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CBS COURSE OF MYCOLOGY Third edition W. Gams H. A. van der Aa A. J. van der Piaats-Niterink R. A. Samson j. A. Stalpers 1987 third edition CENTRAALBUREAU VOOR SCHIMMELCULTURES BAARN DELFT INSTITUTE OF THE ROYAL NETHERLANDS ACADEMY OF ARTS AND SCIENCES CIP-GEGEVENS KONINKLUKE BIBLIOTHEEK, DEN HAAG CBS CBS course of mycology / W. Gams ... [et al.]. - Baam: Centraalbureau voor Schimmelcuitures.111. . Met lit. opg. LX I ISBN 90-70351-12-9 geb. SISO 587.2 UDC 582.28 Trefw.: schimmels. pr Copyright © 1987 by Centraalbureau voor Schimmelcuitures, BAARN I' All rights reserved. No part of this work covered by the copyright hereon may be reproduced or used in any form or by any means - graphic, electronic, or mechanical, including photocopying, recording, 1 iaping, or information storage and retrieval systems - without permission of the publisher. Published and distributed by Centraalbureau voor Schimmelcuitures, P.O. BOX 273,3740 AG BAARN, The Netherlands. pxsr ! ;pg-z.9T«f gz-o CBS COURSE OF MYCOLOGY CONTENTS I. INTRODUCTION 1 II. METHODS 3 A. Aseptic working 3 B. Preparation of media 3 C. Choice of media, incubation 6 D. Isolation techniques and ecological groups of fungi 7 1. Single-spore cultures 7 2. Water moulds g 3. Soil fungi 9 4. Fungi on living plants 13 5. Fungi on seeds 14 6. Fungi with fleshy sporocarps 15 7. Fungi on decaying wood 15 8. Fungi on dung (coprophilous fungi) 15 9. Entomogenous fungi ]g 10.Thermophilic and thermotolerant fungi 16 E. Microscopic examination (light microscopy) 16 !. Optical equipment 16 2. Mounting fluids 17 3. Preparations 17 4. Permanent mounts 19 5. Other stains 19 F. Submicroscopical techniques 20 G. Preservation of living cultures 20 H. Herbarium techniques 21 III. THE FUNGAL SYSTEM 22 Introduction 22 Nomenclature 22 IV. THE DIVISIONS AND CLASSES OF FUNGI 25 Mvxomycota 25 Chytridiomycota 27 Oomycota 30 Zygomycota 36 Ascomycota 42 Hemiascomycetes 42 Ascomycetes 45 Basidiomycota 53 Heterobasidiomycetes 56 Homobasidiomycetes 60 Deuteromycota 64 V. REFERENCES 86 Methods 86 a) Textbooks 86 b) Some special papers 86 c) Soil fungi • 87 d) Seed fungi 89 e) Coprophilous fungi 89 Guide to the taxonomical literature 89 a) Textbooks and general works 89 b) Myxomycetes 92 c) Chytridiomycetes 93 d) Oomycetes 94 e) Zygomycetes 95 f) Hemiacomycetes 97 g) Ascomycetes 97 h) Basidiomycetes 106 i) Deuteromycetes 118 Applied Mycology 125 a) Fungi in biodeterioration 125 b) Fungi in food 126 c) Mycotoxins 126 d) Antibiotics 128 e) Medical mycology 128 The most important periodicals 130 VI. INDEX OF TECHNICAL TERMS ■ 133 OUTLINE OF THE FUNGAL SYSTEM inside back cover St I. INTRODUCTION The mycology course given every year at the Centraalbureau voor Schimmel cuitures at Baarn is mainly intended as an introduction to systematic mycology with emphasis on working with living pure cultures. Practical work with selected examples of the various fungal groups helps to build up a knowledge of the fungal system. This guiding text contains a fuller description of the techniques used than can be given in the short introductory lectures, with indications of how to deal with and to identify the demonstrated fungi. The study of pure cultures has many advantages over that of working with fungi on natural substrates and is usually essential for a reliable identification of zygomycetes, ascomycetes and deuteromycetes. The work with pure cultures re quires careful manipulation and working under sterile conditions. With other groups of fungi (myxomycetes, some aquatic fungi, some ascomycetes and, in particular, most of the basidiomycetes). the whole development can be observed only on the natural substrates or in mixed cultures. A range of parasitic fungi cannot be grown in vitro at all. Some representatives of these groups wiil be demonstrated on the natural substrate. The arrangements of the fungal system is subjected to many changes as a consequence of intensified research in recent years. In each textbook a different arrangement is presented. For this introduction