PART I. PHYSIOLOGY 1 ISOLATION 1 CULTURE MEDIA 2

MAINTENANCE OF STOCK CULTURES 2 PHYSIOLOGY: NUTRITION AND ENVIRONMENT 3 USE OF IMPERFECT FUNGI TO ILLUSTRATE BIOLOGICAL PRINCIPLES 4

PART I. TAXONOMY AND IDENTIFICATION 6

THE SACCARDO SYSTEM OF CLASSIFICATION 6 FAMILIES OF MONILIALES 7

KEY TO GENERA 8

MUCORALES 8 MONILIALES 9 HELICOSPORES 9 NOT HELICOSPORES 10 MONILIACEAE 10 DEMATIACEAE 17

TUBERCULARIACEAE 25 STILBACEAE 26

SPHAEROPSIDALES 28 MELANCONIALES 3 MYCELIA STERILIA 34

SIMPLIFIED KEY TO SOME SELECTED COMMON GENERA 35 THE HUGHES-TUBAKI-BARRON SYSTEM OF CLASSIFICATION 40 vii http://arab2000.forumpro.fr

ALTERNATE KEY TO SERIES AND GENERA 41 ARTHROSPORAE 4 MERISTEM ARTHROSPORAE 4 ALEURIOSPORAE 45 ANNELLOSPORAE 48 BLASTOSPORAE 48 BOTRYOBLASTOSPORAE 50 - POROSPORAE * 51 SYMPODULOSPORAE 52 PHIALOSPORAE 5

DESCRIPTIONS AND ILLUSTRATIONS OF GENERA 59

REFERENCES 198 GLOSSARY 212 INDEX TO GENERA 216 vill http://arab2000.forumpro.fr

The Deuteromycetes or Fungi lmperfecti (former taxonomic designations) are an anomalous, heterogeneous assemblage of asexual ascomycetes and basidiomycetes which no longer have formal taxonomic status. These fungi were traditionally considered as lesser fungi because they lacked the perfect stage—sexual reproduction. The absence of asci (ascomycetes) and basidia (basidiomycetes) prevented their assignment to a natural taxon and necessitated .artificial non-sexual characteristics to describe and classify them. This genetic inability of many imperfects to reproduce sexually is considered a primitive condition and in contemporary mycology presents a taxonomic quandary. Alexopoulus et al, 1996, provide excellent scientific rationale for excluding imperfect fungi from contemporary fungal systematics, and discuss considerations needed to develop logical and valid taxonomic approaches to determine their phylogeny (1). Consequently, the taxons which previously were recognized as taxonomically valid for the deuteromycetes (imperfect fungi), are used in this book only to facilitate their identification.

The imperfects are important eucaryotic microorganisms (possessing nuclei and organelles) which affect humans and most other life forms in a myriad of ways. The need to determine their identities is paramount in research, industry, medicine, plant pathology and in many other disciplines. Imperfect fungi are identified according to their conidial or non-sexual states. Nevertheless, many imperfects possess sexual structures of known ascomycetes or basidiomycetes, whereas others produce no conidia and/or sexual structures. Roper, 1966, described a parasexual cycle in which genetic recombination can occur in hyphae (16). This observation suggests that some fungi may never have possessed sexual structures or required sexual reproduction for genetic exchange. However, while there is little data which substantiates that pansexuality occurs under natural conditions today, it could have occurred during the origin and evolution of these fungi.

When sexual structures are associated with the conidial state, a valid taxonomic status can be ascribed.

However, this often does not occur, and for practical purposes is not important. Although the scientific name of the sexual state constitutes a valid taxonomic designation, the imperfect name is retained for practicality and for conventional use. Therefore, to identify the imperfect fungi, it is necessary to know their conidial morphologies regardless of whether the sexual state is also present in culture or in nature.

The deuteromycetes constitute an important group of fungi which require continued study despite their obscure and confounding systematic relationships both to themselves and to other fungi. Barron, 1968 (2), Hunter and Barnett, 1973 (10), Hunter « tf al., 1978 (1), and Alexopoulus et al. (1) provide additional information on many aspects of the morphology, sporulation, growth, ecology and economic importance of imperfect fungi.

Scanning electron and light photomicrographs are provided on several of the following pages. They show conidia, conidiophores, and hyphal structures found on many different kinds of imperfect fungi. Compare them with like illustrations in the book to better understand how these structures are important in identifying imperfect fungi.

