
Sararanga 51, 52, 57, 58, 58 113–114
saxicolous lichens 116–117 sphingolipid synthesis 180–182
scale insects 137 Sporoschisma 43, 46
scalp infections 150–151 sporotrichosis 145–146, 155–156,
Schultes, R.E. 91 158
Index 203
Spruce, Richard (1817–1893) 90–91 Trans-Amazonian highway 95–96

Stahel, G. 84, 86, 92, 93, 105, 106 see also anthropogenic factors;
subcutaneous mycoses 154–157 migration
substrata, lichens 115 trans-Andean oil pipeline 101
superficial infections 154 see also anthropogenic factors;
superficial mycoses 146–154 migration
Surinam 92, 95 travellers 145–163
Surumoni 120 tree crops xiii, 83–107
sustainable development contribution Trichoderma stromaticum 97–98
6–7 Trichomycetes 135
systematics xii, 13–47 Trichophyton 147, 148, 149, 150
systemic mycoses 157–162 Tricovab 98
Trinidad 97
tropical fungi defined 168–174
taxonomy Tropical Mycology 2
base-line xii, 13–30 Tubeufia cyclindothecia 45–47, 46, 47
literature, lichens 117–118 Turrialba Research Station, Costa Rica
mildew xiii, 64, 65–67 91
misplaced species 136–137
placement, rubber leaf blight
pathogen 106 UK apple germplasm collection 107
reassessment, moulds 33–39 Ule, Ernesto 84
witches’ broom disease 92 ultrastructure 68
teleomorph understorey community 120
anamorph connections 41–42, 44,
45, 46, 47
Cordyceps 137 Van Hall, C.J.J. 98
Fusicladium 85 Verrucalvaceae 64, 69–70
genera 139 vertebrates xii, 133–142
Hypocrella 136 Verticillium 138
Ingoldian hyphomycetes 45 Vezda, Antonin 116
new species 42 Viridiofungin A 180–181
retained 138
stage, lignicolous freshwater higher water moulds 77
fungi 41–47 Weber, H.J. 33
see also anamorph; reproduction White Agriculture 7
temperate taxa xii–xiii Wilson, Edward 8
termites 3, 123–124 witches’ broom disease 91–98, 102,
Thailand 41–47, 120 107
thalli 119
Thelotremataceae 119
Theobroma 91–98, 100, 101, 104, 105 Xylariales 173
timber species 42
Tinea nigra 154
yeasts 146, 151–154, 159, 161
tissue malformations, rubber host 86,
see also ascomycetes; mildew; moulds
106
Torrubiella 137, 138
Trachysphaera 76 Zygomycota 134, 135

Micología Tropical - Micromycetes

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Tropical Mycology:

CABI Publishing CABI Publishing

Library of Congress Cataloging-in-Publication Data

ISBN 0 85199 543 8

Typeset in Photina by Wyvern 21 Ltd.

1 Why Study Tropical Fungi? 1

2 Key to Tropical Species of Nectria-like Fungi 13

3 A Reassessment of the Taxonomy of Some Tropical Sooty Moulds 33

4 Lignicolous Freshwater Higher Fungi with Reference to their

5 The Pandanaceae – Does it Have a Diverse and Unique Fungal

6 Aspects of Graminicolous Downy Mildew Biology: Perspectives for

7 Invasive Neotropical Pathogens of Tree Crops 83

8 Lichens of Tropical Forests 113

9 The Importance of Invertebrate-pathogenic Fungi from the Tropics 133

10 Tropical Mycoses: Hazards to Travellers 145

11 Recent and Future Discoveries of Pharmacologically Active

It is with great pleasure that this volume is dedicated to Dr Reginald W.G.

Nigel L. Hywel-Jones, BIOTEC–Mycology, Yothi Laboratories, 73/1 Rama 6 Road,

papers which offer a wide spectrum of information, linking results from

pathogenic on spiders and mites and that entomogenous or even entomopath-

Departamento de Biología Vegetal II, Facultad de Farmacia,

© CAB International 2002. Tropical Mycology, Vol. 2, Micromycetes 1

Because they are . . .

(1) . . . a major component of biodiversity

(2) . . . essential to the survival of other organisms

Fungi can be essential to the survival of other organisms at the individual,

appreciated (Bacon and White, 2000), connections and nutrient transfers

(3) . . . crucial in global ecological processes

(4) . . . a source of novel bioactive compounds

(5) . . . a source of biocontrol agents

The biocontrol of insects and weeds is high on the agendas of developed

(6) . . . a source of plant pathogens

(7) . . . a threat to human health

There is always a need to know the poisonous fungi in an area, to document

(8) . . . able to contribute to sustainable development

(9) . . . a part of human culture

Certain mushrooms have long histories as a part of human cultures: in

(10) . . . and there

In 1992 I forecast that, for tropical mycology to progress, the prerequisite

Fungi historically identiﬁed as Nectria are a conspicuous element of the

© CAB International 2002. Tropical Mycology, Vol. 2, Micromycetes 13

Key to Common Tropical Species of Nectria, Genera

Species with white, yellow, orange or brown perithecia, not turning

1. Ascospores averaging 15 µm or less in length ..........................................2

4. Perithecial hairs cylindrical, hyphal; ascospores 5.5–7 ¥

pale golden hyphae; ascospores 7.5–10 ¥ 1.5–2.5 µm,

22. Ascospores averaging 5–6 µm in width.............................33

30. Perithecia yellow to orange or brown, smooth to slightly

around the apex; on herbaceous and woody debris...............

48. Ascospores smooth or only faintly striate..........................50

67. Perithecia completely immersed in stroma, stroma appearing

Species with yellow-orange, orange, red to purple perithecia, becoming

chaetopsinae-polyblastiae (Samuels) Rossman & Samuels

19. Ascospores smooth, 13–18 ¥ 5–8 µm; anamorph Fusarium

27. Ascospores smooth; anamorph Cylindrocladium; on decaying leaves,

45. Ascospores at most pale yellow-brown, striations ﬁne, sometimes difﬁ-

53. Ascospores 15–19.5 ¥ 6.5–8 µm, roughened, rarely smooth;

61. Ascospores yellow-brown, smooth, 18–21 ¥ 6.5–7.5 µm; perithecia

71 (66). Perithecia typically formed at the base of red synnemata, superﬁ-

© CAB International 2002. Tropical Mycology, Vol. 2, Micromycetes 33

phylloplane habitat, and this creates superﬁcial morphological similarities,

A total of 272 isolates, including herbarium specimens and living cultures,