we follow mainly von Arx (1974), Ainsworth et al. (1973/74), Alexopoulos (1962 and 1979), Muller & Loeffler (1982) and Gams (1979). The principal steps of a morphological study are: (1) a cursory observation with the naked eye and low power of the microscope, (2) detailed study of squash mounts or sections, (3) preparation of an accurate description with drawings (pre ferably with a camera lucida), and (4) consultation of the literature for identi fication. It is particularly important to make drawings at a large scale. In some cases comparison with well identified cultures, specimens or drawings is indis pensable for a reliable identification. This guiding text consists of two parts: a) descriptions of general methods and b) short characterizations of the fungal groups demonstrated (with some spe cial techniques). The list of methods is limited mainly to cultural work and is not intended to be complete; for more exhaustive treatments the reader is referred to Booth (1971) and Stevens (1974). Some chapters have received particular atten tion: the ecology of soil fungi is treated in some detail; a text on nomenclature may serve as a guide to a critical evaluation of taxonomic literature in general; it will also explain why fungal names still often need to be changed. Certain groups of fungi are more fully treated than others, because of the emphasis on culture work; on the other hand, larger fungi and plant-parasitic fungi had to be underrepresented for the same reason. Recognition of the mode of conidium formation in deuteromycetes usually causes the greatest difficulties to beginners. Because acquaintance with these complicated structures is important for a reliable identification of the most commonly occurring moulds, many details are given in order to make the terminology understandable; again, it is not our intention to list all modes of conidium ontogeny. It is important not to rely only on one or two textbooks. Therefore a guide to the recent taxonomic literature has been incorporated. As a background to applied mycology, lists of recent references dealing with fungi in biodeterioration 1 and in foods, with mycotoxins, antibiotics, and medical mycology, have been appen ded. The writers are indebted to the authors and copyrightjiolders of the illustrations used for permission of reproduction. Dr. J. T. Mills, Canada Agriculture Research Station, Winnipeg, kindly corrected the English text of the first edition. The second edition (1983) of this booklet differed from the first one (1975) by some changes in the systematic arrangement, improved illustrations and updated references. The present, third edition has been further updated and A. W. A. M. de Cock joined the authors in taking care of the zoosporic fungi. Baarn, December 1986. O. METHODS A. ASEPTIC WORKING For transferring fungus cultures, aseptic working and sterility are essential. Inoculation and transfer is carried out with flame-sterilized autoclaved tools (Fig. 1). Media are usually autoclaved at I atm. (120°C) for 20-30 min, while glass Petri dishes are kept in an oven at 160°C for 3 h. For sterilizing working tables and the atmosphere, the benches can be wiped with a 4% formalin solution the evening before the work, or some concentrated formalin is evaporated by pouring it over a spoon-full of KMnO powder; the atmosphere can also be decontaminated with Aerosept fumes or spraying a diluted Tego solution, while Tego, ethanol or a 1% Zephirol solutions can be used for cleaning the benches. Idea! for obtaining clean cultures is the use of laminar-flow clean bench. Fig. 1. Equipment for inoculation: a. needle holder with pointed needle, b. flat tened needle, c. loop. d. streaking a loop over a Petri dish (from von Arx. Piizkunde. 