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The imperfect fungi or deuteromycetes have been classified according to principles established by

Saccardo in Sylloge fungorum (17). While this taxonomic system is no longer valid, it is still the best way to learn the mycology that is necessary for identifying the imperfect fungi. It is also the primary means used in this book to identify imperfect fungi. The scientific names of imperfect fungi are still used, albeit, only in a non-taxonomic sense, and as a necessity to know their practical importance in the biosphere. The Hughes-Tubaki-Barron System (conidial ontogeny) has also been used as a way of classifying and identifying these fungi (2, 9, 18). Details pertaining to this system are provided on pages 40-4 and related identification keys are found on pages 4-57. The use of conidial and conidiophore ontogeny for identifying deuteromycetes should be used by individuals who are well versed in mycology. The shape, pigmentation, and septation of conidia are important characteristics in the Saccardo System but reduced to secondary importance in the Hughes-Tubaki-Barron System.

To better understand the Saccardo System, common and economically-important imperfect fungi of the four form orders will be presented. Following the Saccardoan System, the species of the form orders can be separated into four distinct groups of fungi. This provides a basis from which to begin a search (appropriate key) for the identity of an unknown fungus. The form orders are as follows: (I) Moniliales - Conidiophores and conidia occurring free and distributed over the mycelium. Conidiophores may be separate, in clusters, or in tightly-packed groups. Illustrative examples and accompanying descriptions of many of the diverse genera in this group are provided from pages 68 through 161; (2) Sphaeropsidales - Conidiophores and conidia contained within asexual fruiting bodies called pycnidia. See pages 162 through 187 for descriptions and illustrations of pycnidia-producing fungi. (3) Melanconiales - Conidia typically produced under natural conditions in an acervulus, an open saucer-shaped fruiting body. In culture, conidiophores may be single or in compact groups similar to sporodochia of the Mormiaies. These fungi can be found on pages 188 through 194; (4) Mycelia Sterilia - Species in this form order are genetically incapable of producing conidia or any kind of reproductive cells. Sclerotia or other survival structures occur in the mycelium. Descriptions and illustrations of the three species depicted in this book are provided on pages 196 and 197.


Conidiophores ot Paecilomyces sp. with typical flask- Conidia of Trichoderma sp. emerging from apices ot the shaped phialides and catenulate conidia. conidiophores.

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Two of the spomlating form orders, Moniliales and Sphaeropsidales can be separated into several form families. Characteristics are predicated upon such artificial features as color, shape, and consistency of the pycnidium in the Sphaeropsidales, or color of the conidia and presence of synnemata or sporodochia in the Moniliales. The form family taxon is not used in Mycelia Sterilia and only one form family exists in the Melanconiales.

There are at least 1,400 form genera of imperfect fungi and several thousand species. The most common in nature and the most economically important are found in the form order Moniliales. Some are pathogens of plants, animals and humans, some produce toxins, while others are important in the production of antibiotics and other chemicals. In the Saccardo System, it is the color and morphology of the conidia which are used to separate form genera into sections. For example, one-celled hyaline (devoid of any color) conidia are called hyalospores; colored, one-celled conidia are phaeospores; didymospores are two-celled; and transversely septate conidia with three or more cells are phragmospores. Add hyalo to phragmospore (hyalophragmospore) and it is a hyaline, transversely septate conidium; cylindricallyspiraled, one to several cell formations are helicospores, regardless of the presence or absence of color. Problems encountered when using the Saccardo system are variations in type of fruiting body (acervulus, sporodochium, and pycnidium), conidium color and conidium morphology. These structures can vary on different media and in their response to varying environmental conditions. Consequently, what is described in the keys may differ slightly to significantly when the fungus in question is grown on different media or when it is incubated at different temperatures. Nevertheless, time and experience will negate these factors. Therefore, because of its simplicity and practicality, the Saccardo System is still the best way for students and others to study and identify imperfect fungi.