1967). For transferring fungal cultures, fruit-bodies or conidial structures should be used if possible. Conidia or spores are usually streaked across the agar surface (Fig. Id). A general rule is to use as little inoculum as possible (exception some basidiomycetes). Cultures which produce a great number of dry conidia, like Penicilliumaad Asper gillus, can be transferred with the use of a wetted inoculum (needle wetted with agar, or conidial suspension in water); Petri dish cultures of Penicillium, Aspergillus and many other fungi are inoculated on three points, c. 2.5 cm apart; during inoculation and incubation they are kept upside down to prevent spread of conidia all over the plate. If possible, do not transfer sterile mycelium or agar blocks of sporul ating cultures, because this leads to degeneration and loss of sporulation capacity. B. PREPARATION OF MEDIA General procedure: Dissolve 15-20 g agar in 1 litre boiling water with the other ingredients in an Erlenmeyer conical flask of twice the volume of the medium or in an enamelled casserole. The molten agar is distributed in test tubes (10 ml for slopes in tubes, 15 ml for pouring Petri dishes) which are sterilized at an overpressure of 1 atm. (120°C) unless otherwise indicated. The subsequent recipes are for 1 1 with 15 g agar. For the suppression of bacterial growth. I ml of antibiotic solutions is added to Petri dishes before pouring out the agar, to give final concentrations of: penicillin-G 50 ppm, rifampicin 5 ppm streptomycin 30-50 ppm, chloramphenicol 50 ppm aureomycin 10-50 ppm. novobiocin 100 ppm neomycin 100 ppm. or vanomycin 10 ppm,. Of these compounds only chloramphenicol withstands autoclaving without loss of activity. 1. Carnation leaf agar (CLA, Fisher et at, 1982) Leaves of cultivated carnation (take care to get a fungicide-free batch) are cut into square pieces, gently dried and sterilized either by means of gamma irradia tion or propylene oxide. The sterile pieces are stored until usage. Spread 3-5 pieces in a small Petri dish with water agar. A suitable medium for identification of Fusarium. 2. Cherry-decoction agar Add 1 1 water to 200 g pulp of sour stone cherries. Heat to boiling and simmer gently for 2 h. Strain through cloth and sterilize at an overpressure of 0.5 atm. (110°C) for 30 min. Dissolve 20 g agar in 800 ml water and sterilize. Add 200 g cherry extract, distribute the mixture over presteriiized tubes and sterilize for 5 min at 0.1 atm. (102°C). pH=3.8-4.6. 3. CMC-agar 10 g carboxymethyl cellulose, 3 g NaNO , I g KH^PO , 0.5 g KC1. 0.5 g MgSO •7H,0, 0.01 gFeS04-7H10, 0.5 g yeast extract. ‘ A suitable medium for isolating fungi' from washed soil particles and for inducing sporulation in cellulolytic fungi. 4. Commeal agar (CMA) Add 60 g freshly ground cornmeal to 1 1 water, heat to boiling and simmer gently for 1 h. Strain through cloth and sterilize for 1 h at 1 atm. overpressure. Fill up to 1 1. 5. Czapek agar 30 g saccharose, 2 or 3 g NaNO,, 1 g K.HPO^, 0.5 g KC1, 0.5 g MgSO -7H 0, 0.01 gFeS04-7H20. 6. Czapek-Yeast autolysate agar (CYA) 1 g K,HP04, 10 ml Czapek concentrate, 5 g yeast autolysate or extract, 30 g sac charose. For identification of PeniciUmm (Pitt, 1979). 7. Dichloran-Glycerol medium (DG18) 5 g peptone, 10 g glucose, 1 g KH,P04. 0.5 g MgS04-7H,0. 2 mg Dichloran. Used particularly for food-borne fungH 8. Hay-infusion agar Sterilize 50 g hay in I I water at 120°C (I atm.) for 30 min. Strain through cloth, fill up to 1 1 and adjust the pH to 6.2 with K.,HP04. 9. Lindane agar (Newton & Nibley, 1956) Dissolve 750 mg lindane 100% in 10 ml acetone. Add 1 ml of this solution to 1 1 malt agar. This medium is used to rescue mite-infested cultures. 10. Littman’s oxgall agar 10 g peptone. 10 g dextrose. 15 g oxgall. 20 g agar and 0.01 g crystal violet. Used to isolate dermatophytes and other Gymnoascaceae. 11. McCIaiy’s medium (acetate medium) 1 g glucose, 1.8 g KC1. 2.5 g yeast extract, 8.2 g sodium acetate trihydrate. (Used for inducing ascospore formation in some yeasts). 12. Malt-extract agar 4% (2%) (MEA) Add water to malt extract from the brewery until it contains 10% sugar (meas urement with areometer). Mix 400 ml (200 ml) of this solution witii 15 g agar and 600 (800) ml water. Malt agar may also conveniently be prepared with malt syrup (10-40 g/1) or malt powder (10-20 g/I). 13. Malt-peptone agar 20 g powdered malt extract. 1 g peptone. 20 g glucose. Blakeslee’s formula, used for PemciUium. 14. Modess agar 0.5 g KH,P04. 0.5 g MgSO H,0. 0.5 g NH4CI. 10 drops 1% FeCl 5 g glu cose, 5 g malt extract, 20 g agar and 1 I water. Adjust pH to 5.7 with HC1. Sterilize for 30 min at an overpressure of 0.5 atm. (110°C). 15. Oatmeal agar (OA) Heat 30 g oat flakes in 1 1 water to boiling and simmer gently for 2 h. Filter through cloth and sterilize for 1 h at 1 atm. Fill up to 1 1. When using powdered oatmeal, filtering is superfluous. Lupin stems may be placed in slants with oat meal agar. 16. Oxytetracydine-Glucose-Yeast extract agar (OGY) 5 g yeast extract, 20 g glucose. 0.1 mg biotin. To 500 ml of the cooled medium the contents of one vial of oxytetracycline is added after rehydration in 10 ml water. Commonly used in food mycology. 17. Potato-carrot agar (PCA) 20 g carrots and 20 g potatoes are washed, pealed, chopped, boiled and simmered for 1 h in I 1 water, boiled again for 5 min. and filtered off. 18. Potato-dextrose agar (PDA) Add 200 g scrubbed and diced potatoes to I I water and boil for I h. Let it pass through a fine sieve, add 20 g dextrose and boil until dissolved. Do not use new potatoes! 19. Potato-sucrose agar (PSA) Same recipe as above, but replace dextrose by sucrose. / - r~—ma ■ 20. Sabouraud agar 40 g maltose (or glucose) and 10 g peptone. ‘TBriS 21. SNA (Synthetic meagre agar = synthetischer nahrstoflarmer Agar, Nirenberg) --W J g KH2PO 1 g KN03, 0.5 g MgS04-7H.,0, 0.5 g KCl, 0.2 g glucose, 0.2 g saccharose, 1 1 distilled water. Pieces of filter'paper or lens tissue may be added as . carbon source. Suitable for identifying Fmarium. CtSra :3^ 22. Soil-extract agar (SEA) Mix approximately equal quatities per weight of soil and water and autoclave for - -.-=»s 30 min at 1 atm. Allow the soil to sediment and filter the supernatant through t" ■ 7 'rj’rrri Pv-ssv' ^*ter PaPer- Add 15 g agar to 1 1 soil extract. 23. Tiyptone-glucose agar (Whisler’s formula, modified) 5 g tryptone. 3 g glucose. 0.2 mg thiamine-HCl, 280 mg KH^PO^. 260 mg (NH4)2S04, 100 mg MgCl2-6H,0, 60 mg CaClv 24. V-8-juice agar 200 ml V-8 juice, 3 g CaC03, 20 g agar, 1 1 water. Sterilize 30 min at 0.5 atm. 25. YpSs agar (Emerson’s medium. 1958) Boil 1 g K,HP04, 0.5 g MgSO}-7H,, 0.4 g yeast extract, 15 g soiuble starch in 1 1 water “until the ingredients are dissolved: add 15 g agar and fill up with water t0 1 1. 26. Natural media Many fungi sporulate better on sterilized plant material, e.g. lupin stems, pieces of carrots, twigs of various trees, which are placed in test tubes with 3 ml water and sterilized at 1 atm. for 1 h, or cold-sterilized by adding 1 ml propylene oxide to a closed vessel of 1 1 kept tight overnight. As protection against mite-infestation, the cotton plug of the specimen tubes can be treated at the margin of the tube with the following solution: 500 ml ethanol 96%, 450 ml water, 50 ml glycerol, 10 g mercuric chloride and a dye (e.g. eosin). The solution is very poisonous and must be handled with care! Smith (1967) recommended adding a few drops of Cypro (pyrethrin+piperonyl butoxide) or Kel- thane to the cotton plug of a tube or to the lid of a reversed petri dish, which : serves to clean mite-infested fungal cultures. For the latter purpose Lindane-agar 1 (no. 7) can also be used. Mites in fungal cultures can also be killed by placing the tube or Petri dish in a domestic microwave oven (Magnetron) for 1-12 sec (Pietrini, 1983). C. CHOICE OF MEDIA, INCUBATION j The choice of media is determined by the purpose of the investigation: iso lation (rapid growth or reduction of too rapid spread, transparency) - determina tion (good sporulation. standardization according to published descriptions) - preser vation - or biochemical tests. For the latter fully synthetic media often are necessary. CXOEj For purposes, of isolation and preservation numerous natural substrates are conven ient. Many fungi behave better on insoluble substrates, such as starch or cellulose which are gradually hydrolysed by extracellular enzymes. Oatmeal or cornmeal agar i-... ciog 6 ■ iT .