Most species of deuteromycetes reside in this form order and are grouped into four form families (see page 7). This is the only form order in which form families are described in this book. Form families Moniliaceae and Dematiaceae have species which are delimited by one or more of the following


Conidia In basipetal chains radiating from the apex of an Aspergillus sp. conidiophore.

xi http://arab2000.forumpro.fr characteristics: conidial septation; conidiophore appearance and branching; conidial morphology; true and pseudomycelium (some imperfects are yeasts without true hyphae); the manner in which the conidia are produced; presence of chlamydospores and morphology; conidia produced in chains or in a head; presence or absence of mucilage; conidial number and arrangement at apex of the conidiophore; conidia produced on conidiophore or mycelium; and exogenous or endogenous production of conidia. Refer to page 68 through page 145 for numerous examples of the Moniliaceae and the Dematiaceae. Note that imperfects in this form order with hyaline conidia are members of the Moniliaceae; those with pigmented conidia and/or conidiophores reside in form family Dematiaceae. The reason that the fungi of these two form families are discussed together is because the only difference between the species is the color of their conidia and conidiophores. This seemingly obvious color difference is at times difficult to determine in culture and under the microscope. However, careful use of the microscope, diligence and experience in identifying these and other fungi, will in time allow orje to make accurate determinations of pigmentation, along with many other pertinent fungal characteristics.

Many of the more common fungi are found in the form families Moniliaceae and Dematiaceae.

Species of Aspergillus (page 95), Penicillium (page 95), Alternaria (page 132) and Stemphylium (page 132) are routinely isolated from the air and numerous other substrates. These genera and several other species of the Moniliaceae are discussed here. Aspergillus fumigatus is an opportunistic pathogen of humans and other animals and is responsible for the human disease aspergillosis, a pulmonary disorder. Penicillium chrysogenum and closely related species are the sources of penicillin, an important antibacterial antibiotic, which has saved countless humans from death and serious illness for many decades. Other species of Penicillium are responsible for the contamination of food and clothing. Gl'tocladium spp. (page 93) are similar to the penicillia, but differ at maturity by having the spore mass encompassed by mucilage. One species, G. roseum is a good example where identification is confusing because it produces two different conidial types, one being the Gliocladium type and the other that of Veriicillium albo-atrum (page 92). Fortunately, this is unusual, but warns one to not always consider fungal cultures contaminated when two distinct conidial types occur in the same culture. Verticillium albo-atrum is a destructive plant pathogen that causes a wilt of some economically-important plants. Monilia (page 73) cinerea var. americana, the pathogen of brown rot of peach and other fruits, is often found as a contaminant of microbial cultures. Geotrichum candidum (page 68) is the causative agent of geotrichosis, a human disease which can occur orally, in the intestine and as a pulmonary disease. Species of the genus Candida (page 71) are common in the Moniliaceae. Note that this fungus is not always filamentous, but can possess yeast-like cells. An important species C. albicans, is an opportunistic human pathogen causing oral and vaginal diseases and may become systemic. This filamentous yeast can be differentiated from other Candida spp. by the production of S to 12 pm spherical chlamydospores on corn meal agar.


One-celled Gliocladium sp. conidia in mucilaginous masses on penicillate branches of conidiophores. xii http://arab2000.forumpro.fr

Many species having pigmented conidia and/or conidiophores, reside in the form family Dematiaceae.

Many of these species are also common and/or economically-important fungi. Stachybotrys (page 89), a soilborne saprotroph, has pigmented single-celled conidia and conidiophores that slime down to form glistening beads. Cladosporium (page 107) is prevalent in the air, and some species are plant or human pathogens. This fungus has a highly branched conidiophore and one-or two-celled conidia that occur in chains. Since all conidia of one species are not always of the same cell number or size, purity of a culture cannot be determined by this means. Aureobasidium (page 71) is a filamentous yeast, hyaline when young, becoming dark with age. Aureobasidium is often confused with species of Candida, but pigmentation appears in its hyphae which is not found in Candida. One species, A. pullalans is saprotrophic, but can become an opportunistic pathogen of plants. This same fungus is also known to be a major agent in the deterioration of painted surfaces. Many species of Helminthosporium (page 125) are well known to plant pathologists as pathogens of grasses. These fungi produce dark cylindrical conidia, which are multiseptate and usually have rounded ends. The conidia of Bipolaris (page 127) and Dreschlera (page 123) are nearly identical to those of Helminthosporium but differ in the mode of conidial formation. The ends of the conidia vary only slightly making the differentiation of species between Bipolaris, Dreschlera and Helminthosporium difficult. Illustrations along with the keys are most helpful in correctly identifying species of these three genera. The most commonly encountered fungus in the Dematiaceae is Alternar'ta (page 133), which produces large muriform conidia, often borne acropetally in chains. Isolates of this fungus are readily recovered from air, soil, decaying vegetation and from diseased potatoes and tomatoes.